海绵状静脉畸形的实验与临床研究
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摘要
海绵状静脉畸形源于血管形态发生的异常,扩张的静脉包含薄的血管壁和异常的平滑肌。病变表现为蓝色、柔软、可被压缩,或可触及坚硬钙化的静脉石。静脉畸形包括了小范围局限性的皮肤静脉畸形以及涉及重要结构的多部位多层次的弥漫性畸形。因静脉畸形有不断发展的特征,使治疗尤为困难；随时间推移,尤其是在青春期,畸形会增大；静脉畸形治疗后容易复发。经皮穿刺硬化疗法作为治疗静脉畸形的一线疗法,和手术相比最大的优点是没有外部瘢痕、很少有严重并发症,一般情况下比切除更安全有效。硬化疗法是把组织硬化剂注射到病变处,引起细胞破坏、血栓形成以及强烈的炎症反应,局部纤维化导致病变收缩。75-90%的患者有良好的结果,包括病变体积的缩小和症状的减轻。硬化治疗常需持续多次。
在我国平阳霉素硬化治疗静脉畸形作为一种常规治疗方式,疗效确切。为指导应用,中华口腔医学会口腔颌面外科专业委员会脉管性疾病学组2011年建议了平阳霉素治疗静脉畸形方法,其中利多卡因作为一种联合用药与平阳霉素配制成一定浓度药液注入畸形静脉中。利多卡因一方面作为酰胺类局麻药物,用于不同部位以阻断感觉神经而产生麻醉,其穿透性和扩散性强。治疗静脉畸形可减轻平阳霉素栓塞过程中因疼痛引起的反射性血管痉挛,使硬化药物更充分的填塞病灶。另一方面,利多卡因作为钙调蛋白阻滞剂对抗肿瘤药物有增敏作用。
利多卡因、平阳霉素注入到畸形静脉中对血管内皮细胞的作用如何?利多卡因是否在平阳霉素治疗静脉畸形过程中有协同作用?平阳霉素和利多卡因浓度的改变对临床治疗效果有何影响?
本课题分为两个部分,第一部分采用昆明小鼠脾脏作为人海绵状静脉畸形的动物模型,通过石蜡切片光镜观察、超薄切片电镜观察、末端转移酶介导的缺口末端标记法(TUNEL法)标记凋亡细胞,Image-Pro Plus6.0病理细胞图像分析系统比较细胞凋亡率,实时荧光定量PCR检测细胞凋亡因子半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达,观察不同浓度利多卡因、不同浓度平阳霉素在不同时间对脾脏血管内皮细胞的毒性作用以及平阳霉素与不同浓度利多卡因联合用药时在不同时间对血管内皮细胞的毒性作用,探讨利多卡因是否对平阳霉素治疗海绵状静脉畸形有协同作用。第二部分回顾性分析平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形,探讨利多卡因浓度对疗效的影响；回顾性分析不同浓度平阳霉素治疗浅表海绵状静脉畸形,探讨平阳霉素浓度对治疗效果的影响,指导临床用药；回顾性分析低浓度平阳霉素治疗浅表静脉畸形,总结治疗效果。
第一部分利多卡因影响平阳霉素治疗海绵状静脉畸形的实验研究
实验1不同浓度利多卡因对小鼠脾脏血管内皮细胞的影响
目的探讨利多卡因对血管内皮细胞的细胞毒性。
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只,为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.1%利多卡因组、0.2%利多卡因组、0.4%利多卡因组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。将小鼠用10%水合氯醛(3ml/kg)腹腔注射麻醉后,常规消毒、铺巾。分层切开皮肤、肌层、腹膜,切口长约1.0cm,将胃翻起显露其背侧的脾脏,钝性牵拉固定,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射不同浓度盐酸利多卡因溶液0.3ml,边注射边匀速退针,稍加压迫针孔止血,分层缝合,继续饲养。术后第2、5、8、14d处理对照组的4只小鼠,实验组B、C、D中的各6只小鼠。麻醉后,按照原切口再次开腹,游离并切断结扎脾脏周围血管及系膜,取出脾脏,去除脾脏头尾,将脾脏中段分割后,分别投入10%中性福尔马林及2.5%的戊二醛溶液中。HE染色,观察不同浓度、不同时间标本变化；TUNEL法检测细胞凋亡,每例标本制切片2张,用Image-Pro Plus6.0病理细胞图像分析系统,将各组TUNEL标记切片进行分析。在400×光镜下,每张切片随机选择6个高倍视野的图片,分别计数每张切片中TUNEL阳性细胞比率(=6个高倍视野的阳性细胞数／细胞总数×100%)并计算其平均值。电镜下观察血管内皮细胞变化。
统计方法：正态分布计量资料采用均数±标准差(x±S)描述,时间与处理因素的效应分析采用析因设计的方差分析,若交互作用有统计学差异,进一步分析单独效应。满足正态且方差齐的数据,采用单因素方差分析(One-way ANOVA);非正态和(或)方差不齐的数据,采用Kruskal-Wallis H秩和检验；若有统计学意义,用SNK法进一步做两两比较,P<0.05为差别有统计学意义,所有数据用SPSS16.0分析。
结果大体观察,对照组2、5、8、14天外观未见明显改变,均表现为包膜光滑完整,边缘整齐,无肿胀。各实验组与对照组比较无明显差异。
显微镜下,生理盐水组,各时间点为正常脾脏表现：脾脏表面被覆薄层纤维组织,其外为单层间皮细胞；其下红、白髓结构清楚,血窦不扩张,脾窦内衬单层内皮细胞,内皮细胞完整,血窦内充以适量的红细胞,间质无炎细胞浸润,组织细胞不增生；脾小体结构清楚,生发中心不扩张。0.1%利多卡因组：各时间点与对照组相比无明显变化。0.2%利多卡因组：2天组,为正常脾脏表现；5天组,偶见脾窦出血；8天组,间质偶见炎症细胞浸润,偶见局部单核或多核巨噬细胞；14天组,间质细胞浸润较8天组稍多,偶见纤维蛋白渗出,偶见吞噬了细胞碎片的多核巨噬细胞。0.4%利多卡因组：2天组,偶见脾窦扩张,充血,红白髓结构清楚,间质偶见炎症细胞浸润；5天组,脾窦扩张、充血较明显,间质炎症细胞浸润增多；8天组,可见间质细胞浸润,少量纤维蛋白渗出,部分脾索结构略模糊,可见到吞噬了细胞碎片的多核巨噬细胞；14天组,间质细胞浸润、纤维蛋白渗出增多,局部可见到多个吞噬了细胞碎片的多核巨噬细胞。部分红白髓结构模糊。
透射电镜观察：生理盐水组、0.1%利多卡因组、0.2%利多卡因组各时间点脾脏可见大量红髓,血窦清晰,细胞连接完好,红细胞聚于脾窦内且数量较多,淋巴细胞分布均匀,结构完整,细胞器完好,整体无明显组织学改变。镜下未见凋亡小体。0.4%利多卡因组2天组、5天组脾脏结构未见明显改变,8天组、14天组可见脾窦筋膜破坏、可见凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝107.676,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间=4.725,P时间=0.005),时间与组间交互效应有显著性意义(F组间×时间＝13.682,P组间×时间＝0.000)。
单独效应方差分析结果表明,生理盐水组4个时间点凋亡率比较差别无统计学意义(F=0.296,P=0.828)。0.1%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别无统计学意义(X2=3.687,P=0.297)。0.2%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别无统计学意义(X2=4.167,P=0.244)。0.4%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别有统计学意义(X2=19.747,P＝0.000)。其中,14天组>8天组>5天组,14天组>2天组。
单独效应方差分析结果表明,2天组：各组之间比较差别无统计学意义(F=1.386,P=0.279)。5天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(X2=11.186,P=0.011)。其中,D组>A组,D组>B组,D组>C组。8天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(X2=13.717,P=0.003)。其中D组>A组,D组>B组,D组>C组。14天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(χ2=12.753,P=0.005)。其中D组>A组,D组>B组,D组>C组。
结论我们的研究表明,利多卡因在0.1%和0.2%的浓度时,小鼠脾脏血管内皮细胞未见组织学改变,未引起细胞凋亡。随着利多卡因干预浓度的增加、作用时间的延长,利多卡因具有细胞毒性作用,可诱发细胞的凋亡。利多卡因诱发小鼠脾脏内皮细胞凋亡具有明显的时间和浓度依赖性。
实验2不同浓度平阳霉素对小鼠脾脏血管内皮细胞的影响
目的探讨平阳霉素对血管内皮细胞的细胞毒性
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.5mg/ml平阳霉素组、1mg/ml平阳霉素组、2mg/ml平阳霉素组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。按照实验1手术方法找到小鼠脾脏,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射不同浓度平阳霉素溶液0.3ml。术后处理同实验1。统计方法同实验1。
结果大体观察,生理盐水组：各时间点外观未见明显改变。0.5mg/ml平阳霉素组：2天组,脾组织暗红光亮,未见明显变化；5天组,个别标本脾组织颜色稍暗,包膜略有紧张；8天组,部分标本脾组织边缘稍钝,轻度肿胀；14天组,大部分标本出现脾组织颜色变暗,略凹陷,包膜紧张度消失。1mg/ml平阳霉素组：2天组,部分脾组织包膜略紧张,有轻度肿胀；5天组,脾组织色泽变暗,边缘稍钝,有轻度肿胀；8天组,脾组织包膜紧张度消失,部分区域出现微凹；14天组,部分标本脾组织萎缩,边缘凹凸不平有切迹,表面白色条纹样瘢痕,与周围组织有粘连。2mg/ml平阳霉素：2天组,脾组织包膜紧张,边缘稍钝,有不同程度肿胀；5天组,脾组织包膜紧张度消失,颜色变暗,部分区域出现微凹；8天组,脾组织不同程度萎缩,部分标本出现边缘切迹,被膜凹凸表现,表面有白色条纹样瘢痕,与周围组织有粘连；14天组,大部分标本出现脾脏质硬暗红,包膜凹凸皱缩,表面有白色瘢痕,脾脏边缘锐利伴有不规则切迹,与周围组织严重粘连,呈现肉眼可见的明显萎缩。
显微镜下,生理盐水组：各时间点为正常脾脏表现。0.5mg/ml平阳霉素：2天组,个别脾窦出现扩张,充血,少量炎细胞浸润；5天组,间质炎细胞浸润,窦内皮细胞肿胀,个别出现变性,少量纤维蛋白渗出。8天组,脾窦扩张、充血显著,窦内皮细胞肿胀、变性,部分脾索结构模糊,较多炎细胞浸润、纤维蛋白渗出及组织细胞增生；14天组：纤维蛋白渗出、变性,纤维组织增生,红细胞弥散,部分脾窦塌陷毁损,脾小体萎缩形态不均。1mg/ml平阳霉素：2天组,脾窦出血、扩张,少量炎细胞浸润,偶见局部多核巨噬细胞；5天组,脾组织充血显著,脾窦炎细胞浸润,纤维蛋白沉积,内皮细胞肿胀、变性,多核巨噬细胞增多；8天组,脾索结构模糊,纤维蛋白沉积伴有纤维组织的增生,可见新鲜出血；14天组,脾窦塌陷毁损,部分脾小体萎缩、形态不均,纤维蛋白渗出、变性,纤维组织增生,较多单核或多核巨噬细胞云集。2mg/ml平阳霉素：2天组,脾窦扩张,充血,窦内皮细胞肿胀,个别窦内皮细胞及脾索纤维细胞变性,或细胞核浓缩、碎裂,脾索结构略模糊,炎细胞浸润、组织细胞增生；5天组,脾组织充血显著,较多窦内皮细胞及脾索纤维细胞变性,大量细胞核浓缩、碎裂、崩解,较多单核或多核巨噬细胞云集,脾索结构模糊,较多炎细胞浸润、组织细胞增生；8天组,脾窦塌陷毁损,内皮细胞胞核溶解、散碎,大量单核或多核巨噬细胞云集。纤维蛋白渗出、变性,纤维组织增生,红细胞弥散,部分脾小体萎缩形态不均,边缘充血、出血；14天组,脾窦内皮细胞凋亡成片,脾小体塌陷萎缩,纤维组织增生,被膜增厚。
电镜下,对照组各时间表现同实验1。各实验组各时间均可见不同程度的血窦壁破坏,线粒体空泡样变、内质网扩张,凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝9549.392,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间=2499.782,P时间=0.000),时间与组间交互效应有显著性意义(F组间×时间＝279.087,P组间×时间=0.000)。
单独效应方差分析结果表明,对照组：4个时间点凋亡率比较差别无统计学意义(F=0.296, P=0.828).0.5mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学差异(F=1144.825,P=0.000)。14天组>8天组>5天组>2天组。1mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学意义(F=1494.281,P=0.000),14天组>8天组>5天组>2天组。2mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学意义(F=963.028,P=0.000),14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：各组之间凋亡率比较差别有统计学意义(F=1232.831,P=0.000)。D组>C组>B组>A组。5天组：各组之间凋亡率比较差别有统计学意义(F=1201.614,P=0.000)。D组>C组>B组>A组。8天组：各组之间凋亡率比较差别有统计学意义(F=2134.600,P=0.000)。D组>C组>B组>A组。14天组：各组之间凋亡率比较差别有统计学意义(F=14886.604,P=0.000)。D组>C组>B组>A组。
结论平阳霉素在0.5mg/ml浓度下可引起细胞明显凋亡,随药物浓度增加,作用时间的延长,凋亡细胞增多,染色加深。PYM引起细胞凋亡的程度,具有明显的浓度和时间依赖性。
实验3平阳霉素与不同浓度利多卡因对小鼠脾脏血管内皮细胞的影响
目的探讨利多卡因是否对平阳霉素治疗海绵状静脉畸形有协同作用
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只,为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.5mg/ml平阳霉素组、0.5mg/ml平阳霉素+0.1%利多卡因组和0.5mg/ml平阳霉素+0.2%利多卡因组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。按照实验1手术方法找到小鼠脾脏,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射0.5mg/ml平阳霉素溶液或平阳霉素利多卡因混合液0.3ml。按照实验1方法在各个时间点取出小鼠脾脏。去除脾脏头尾,将对照组脾脏中断分割后,分别投入10%中性福尔马林及2.5%的戊二醛溶液中；将实验组脾脏中断分为三份,一份迅速置入冻存管,放入液氮中冻存；另两份分别投入10%中性福尔马林及2.5%的戊二醛溶液中。液氮中冻存组织用于凋亡蛋白Caspase-3的实时荧光定量(RT-PCR)检测。统计方法同实验1。
结果大体观察,对照组：2、5、8、14天外观未见明显改变。0.5mg/ml平阳霉素组：各时间点外观同实验2同等浓度平阳霉素组表现。0.5mg/ml平阳霉素+0.1%利多卡因组：各时间点与0.5mg/ml平阳霉素组相比外观无明显变化。0.5mg/ml平阳霉素+0.2%利多卡因组：2天组,脾组织暗红光亮色,部分标本包膜略紧张,略有轻度肿胀；5天组,脾组织色泽变暗,边缘稍钝；8天组,脾组织包膜紧张度消失,颜色变暗,部分区域出现微凹；14天组,部分标本脾组织萎缩,边缘凹凸不平有切迹。
显微镜下,对照组,各时间点为正常脾脏表现。0.5mg/ml平阳霉素组,各时间点同实验2同浓度平阳霉素组表现。0.5mg/ml平阳霉素+0.1%利多卡因组,各时间点与0.5mg/ml平阳霉素组相比无明显变化。0.5mg/ml平阳霉素+0.2%利多卡因组：2天组,部分脾窦出血、扩张,少量炎细胞浸润,偶见局部多核巨噬细胞；5天组,脾组织充血显著,脾窦炎细胞浸润,少量纤维蛋白沉积,内皮细胞肿胀、变性,多核巨噬细胞增多；8天组,脾索结构模糊,纤维蛋白沉积伴有纤维组织的增生,可见新鲜出血；14天组：部分脾窦塌陷毁损,部分脾小体萎缩、形态不均,纤维蛋白渗出、变性、增生,较多单核或多核巨噬细胞云集。
电镜观察,对照组各时间表现同实验1,各实验组各时间均可见不同程度的血窦壁破坏,线粒体空泡样变、内质网扩张,凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝1510.583,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间＝917.257,P时间＝0.000),时间与组间交互效应有显著性意义(F组间×时间＝88.540,P组间×时间=0.000)。
单独效应方差分析结果表明,生理盐水组4个时间点凋亡率比较差别无统计学意义(F=0.296,P=0.828)。0.5mg/ml平阳霉素组：采用秩和检验,4个时间点凋亡率比较差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.1%利多卡因组：采用方差分析,4个时间点凋亡率比较差别有统计学差异(F=876.169,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.2%利多卡因组：采用方差分析,4个时间点凋亡率比较差别有统计学差异(F=21.600,P=0.000)。14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.360,P=0.001)。其中,D组>C组>A组；D组>B组>A组。5天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.202,P=0.001)。其中,D组>C组>A组；D组>B组>A组。8天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.715,P=0.001)。其中,D组>C组>A组；D组>B组>A组。14天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.146,P=0.001)。其中,D组>C组>A组；D组>B组>A组。
各实验组Caspase-3拷贝数进行析因方差分析,不同组间Caspase-3拷贝数比较差异显著(F组间=9549.392,P组间＝0.000),不同时间点Caspase-3拷贝数比较差异显著(F时间＝2499.782,P时间＝0.000),时间与组间交互效应有显著性意义(F组间×时间=279.087,P组间×时间＝0.000)。
单独效应方差分析结果表明,0.5mg/ml平阳霉素组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.1%利多卡因组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.2%利多卡因组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=15.158,P=0.001)。D组>C组>B组。5天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=11.415,P=0.003)。其中,D组>C组；D组>B组。8天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=11.474,P=0.003)。其中D组>C组；D组>B组。14天组：采用方差分析,各组之间比较caspase-3拷贝数差别有统计学意义(F=127.582,P=0.000)。其中,D组>C组；D组>B组。
结论0.2%的利多卡因在平阳霉素导致的细胞凋亡中起到协同作用,并且随时间延长作用增加。提示利多卡因在配合平阳霉素治疗海绵状静脉畸形的过程中,不仅是起到封闭和止痛作用,而且对平阳霉素细胞毒性具有协同作用,且具有浓度及时间依赖性。本实验数据可以假定利多卡因在人体内会起到类似的作用。然而,由于动物模型实验的有限性,尽管观察了利多卡因对平阳霉素细胞毒性作用的协同作用,对于临床的真正影响及指导需要进一步的研究。
第二部分平阳霉素硬化治疗海绵状静脉畸形的临床研究
第一节平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形的对照研究
目的比较平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形的临床疗效,探讨利多卡因浓度对治疗效果的影响。
方法自2008年6月-2010年6月,我们采用2mg/ml平阳霉素、2mg/ml平阳霉素联合0.5%利多卡因及2mg/ml平阳霉素联合1%利多卡因,分别治疗海绵状静脉畸形各40例。所有患者知情同意。建立患者标准化数据收集表格,包括患者年龄、性别、病变部位、大小、病史专科检查、注射药量,治疗次数、临床反应、副作用以及随访。患者治疗前后拍摄数码照片。注射方法：严格消毒后,用5号针头从距病变边缘0.5cm处正常皮肤处进针,针尖水平穿刺到静脉畸形处,回抽出血确定注射器刺入的静脉区域,缓慢注射至局部肿胀,稍显苍白,注意勿使局部注药后张力过大。对面积稍大的静脉畸形,一处进针药液不能浸润到的部位,可采用多点注射。每次注射的药液体积是1.5-6.0ml。治疗后局部压迫穿刺点3min以防药液外溢。根据病变情况决定治疗的次数,2次治疗间隔15d。每次治疗后留观0.5h。三组治疗操作均为一人独立完成。
统计学方法三组性别比较采用卡方检验,年龄、病变面积和治疗次数比较采用完全随机设计资料的方差分析,三组间的疗效比较采用Kruskal-Wallis H秩和检验,三组间的不良反应比较采用卡方检验,P<0.05为差别有统计学意义。所有数据用SPSS16.0分析。
结果三组患者性别、年龄、病变面积差异均无统计学意义(P>0.05)。2mg/ml平阳霉素联合1%利多卡因治疗组的平均治疗次数有降低趋势,但三组治疗次数差异无统计学意义(P=0.586)。经过注射治疗后,随访12-16个月,均未见有肺纤维化及肝肾功能损害现象,三组间的疗效差异无统计学意义(P=0.904)。三组患者治疗后病变部位均有不同程度的肿涨和疼痛,在临床观察中2mg/ml平阳霉素组疼痛表现程度远大于另外两组。各组中均有发热、胃肠道反应及局部水疱或皮肤溃疡、坏死发生。三组不良反应差异无统计学意义(P=0.875)。
结论利多卡因对平阳霉素治疗海绵状静脉畸形未表现出协同作用。
第二节不同浓度平阳霉素治疗海绵状浅表静脉畸形的对照研究
目的比较不同浓度平阳霉素治疗海绵状浅表静脉畸形的临床疗效。
方法自2008年2月-2010年2月,我们采用低浓度平阳霉素(0.5mg/ml)及常规浓度平阳霉素(2mg/ml)分别治疗体表单发、局限性海绵状静脉畸形各32例。所有患者知情同意。建立患者标准化数据收集表格,包括患者年龄、性别、病变部位、大小、病史专科检查、注射药量,治疗次数、临床反应、副作用以及随访。常规消毒后,4号半针头从距病变边缘0.5cm处正常皮肤粘膜处进针,回抽出血确定注射器刺入的静脉区域,缓慢注射至局部肿胀,稍显苍白。对面积稍大的静脉畸形,一处进针药液不能浸润到的部位,可采用多点注射。每次注射的药液体积是0.7-6.0ml。治疗后局部压迫穿刺点3min以防药液外溢。根据病变情况决定治疗的次数,2次治疗间隔15d。每次治疗后留观0.5h。两组治疗操作均为一人独立完成。
统计学方法两组性别比较采用卡方检验,年龄和病变面积比较采用t检验,两组间的疗效比较采用Mann-Whitney U秩和检验,两组间的不良发应采用卡方检验,P<0.05为差别有统计学意义,所有数据用SPSS16.0分析。
结果两组患者性别、年龄、病变面积差异均无统计学意义(P>0.05)。经过1-6次注射治疗后,随访12-16个月,均未见有肺纤维化及肝肾功能损害现象,两组间的疗效差异无统计学意义(P=0.306)。两组患者治疗后病变部位均有不同程度的肿胀和疼痛,在临床观察中低浓度组表现程度远小于正常浓度组。两组不良反应差异无统计学意义(P=0.198)。
结论低浓度平阳霉素硬化治疗重要功能器官的单发、局限性浅表静脉畸形,局部肿胀轻、并发症少,安全有效,患者耐受性好,是一种较好的治疗方式。
第三节低浓度平阳霉素联合地塞米松硬化治疗唇部浅表静脉畸形
目的观察低浓度平阳霉素加地塞米松硬化治疗唇部浅表静脉畸形的临床疗效。
方法2007年1月-2011年1月,应用低浓度平阳霉素(0.5mg/ml)联合地塞米松(1mg/ml)硬化治疗唇部浅表静脉畸形患者68例,其中男29例,女39例,年龄13～69岁；病变范围最小0.5×0.8cm,最大3×2.4cm。根据病变情况决定治疗的次数,2次治疗间隔15d。
结果68例患者最少注射2次,最多注射8次,治疗后随访12～32个月,平均随访21个月。患者外观满意,没有复发倾向。根据疗效评价标准,66例患者治愈,2例患者有效。
结论低浓度平阳霉素联合地塞米松硬化治疗唇部浅表静脉畸形是安全有效的治疗方法。
第四节低浓度平阳霉素注射治疗龟头部静脉畸形
目的探讨龟头部静脉畸形的治疗方法。
方法对2002年1月～2010年10月收治29例龟头部静脉畸形患者,采用低浓度平阳霉素(0.5mg/ml)经皮穿刺注射治疗病变区域。年龄16～33岁,龟头部病变为蓝色、突起的、不规则肿物,指压后可缩小,彩色多普勒超声检查证实为血管样病变,与尿道海绵体或阴茎海绵体之间有清楚的界限。每次治疗注射的药液体积是1～3ml,并在阴茎体部病变区近心端置于止血带,自注射时到注射后5分钟阻断龟头部的血液回流,以保持静脉畸形体内的药物浓度及含量。治疗后外用抗生素软膏,纱布适度加压包扎。根据病变情况决定重复治疗的次数,2次治疗间隔15天。
结果16例患者治疗3次,8例4次,5例6次。治疗后随访12～38个月,平均随访时间为18个月。26名患者治愈,3名患者有效,均没有复发倾向。患者均具有正常的阴茎感觉、勃起和排尿功能,外观满意。
结论低浓度平阳霉素注射治疗是龟头部静脉畸形安全有效的首选的治疗方法。
Venous malformation(VM) results from an error in vascular morphogenesis; veins are dilated with thin walls and abnormal smooth muscle. Consequently, lesions expand, flow stagnates, and clotting occurs. Lesions are blue, soft, and compressible; hard calcified phleboliths may be palpable. VMs may range from small localized skin lesions to diffuse malformations involving multiple tissue planes and vital structures. A VM is especially problematic because it is progressive; it enlarges over time, particularly during adolescence; and often reexpands after treatment.Consequently, most patients who present with congenital lesions will ultimately require intervention.
The first-line treatment of problematic VMs is sclerotherapy, which is generally safer and more effective than resection. Sclerotherapy involves the injection of a sclerosant into the malformation, which then cause cellular destruction, thrombosis, and intense inflammation. Scarring leads to shrinkage of the lesion. Good to excellent results are obtained in75%to90%of patients, including reduction in the size of the malformation and alleviation of the symptoms. Often, multiple treatments are required.
In our country, Pingyangmycin sclerotherapy as a conventional treatment method for the treatment of venous malformation is observed to have exact curative effect. In order to guide application, therapeutic method of venous malformation with Pingyangmycin and lidocaine was proposed by committee of Oral and Maxillofacial Surgery, Branch Association of stomatology, China medical Association in2011.On one hand, Lidocaine as amide local anaesthetics can be used at different sites to block sensory nerve and lead to anesthesia, with strong penetrability and diffusivity. During the treatment of venous malformation, it can help to alleviate reflective vasospasm caused by the pain due to embolisation with Pingyangmycin and promote the medicine to fill up the nidus more completely. On the other hand, Lidocaine as calmodulin inhibitor has sensitivity-increasing effect for antineoplastic drug.
This subject adopts the spleen of Kunming mouse as the animal model for human cavernous venous malformation to observe toxic effects of Lidocaine of different concentrations and Pingyangmycin of different concentrations to splenic vascular endothelial cells at different time, and toxic effects of combined medicine of Pingyangmycin and Lidocaine of different concentrations to vascular endothelial cells at different time, and to explore whether Lidocaine has synergic action to Pingyangmycin for the treatment of cavernous venous malformation by means of paraffin section optical microscopic observation, ultrathin section electron microscopic observation, labeling apoptotic cells in terminal transferase mediated nick end labeling method (TUNEL method), comparing apoptosis rate according to Image-Pro Plus6.0pathologic cells'image analysis system, and detecting expression of cysteinyl aspartate specific protease-3(Caspase-3) of apoptosis factor by real-time quantitative PCR technology. Retrospectively analyze treating cavernous venous malformation with Pingyangmycin and Lidocaine of different concentrations to explore the influence of Lidocaine for curative effect in aspect of clinic medication proportion; retrospectively analyze treating superficial cavernous venous malformation with Pingyangmycin of different concentrations to explore the influence of Pingyangmycin concentration for therapeutic effect and guide clinic medication; retrospectively analyze treating superficial venous malformation with Pingyangmycin of low concentration to conclude therapeutic effect.
Part I Experimental Study on Influence of Lidocaine for the Treatment of Venous Malformation by Pingyangmycin
Experiment1Influence of Lidocaine of Different Concentrations for Mouse Splenic Vascular Endothelial Cells
Objective:To explore cytotoxicity of lidocaine to vascular endothelial cells.
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.1%Lidocaine group,0.2%Lidocaine group, and0.4%Lidocaine group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Mice was narcotized with10%chloral hydrate (3ml/kg) by intraperitoneal injection and disinfected. Surgical incision into the abdominal wall, turn up the stomach to reveal the spleen, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to contrast group and inject0.3ml Lidocaine Hydrochloride of different concentrations to experimental group. Withdraw injector at uniform speed while injecting, slightly press the pinhole of injector to stop bleeding, sew by layers, and continue to feed. Process4mice in control group on the2nd,5th,8th, and14th day after surgery, and6mice in experimental group B, C, and D. Open the peritoneal cavity along original cut after anesthesia, dissociate and cut off blood vessel and ligate mesentery around spleen, fetch the spleen, remove its head and tail, divided the middle section of the spleen into two parts., put into10%neutral formalin or2.5%glutaraldehyde solution respectively. Observe specimen changes at different concentrations and different times after HE staining; detect the apoptosis in TUNEL method by the following steps:prepare2sections for each specimen and analyze TUNEL labeling section of every group by Image-Pro Plus6.0pathologic cells' image analysis system. Randomly select6images under optical microscope at400times in every section.count TUNEL positive cell ratio (=6positive cell counts at high magnification/total cell counts×100%) in each section and calculate its mean value. Observe the changes of vascular endothelial cells under electron microscope.
Statistical Method:Normal distributed measurement data is described by mean±standard deviation (χ±s).Factor analysis was used to test the effect of interaction between group and time. If the effect of interaction was statistically significant then analyzed the effect alone. If the data are normal distribution and equal variance, then analyze by one-way ANOVA; if the data are abnormal distribution and (or) heterogeneity of variance, then adopt Kruskal-Wallis H Rank sum test; and if there is statistical significance, further make multiple comparison in SNK method, in which, if P﹤0.05, the disparity has statistical significance. The data were analyzed by SPSS software16.0.
Result:The appearances of physiological saline group have no significant changes on the2nd,5th,8th, and14th day, through general observation, manifesting that the capsulae is smooth and complete, the edge is tidy and without swelling. There is no obvious difference between every experimental group and control group.
Under microscope, physiological saline group shows normal spleen performances at every time point:the spleen surface is with a thin layer of fibrous tissue, the outer layer are monolayer mesothelial cells; under which the red and white pulps are clear, blood sinus is not dilated, the splenic sinus is lined with monolayer endothelial cells which are complete, the blood sinus is filled with appropriate amount of erythrocyte, there are no inflammatory cells infiltrated in mesenchyme, and the histiocyte has no hyperplasia; the splenic corpuscle has clear structure and the germinal center is not dilated.0.1%Lidocaine group:there is no obvious difference compared with contraol group at every time point.0.2%Lidocaine group:in2d day group, it shows a normal spleen performance; in5th day group, the splenic sinus bleeds occasionally; in8th day group, there are occasionally inflammatory cells infiltrated in mesenchyme and there appears local mononuclear or polynuclear macrophage occasionally; in14th day group, the infiltrated inflammatory cells are more than that in8th day group, there are fibrous protein exuding occasionally, and there appears polynuclear macrophages that swallow cell debris occasionally.0.4%Lidocaine group:in2nd day group, the splenic sinus is occasionally dilated and hyperemic, the red and white pulps are clear, and there are occasionally inflammatory cells infiltrated in mesenchyme; in5th day group, splenic sinus is dilated and obviously hyperemic, and inflammatory cells infiltrated in mesenchyme is increased; in8th day group, there exists interstitial cell infiltration, there is a small quantity of fibrous protein exuding, partial spleen trabecular structure is slightly obscure, and there appears polynuclear macrophages that swallow cell debris; in14th day group, the interstitial cell infiltration and fibrous protein exudation are increased, there appears fewer polynuclear macrophages that swallow cell debris locally, and the structures of partial red and white pulps are obscure.
Transmission Electron Microscope Observation:It was revealed that three groups (A, B and C group)showed the similar represent at the different time:red pulp in large quantities,integrate structure of blood sinus, good cell junction, many red blood cell in blood sinus, lymphocytes dispersed well and integrate organelles. No apoptotic bodies was observed under electronic microscope. In the D group, there was no obvious histology change of spleen at the2nd and5th day,but failure of blood sinus and apoptotic bodies can be observed at8th and14th day.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance (F组间=107.676, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=4.725, P时间=0.005) and the effect of interaction between group and time was statistically significant.(F组间×时间=13.682, P组间×时间=0.000)
The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828). Both B (χ2=3.687, P=0.297)and C (χ2=4.167, P=0.244)group showed the same result as A in the tests. The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group D (x2=19.747, P=0.000).14th day group>8th day group>5th day group, and14th day group>2nd day group.
Apoptosis rate comparison between all groups at different time points:in2nd day group, the comparison differences between every group have no statistical significance (F=1.386, P=0.279); in5th day group,8th group, and14group, the apoptosis rates of0.4%Lidocaine group are rising compared with other groups, D group> A group (P<0.05), D group>B group (P<0.05) and D group>C group (P <0.05).
Conclusion:When Lidocaine is of the concentrations of0.1%and0.2%, mice splenic vascular endothelial cells has no histological changes and all physiological functions work well, which cannot lead cell apoptosis. But as the intervention concentration of Lidocaine is increased and the action time is prolonged, the Lidocaine will have cytotoxicity and lead to cell apoptosis. Lidocaine inducing mice splenic vascular endothelial cells apoptosis has a time and concentration dependence.
Experiment2Influence of Pingyangmycin of Different Concentrations for
Mouse Splenic Vascular Endothelial Cells
Objective:Explore Cytotoxicity of Pingyangmycin to Vascular Endothelial Cells.
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.5mg/ml Pingyangmycin group,1mg/ml Pingyangmycin group, and2mg/ml Pingyangmycin group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Find mouse spleen according to the method of experiment1, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to control group and inject0.3ml Pingyangmycin of different concentrations to experimental group. Treatment after surgery is the same as experiment1and the statistical method is the same as experiment1.
Result:The appearances of physiological saline group have no significant changes.0.5mg/ml Pingyangmycin group:in2nd day group, the splenic tissue is dull-red and bright, without obvious change; in5th day group, individual specimen has slightly dark color, and the capsulae is a little strained, in8th day group, the margin of partial specimen is slightly blunt and with mild swelling; in14th day group, most specimens become dark, slightly sunk, and the turgidity of splenic capsula disappears; lmg/ml Pingyangmycin group:in2nd day group, partial capsula serosa lienis is slightly strained and slightly swelling; in5th day group, capsula serosa lienis become dark, the margin is slightly blunt and with mild swelling; in8th day group, the turgidity of capsula serosa lienis disappears, and a slight irregularity in the surface of the partial area.; in14th day group, part of specimen atrophy, the margin is uneven and has incisure, there is white stripe cicatrix on surface, and adheres to the surrounding tissues.2mg/ml Pingyangmycin:in2nd day group, the capsula serosa lienis is strained, the margin is slightly blunt and with swelling of different degrees; in5th day group, the turgidity of capsula serosa lienis disappears, the color becomes dark, and a slight irregularity in the surface of the partial area; in8th day group, the spleen atrophy at different degrees, partial specimen has margin incisure, the capsula has concave-convex, there is white stripe cicatrix on surface, and adheres to the surrounding tissues; in14th day group, most specimens have hard and dull-red spleens, the capsulas are concave-convex and atrophy, there is white cicatrix on surface, spleen margins are sharp and have irregular incisures, and adhere to the surrounding tissues and are remarkable macroscopic shrink.
Under microscope, physiological saline group shows normal spleen performances at every time point.0.5mg/ml Pingyangmycin:in2nd day group, individual sinus lienis is dilated and hyperemic, and there exists little inflammatory cell infiltration; in5th day group, there exists interstitial inflammatory cell infiltration, sinusoidal endothelial cells are swelling, individuals are degeneration and with little fibrous protein exudation, in8th day group, splenic sinuses are dilated and obviously hyperemic, sinusoidal endothelial cells are swelling and degeneration, part of splenic cords are obscure, and there exists more inflammatory cell infiltration, fibrous protein exudation, and histiocytosis; in14th day group, there exist fibrous protein exudation, degeneration, and fibroplastic proliferation, erythrocytes diffuse, part of splenic sinuses are sunk and destroyed, and splenic corpuscle atropies and has uneven forms, lmg/ml Pingyangmycin:in2nd day group, splenic sinus is hyperemic and dilated, there exists little inflammatory cell infiltration, and there appear polynuclear macrophages occasionally; in5th day group, splenic tissue is obviously hyperemic, and has inflammatory cell infiltration, fibrous protein deposits, endothelial cells swell and degeneration, and polynuclear macrophages increase; in8th day group, splenic cords are obscure, fibrous protein deposits with hyperplasia of fibrous tissue, and it is clear to see fresh bleeding; in14th day group, splenic sinuses are degeneration and destroyed, splenic corpuscle atrophise and has uneven forms, there exist fibrous protein exudation, degeneration, and more mononuclear or polynuclear macrophages gather.2mg/ml Pingyangmycin:in2nd day group, the splenic sinus is dilated and hyperemic, sinusoidal endothelial cells swell, individual sinusoidal endothelial cell and splenic cord fibrocyte degenerates, or cell nucleus concentrates, crushes, the splenic cord structure is obscure, and there exists little inflammatory cell infiltration and histiocyte hyperplasia; in5th day group, the splenic tissue is hyperemic obviously, more sinusoidal endothelial cells and splenic cord fibrocyte degenerate, a large number of cell nucleuses concentrate, crush, and disintegrate, much more mononuclear or polynuclear macrophages gather, the splenic cord structure is obscure, and there exists more inflammatory cell infiltration and histiocyte hyperplasia; in8th day group, splenic sinuses are destroyed, endothelial cell nuclei dissolves and shatters, and a large number of mononuclear or polynuclear macrophages gather, there exist fibrous protein exudation, degeneration, fibrous tissue proliferates, erythrocyte diffuse, part of splenic corpuscles shrink and with uneven forms, and the margin is hyperemic and bleeding; in14th day group, splenic sinus endothelial cell apoptosis increases, splenic corpuscle collapses and shrink, fibrous tissue proliferates, and the capsula is thicken.
Under Electron Microscope, It was revealed that groups A showed the represent as experiment1.Failure of blood sinus, apoptotic bodies, expanded Endoplasmic Reticulum and structural change of mitochondrion can be observed in varying degrees in group B,C and D at the different times.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance ((F组间=9549.392, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=2499.782, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=279.087, P组间×时间=0.000). The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828) The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group B (F=1144.825, P=0.000),C (F=1494.281, P=0.000)and D(F=963.028, P=0.000),14th day group>8thday group>5th day group,>2nd day group. Apoptosis rate comparison between all groups at different time points:in2nd (F=1232.831, P=0.000),5th (F=1201.614, P=0.000),8th (F=2134.600, P=0.000) and14th (F=14886.604, P=0.000) day group, the comparison differences between each group have statistical significance. D group>C group>B group> A group.
Conclusion:Pingyangmycin can lead to obvious vascular endothelial cell apoptosis at the concentration of0.5mg/ml. Its cytotoxicity to vascular endothelial cell shows obvious time and concentration dependence.
Experiment3Influence of Pingyangmycin and Lidocaine of Different Concentrations for Mouse Splenic Vascular Endothelial Cells
Objective:To Explore Whether Lidocaine Has synergistic effect on Pingyangmycin for Treatment of Cavernous Venous Malformation
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.5mg/ml Pingyangmycin group,0.5mg/ml Pingyangmycin+0.1%Lidocaine group, and0.5mg/ml Pingyangmycin +0.2%Lidocaine group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Find mouse spleen according to the method of experiment1, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to control group and inject0.5mg/ml Pingyangmycin solution or mixed solution of Pingyangmycin and Lidocaine0.3ml. Fetch mouse spleen at every time point according to the method of experiment1. Remove its head and tail, divide the middle section of the spleen into two parts in group A, then put into10%neutral formalin and2.5%glutaraldehyde solution respectively; divide the middle section of the spleen into three parts in experimental group, place one part is preserved in liquid nitrogen; put the other two parts into10%neutral formalin and2.5%glutaraldehyde solution respectively. The liquid nitrogen frozen tissue is used for real-time quantitative (RT-PCR) check of proapoptotic protein Caspase-3. The statistical method is the same as that in experiment1.
Result:The appearances of group A have no significant changes on the2nd,5th,8th, and14th day. Group B:appearances at every timing point are the same as the performances of Pingyangmycin group of the same concentration in experiment2. No significant changes were observed in group C compared with the group B. group D: in2nd day group, the splenic tissue is dull-red and bright, partial capsula serosa lienis is slightly strained and slightly swelling; in5th day group, spleen become dark and the margin is slightly blunt; in8th day group, the turgidity of capsula serosa lienis disappears, the color becomes dark, and slight irregularity in the surface of the partial area.; in14th day group, part of specimen the margin is uneven and has incisure.
Under microscope, group A shows normal spleen performances at every timing point. Group B, the performances at every time point are the same as the performances of Pingyangmycin group of the same concentration in experiment2. group C:The performances at every timing point have no obvious changes compared with that of group B. group D:in2nd day group, partial splenic sinus is dilated and hyperemic, there exists little inflammatory cell infiltration, and there appear polynuclear macrophages occasionally; in5th day group, splenic tissue is obviously hyperemic, splenic sinus has inflammatory cell infiltration, little fibrous protein deposits, endothelial cells swell and denaturize, and polynuclear macrophages increase; in8th day group, splenic cords are obscure, fibrous protein deposits with hyperplasia of fibrous tissue, and it is clear to see fresh bleeding; in14th day group, part of splenic sinuses are destroyed, partial splenic corpuscle shrinks and has uneven forms, there exists fibrous protein exudation, degeration, and proliferation, and more mononuclear or polynuclear macrophages gather.
Under Electron Microscope, It was revealed that groups A showed the represent as experiment1.Failure of blood sinus, apoptotic bodies, expanded Endoplasmic Reticulum and structural change of mitochondrion can be observed in varying degrees in group B, C and D at the different times.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance (F组间=1510.583, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=917.257, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=88.540, P组间×时间=0.000). The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828) The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group B (χ2=21.600, P=0.000),C (F=876.169, P=0.000) and D (F=21.600, P=0.000),14th day group>8thday group>5th day group,>2nd day group. Apoptosis rate comparison between all groups at different time points:in2nd (χ2=17.360, P=0.0011,5th (χ2=17.202, P=0.001),8th (χ2=17.715, P=0.001) and14th (χ2=17.146, P=0.001) day group, the comparison differences between each group have statistical significance. D group>C group>A group, D group>B group> A group.
Factor analysis was used to test the caspase-3copy numbers, the comparisons of three groups have statistical significance (F组间=9549.392, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=2499.782, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=279.087, P组间×时间=0.000).The results of analysis show that the difference of caspase-3copy numbers had statistics significance at four time points in the group B(χ2=21.600, P=0.000),C (χ2=21.600, P=0.000) and D (F=21.600, P=0.000),14th day group>8th day group>5th day group,>2nd day group. Caspase-3copy numbers comparison between all groups at different time points:in2nd day group, the comparison differences between all groups have statistical significance (χ2=15.158, P=0.001), group D>group C>group B. In5th day group (χ2=11.415, P=0.003),8th day group (χ2=11.474, P=0.003), and14th day group (F=127.582, P=0.000), the comparison between all groups, group D>group C; group D> group B.
Conclusion:When applying0.5mg/mI Pingyangmycin and0.1%Lidocaine, the apoptosis rate of splenic endothelial cell has no obvious change compared with only applying Pingyangmycin, and Lidocaine does not have synergistic action for apoptosis induced by Pingyangmycin; when applying0.5mg/ml Pingyangmycin and0.2%Lidocaine, the apoptosis rate of splenic endothelial cell is obviously increased compared with only applying Pingyangmycin and is obviously increased compared with applying0.5mg/ml Pingyangmycin and0.1%Lidocaine, Lidocaine has synergistic action for apoptosis induced by Pingyangmycin and the action improves as time goes on. It implies that Lidocaine's synergistic action to Pingyangmycin has time and concentration dependence.
Part II The Clinical Study on Pingyangmycin Sclerotherapy of Cavernous Venous Malformation
Section I Effect of Lidocaine in Defferent Concentration with Pingyangycin on Cavernous Venous Malformation
Objective To investigate the clinical efficacy of Lidocaine in defferent concentration with Pingyangycin oncavernous venous malformation.
Method Patients with cavernous venous malformation were treated with Lidocaine in defferent concentration with Pingyangycin in our department from June2008to June2010. Patients were divided into three groups:treated with1mg/ml Pingyangmycin (group A), treated with lmg/ml Pingyangmycin with0.5%Lidocaine (group B), treated with lmg/ml Pingyangmycin with1%Lidocaine (group C). All of the patients presented with a measurable lesion. Informed consent was given to the use of drug as injection therapy. A standardised data collection sheet recorded patient details including:age, sex, weight, location of lesion, size, clinical history, special investigations, dosage, clinical response, side effects and follow-up. Colour photographs were taken of every patient before, during, and after completion of treatment. All patients underwent direct intralesional injection of different solution.A needle connected to a syringe containing liquid medicine was inserted into the venous space, and the intraluminal position of its tip was confirmed by blood aspiration. The sclerosant was then injected slowly into the lesion. The volume of the solution varied from1.5to6.0ml per injection.They were observed0.5h and ischarged home. Treatment was performed in several stages. The applications were carried out after intervals of15days. Procedure was done by one operator.
Utilizing Chi-square test, analysis of variance and rank sum texst to analyse the collected data in SPSS16.0software. If P<0.05, the difference has statistical significance.
Result The ages, sexes and clinical characteristics of patients with cavernous venous malformation were not significantly different (P>0.05). There were decreasing trend of mean therapeutic times in group C, but therapeutic times in three groups had no significant difference (P=0.586). Twelve to16months followed up after termination of the therapy. All patiens had no functional lesion of liver and kidney and pulmonary fibrosis. Curative effect in three groups had no significant difference (P=0.904).All patients showed different degree hypertrophy and pain and the postoperative pain degrees of group A is more than group B and group C by clinical observation. Adverse reatction and complication in patients of the three groups:fever, gastrointestinal reatction, local blister, skin ulcer and local necrosis. The rate of complications and drug adverse reactions were similar in two groups (P=0.875)
Conclusion Lidocaine has no synergistic effect with Pingyangmycin in the process of treating cavernous venous malformation.
Section II Comparison among treatment of cavernous venous malformation with Pingyangmycin of different concentration
Objective To compare the clinical efficacy of cavernous venous malformation treated with Pingyangmycin of different concentration
Method Patients with cavernous venous malformation were treated with Pingyangycin of defferent concentration in our department from February2008to February2010. Patients were divided into two groups:treated with0.5mg/ml Pingyangmycin (group A), treated with2mg/ml Pingyangmycin (group B),32patients in each group. All of the patients presented with a measurable lesion. Informed consent was given to the use of drug as injection therapy. A standardised data collection sheet recorded patient details including:age, sex, weight, location of lesion, size, clinical history, special investigations, dosage, clinical response, side effects and follow-up. Colour photographs were taken of every patient before, during, and after completion of treatment. All patients underwent direct intralesional injection of different solution. A needle connected to a syringe containing liquid medicine was inserted into the venous space, and the intraluminal position of its tip was confirmed by blood aspiration. The sclerosant was then injected slowly into the lesion. The volume of the solution varied from0.7to6.0ml per injection. They were observed0.5h and is charged home. Treatment was performed in several stages. The applications were carried out after intervals of15days. Procedure was done by one operator.
Utilizing Chi-square test, t-test, rank sum test to analyse the collected data in SPSS16.0software. If P<0.05, the difference has statistical significance.
Result The ages, sexes and clinical characteristics of patients with cavernous venous malformation were not significantly different(P>0.05). Cases had treated for1to6times and12to16months followed up after termination of the therapy. All patiens had no functional lesion of liver and kidney and pulmonary fibrosis. All patients showed different degree hypertrophy and pain and that of groupB is more than group A by clinical observation. Curative effect in two groups had no significant difference (P=0.306). The rate of complications and drug adverse reactions were similar in two groups (P=0.198)
Conclusion Sclerotherapy with low concentration Pingyangmycin is proved to be a safe, effective therapy for superficial sporadic, solitary venous malformations of critical organs.
Section III Percutaneous sclerotherapy for superficial venous malformations of the lips with low concentration Pingyangmycin and dexamethasonum
Objective To evaluate the clinical effect of using low concentration Pingyangmycin and dexamethasonum to treat superficial vascular malformation of the lips.
Methods Sixty eight patients with superficial venous malformation of the lips(29males and39females, age range:13-69years)were treated in our department from January2007to January2011.The lesions ranged in size from0.5×0.8cm to3×2.1cm. Pingyangmycin(about0.5mg/ml) and dexamethasonum (about1mg/ml) was injected into the center and periphery of the superficial venous malformation, the injection was repeated every15days when necessary.
Results All patients required multiple therapeutic sessions. At a mean follow-up of21months (range12to32),66patients were cured and2had marked improvement. No patients presented with signs of recurrence.
Conclusion Percutaneous Sclerotherapy with low concentration Pingyangmycin and dexamethasonum is proved to be a safe, effective therapy for superficial venous malformations of the lips.
Section IV Treatment for venous malformations of the glans penis with low concentration Pingyangmycin
Objective To study the therapeutic methods of venous malformations of the glans penis.
Methods From January2002to October2010,29patients, aged16～33years old, were treated with low concentration Pingyangmycin (0.5mg/ml) and the volume of the solution varied from1to3ml per injection.. Physical examination showed light-t
在我国平阳霉素硬化治疗静脉畸形作为一种常规治疗方式,疗效确切。为指导应用,中华口腔医学会口腔颌面外科专业委员会脉管性疾病学组2011年建议了平阳霉素治疗静脉畸形方法,其中利多卡因作为一种联合用药与平阳霉素配制成一定浓度药液注入畸形静脉中。利多卡因一方面作为酰胺类局麻药物,用于不同部位以阻断感觉神经而产生麻醉,其穿透性和扩散性强。治疗静脉畸形可减轻平阳霉素栓塞过程中因疼痛引起的反射性血管痉挛,使硬化药物更充分的填塞病灶。另一方面,利多卡因作为钙调蛋白阻滞剂对抗肿瘤药物有增敏作用。
利多卡因、平阳霉素注入到畸形静脉中对血管内皮细胞的作用如何?利多卡因是否在平阳霉素治疗静脉畸形过程中有协同作用?平阳霉素和利多卡因浓度的改变对临床治疗效果有何影响?
本课题分为两个部分,第一部分采用昆明小鼠脾脏作为人海绵状静脉畸形的动物模型,通过石蜡切片光镜观察、超薄切片电镜观察、末端转移酶介导的缺口末端标记法(TUNEL法)标记凋亡细胞,Image-Pro Plus6.0病理细胞图像分析系统比较细胞凋亡率,实时荧光定量PCR检测细胞凋亡因子半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达,观察不同浓度利多卡因、不同浓度平阳霉素在不同时间对脾脏血管内皮细胞的毒性作用以及平阳霉素与不同浓度利多卡因联合用药时在不同时间对血管内皮细胞的毒性作用,探讨利多卡因是否对平阳霉素治疗海绵状静脉畸形有协同作用。第二部分回顾性分析平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形,探讨利多卡因浓度对疗效的影响；回顾性分析不同浓度平阳霉素治疗浅表海绵状静脉畸形,探讨平阳霉素浓度对治疗效果的影响,指导临床用药；回顾性分析低浓度平阳霉素治疗浅表静脉畸形,总结治疗效果。
第一部分利多卡因影响平阳霉素治疗海绵状静脉畸形的实验研究
实验1不同浓度利多卡因对小鼠脾脏血管内皮细胞的影响
目的探讨利多卡因对血管内皮细胞的细胞毒性。
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只,为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.1%利多卡因组、0.2%利多卡因组、0.4%利多卡因组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。将小鼠用10%水合氯醛(3ml/kg)腹腔注射麻醉后,常规消毒、铺巾。分层切开皮肤、肌层、腹膜,切口长约1.0cm,将胃翻起显露其背侧的脾脏,钝性牵拉固定,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射不同浓度盐酸利多卡因溶液0.3ml,边注射边匀速退针,稍加压迫针孔止血,分层缝合,继续饲养。术后第2、5、8、14d处理对照组的4只小鼠,实验组B、C、D中的各6只小鼠。麻醉后,按照原切口再次开腹,游离并切断结扎脾脏周围血管及系膜,取出脾脏,去除脾脏头尾,将脾脏中段分割后,分别投入10%中性福尔马林及2.5%的戊二醛溶液中。HE染色,观察不同浓度、不同时间标本变化；TUNEL法检测细胞凋亡,每例标本制切片2张,用Image-Pro Plus6.0病理细胞图像分析系统,将各组TUNEL标记切片进行分析。在400×光镜下,每张切片随机选择6个高倍视野的图片,分别计数每张切片中TUNEL阳性细胞比率(=6个高倍视野的阳性细胞数／细胞总数×100%)并计算其平均值。电镜下观察血管内皮细胞变化。
统计方法：正态分布计量资料采用均数±标准差(x±S)描述,时间与处理因素的效应分析采用析因设计的方差分析,若交互作用有统计学差异,进一步分析单独效应。满足正态且方差齐的数据,采用单因素方差分析(One-way ANOVA);非正态和(或)方差不齐的数据,采用Kruskal-Wallis H秩和检验；若有统计学意义,用SNK法进一步做两两比较,P<0.05为差别有统计学意义,所有数据用SPSS16.0分析。
结果大体观察,对照组2、5、8、14天外观未见明显改变,均表现为包膜光滑完整,边缘整齐,无肿胀。各实验组与对照组比较无明显差异。
显微镜下,生理盐水组,各时间点为正常脾脏表现：脾脏表面被覆薄层纤维组织,其外为单层间皮细胞；其下红、白髓结构清楚,血窦不扩张,脾窦内衬单层内皮细胞,内皮细胞完整,血窦内充以适量的红细胞,间质无炎细胞浸润,组织细胞不增生；脾小体结构清楚,生发中心不扩张。0.1%利多卡因组：各时间点与对照组相比无明显变化。0.2%利多卡因组：2天组,为正常脾脏表现；5天组,偶见脾窦出血；8天组,间质偶见炎症细胞浸润,偶见局部单核或多核巨噬细胞；14天组,间质细胞浸润较8天组稍多,偶见纤维蛋白渗出,偶见吞噬了细胞碎片的多核巨噬细胞。0.4%利多卡因组：2天组,偶见脾窦扩张,充血,红白髓结构清楚,间质偶见炎症细胞浸润；5天组,脾窦扩张、充血较明显,间质炎症细胞浸润增多；8天组,可见间质细胞浸润,少量纤维蛋白渗出,部分脾索结构略模糊,可见到吞噬了细胞碎片的多核巨噬细胞；14天组,间质细胞浸润、纤维蛋白渗出增多,局部可见到多个吞噬了细胞碎片的多核巨噬细胞。部分红白髓结构模糊。
透射电镜观察：生理盐水组、0.1%利多卡因组、0.2%利多卡因组各时间点脾脏可见大量红髓,血窦清晰,细胞连接完好,红细胞聚于脾窦内且数量较多,淋巴细胞分布均匀,结构完整,细胞器完好,整体无明显组织学改变。镜下未见凋亡小体。0.4%利多卡因组2天组、5天组脾脏结构未见明显改变,8天组、14天组可见脾窦筋膜破坏、可见凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝107.676,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间=4.725,P时间=0.005),时间与组间交互效应有显著性意义(F组间×时间＝13.682,P组间×时间＝0.000)。
单独效应方差分析结果表明,生理盐水组4个时间点凋亡率比较差别无统计学意义(F=0.296,P=0.828)。0.1%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别无统计学意义(X2=3.687,P=0.297)。0.2%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别无统计学意义(X2=4.167,P=0.244)。0.4%利多卡因组采用Kruskal-Wallis H秩和检验,4个时间点凋亡率比较差别有统计学意义(X2=19.747,P＝0.000)。其中,14天组>8天组>5天组,14天组>2天组。
单独效应方差分析结果表明,2天组：各组之间比较差别无统计学意义(F=1.386,P=0.279)。5天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(X2=11.186,P=0.011)。其中,D组>A组,D组>B组,D组>C组。8天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(X2=13.717,P=0.003)。其中D组>A组,D组>B组,D组>C组。14天组：0.4%利多卡因组凋亡率较其它组升高,采用Kruskal-Wallis H秩和检验,差别有统计学意义(χ2=12.753,P=0.005)。其中D组>A组,D组>B组,D组>C组。
结论我们的研究表明,利多卡因在0.1%和0.2%的浓度时,小鼠脾脏血管内皮细胞未见组织学改变,未引起细胞凋亡。随着利多卡因干预浓度的增加、作用时间的延长,利多卡因具有细胞毒性作用,可诱发细胞的凋亡。利多卡因诱发小鼠脾脏内皮细胞凋亡具有明显的时间和浓度依赖性。
实验2不同浓度平阳霉素对小鼠脾脏血管内皮细胞的影响
目的探讨平阳霉素对血管内皮细胞的细胞毒性
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.5mg/ml平阳霉素组、1mg/ml平阳霉素组、2mg/ml平阳霉素组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。按照实验1手术方法找到小鼠脾脏,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射不同浓度平阳霉素溶液0.3ml。术后处理同实验1。统计方法同实验1。
结果大体观察,生理盐水组：各时间点外观未见明显改变。0.5mg/ml平阳霉素组：2天组,脾组织暗红光亮,未见明显变化；5天组,个别标本脾组织颜色稍暗,包膜略有紧张；8天组,部分标本脾组织边缘稍钝,轻度肿胀；14天组,大部分标本出现脾组织颜色变暗,略凹陷,包膜紧张度消失。1mg/ml平阳霉素组：2天组,部分脾组织包膜略紧张,有轻度肿胀；5天组,脾组织色泽变暗,边缘稍钝,有轻度肿胀；8天组,脾组织包膜紧张度消失,部分区域出现微凹；14天组,部分标本脾组织萎缩,边缘凹凸不平有切迹,表面白色条纹样瘢痕,与周围组织有粘连。2mg/ml平阳霉素：2天组,脾组织包膜紧张,边缘稍钝,有不同程度肿胀；5天组,脾组织包膜紧张度消失,颜色变暗,部分区域出现微凹；8天组,脾组织不同程度萎缩,部分标本出现边缘切迹,被膜凹凸表现,表面有白色条纹样瘢痕,与周围组织有粘连；14天组,大部分标本出现脾脏质硬暗红,包膜凹凸皱缩,表面有白色瘢痕,脾脏边缘锐利伴有不规则切迹,与周围组织严重粘连,呈现肉眼可见的明显萎缩。
显微镜下,生理盐水组：各时间点为正常脾脏表现。0.5mg/ml平阳霉素：2天组,个别脾窦出现扩张,充血,少量炎细胞浸润；5天组,间质炎细胞浸润,窦内皮细胞肿胀,个别出现变性,少量纤维蛋白渗出。8天组,脾窦扩张、充血显著,窦内皮细胞肿胀、变性,部分脾索结构模糊,较多炎细胞浸润、纤维蛋白渗出及组织细胞增生；14天组：纤维蛋白渗出、变性,纤维组织增生,红细胞弥散,部分脾窦塌陷毁损,脾小体萎缩形态不均。1mg/ml平阳霉素：2天组,脾窦出血、扩张,少量炎细胞浸润,偶见局部多核巨噬细胞；5天组,脾组织充血显著,脾窦炎细胞浸润,纤维蛋白沉积,内皮细胞肿胀、变性,多核巨噬细胞增多；8天组,脾索结构模糊,纤维蛋白沉积伴有纤维组织的增生,可见新鲜出血；14天组,脾窦塌陷毁损,部分脾小体萎缩、形态不均,纤维蛋白渗出、变性,纤维组织增生,较多单核或多核巨噬细胞云集。2mg/ml平阳霉素：2天组,脾窦扩张,充血,窦内皮细胞肿胀,个别窦内皮细胞及脾索纤维细胞变性,或细胞核浓缩、碎裂,脾索结构略模糊,炎细胞浸润、组织细胞增生；5天组,脾组织充血显著,较多窦内皮细胞及脾索纤维细胞变性,大量细胞核浓缩、碎裂、崩解,较多单核或多核巨噬细胞云集,脾索结构模糊,较多炎细胞浸润、组织细胞增生；8天组,脾窦塌陷毁损,内皮细胞胞核溶解、散碎,大量单核或多核巨噬细胞云集。纤维蛋白渗出、变性,纤维组织增生,红细胞弥散,部分脾小体萎缩形态不均,边缘充血、出血；14天组,脾窦内皮细胞凋亡成片,脾小体塌陷萎缩,纤维组织增生,被膜增厚。
电镜下,对照组各时间表现同实验1。各实验组各时间均可见不同程度的血窦壁破坏,线粒体空泡样变、内质网扩张,凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝9549.392,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间=2499.782,P时间=0.000),时间与组间交互效应有显著性意义(F组间×时间＝279.087,P组间×时间=0.000)。
单独效应方差分析结果表明,对照组：4个时间点凋亡率比较差别无统计学意义(F=0.296, P=0.828).0.5mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学差异(F=1144.825,P=0.000)。14天组>8天组>5天组>2天组。1mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学意义(F=1494.281,P=0.000),14天组>8天组>5天组>2天组。2mg/ml平阳霉素组：4个时间点凋亡率比较差别有统计学意义(F=963.028,P=0.000),14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：各组之间凋亡率比较差别有统计学意义(F=1232.831,P=0.000)。D组>C组>B组>A组。5天组：各组之间凋亡率比较差别有统计学意义(F=1201.614,P=0.000)。D组>C组>B组>A组。8天组：各组之间凋亡率比较差别有统计学意义(F=2134.600,P=0.000)。D组>C组>B组>A组。14天组：各组之间凋亡率比较差别有统计学意义(F=14886.604,P=0.000)。D组>C组>B组>A组。
结论平阳霉素在0.5mg/ml浓度下可引起细胞明显凋亡,随药物浓度增加,作用时间的延长,凋亡细胞增多,染色加深。PYM引起细胞凋亡的程度,具有明显的浓度和时间依赖性。
实验3平阳霉素与不同浓度利多卡因对小鼠脾脏血管内皮细胞的影响
目的探讨利多卡因是否对平阳霉素治疗海绵状静脉畸形有协同作用
方法将88只雌性昆明小鼠按随机区组设计原则随机分为4组,其中A组16只,为生理盐水对照组；实验组72只,分为B、C、D三组,每组24只,分别为0.5mg/ml平阳霉素组、0.5mg/ml平阳霉素+0.1%利多卡因组和0.5mg/ml平阳霉素+0.2%利多卡因组。每组又随机为4个日程组(2、5、8、14d组),对照组每日程4只,实验组每日程6只。按照实验1手术方法找到小鼠脾脏,用4号半针头沿脾脏长轴方向穿刺,对照组注射生理盐水0.3ml,实验组注射0.5mg/ml平阳霉素溶液或平阳霉素利多卡因混合液0.3ml。按照实验1方法在各个时间点取出小鼠脾脏。去除脾脏头尾,将对照组脾脏中断分割后,分别投入10%中性福尔马林及2.5%的戊二醛溶液中；将实验组脾脏中断分为三份,一份迅速置入冻存管,放入液氮中冻存；另两份分别投入10%中性福尔马林及2.5%的戊二醛溶液中。液氮中冻存组织用于凋亡蛋白Caspase-3的实时荧光定量(RT-PCR)检测。统计方法同实验1。
结果大体观察,对照组：2、5、8、14天外观未见明显改变。0.5mg/ml平阳霉素组：各时间点外观同实验2同等浓度平阳霉素组表现。0.5mg/ml平阳霉素+0.1%利多卡因组：各时间点与0.5mg/ml平阳霉素组相比外观无明显变化。0.5mg/ml平阳霉素+0.2%利多卡因组：2天组,脾组织暗红光亮色,部分标本包膜略紧张,略有轻度肿胀；5天组,脾组织色泽变暗,边缘稍钝；8天组,脾组织包膜紧张度消失,颜色变暗,部分区域出现微凹；14天组,部分标本脾组织萎缩,边缘凹凸不平有切迹。
显微镜下,对照组,各时间点为正常脾脏表现。0.5mg/ml平阳霉素组,各时间点同实验2同浓度平阳霉素组表现。0.5mg/ml平阳霉素+0.1%利多卡因组,各时间点与0.5mg/ml平阳霉素组相比无明显变化。0.5mg/ml平阳霉素+0.2%利多卡因组：2天组,部分脾窦出血、扩张,少量炎细胞浸润,偶见局部多核巨噬细胞；5天组,脾组织充血显著,脾窦炎细胞浸润,少量纤维蛋白沉积,内皮细胞肿胀、变性,多核巨噬细胞增多；8天组,脾索结构模糊,纤维蛋白沉积伴有纤维组织的增生,可见新鲜出血；14天组：部分脾窦塌陷毁损,部分脾小体萎缩、形态不均,纤维蛋白渗出、变性、增生,较多单核或多核巨噬细胞云集。
电镜观察,对照组各时间表现同实验1,各实验组各时间均可见不同程度的血窦壁破坏,线粒体空泡样变、内质网扩张,凋亡小体形成。
血管内皮细胞凋亡率进行析因方差分析,不同组间细胞凋亡率比较差异显著(F组间＝1510.583,P组间＝0.000),不同时间点细胞凋亡率比较差异显著(F时间＝917.257,P时间＝0.000),时间与组间交互效应有显著性意义(F组间×时间＝88.540,P组间×时间=0.000)。
单独效应方差分析结果表明,生理盐水组4个时间点凋亡率比较差别无统计学意义(F=0.296,P=0.828)。0.5mg/ml平阳霉素组：采用秩和检验,4个时间点凋亡率比较差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.1%利多卡因组：采用方差分析,4个时间点凋亡率比较差别有统计学差异(F=876.169,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.2%利多卡因组：采用方差分析,4个时间点凋亡率比较差别有统计学差异(F=21.600,P=0.000)。14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.360,P=0.001)。其中,D组>C组>A组；D组>B组>A组。5天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.202,P=0.001)。其中,D组>C组>A组；D组>B组>A组。8天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.715,P=0.001)。其中,D组>C组>A组；D组>B组>A组。14天组：采用秩和检验,各组之间凋亡率比较差别有统计学意义(X2=17.146,P=0.001)。其中,D组>C组>A组；D组>B组>A组。
各实验组Caspase-3拷贝数进行析因方差分析,不同组间Caspase-3拷贝数比较差异显著(F组间=9549.392,P组间＝0.000),不同时间点Caspase-3拷贝数比较差异显著(F时间＝2499.782,P时间＝0.000),时间与组间交互效应有显著性意义(F组间×时间=279.087,P组间×时间＝0.000)。
单独效应方差分析结果表明,0.5mg/ml平阳霉素组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.1%利多卡因组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。0.5mg/ml平阳霉素+0.2%利多卡因组：采用秩和检验,4个时间点比较caspase-3拷贝数差别有统计学意义(X2=21.600,P=0.000)。14天组>8天组>5天组>2天组。
单独效应方差分析结果表明,2天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=15.158,P=0.001)。D组>C组>B组。5天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=11.415,P=0.003)。其中,D组>C组；D组>B组。8天组：采用秩和检验,各组之间比较caspase-3拷贝数差别有统计学意义(X2=11.474,P=0.003)。其中D组>C组；D组>B组。14天组：采用方差分析,各组之间比较caspase-3拷贝数差别有统计学意义(F=127.582,P=0.000)。其中,D组>C组；D组>B组。
结论0.2%的利多卡因在平阳霉素导致的细胞凋亡中起到协同作用,并且随时间延长作用增加。提示利多卡因在配合平阳霉素治疗海绵状静脉畸形的过程中,不仅是起到封闭和止痛作用,而且对平阳霉素细胞毒性具有协同作用,且具有浓度及时间依赖性。本实验数据可以假定利多卡因在人体内会起到类似的作用。然而,由于动物模型实验的有限性,尽管观察了利多卡因对平阳霉素细胞毒性作用的协同作用,对于临床的真正影响及指导需要进一步的研究。
第二部分平阳霉素硬化治疗海绵状静脉畸形的临床研究
第一节平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形的对照研究
目的比较平阳霉素联合不同浓度利多卡因治疗海绵状静脉畸形的临床疗效,探讨利多卡因浓度对治疗效果的影响。
方法自2008年6月-2010年6月,我们采用2mg/ml平阳霉素、2mg/ml平阳霉素联合0.5%利多卡因及2mg/ml平阳霉素联合1%利多卡因,分别治疗海绵状静脉畸形各40例。所有患者知情同意。建立患者标准化数据收集表格,包括患者年龄、性别、病变部位、大小、病史专科检查、注射药量,治疗次数、临床反应、副作用以及随访。患者治疗前后拍摄数码照片。注射方法：严格消毒后,用5号针头从距病变边缘0.5cm处正常皮肤处进针,针尖水平穿刺到静脉畸形处,回抽出血确定注射器刺入的静脉区域,缓慢注射至局部肿胀,稍显苍白,注意勿使局部注药后张力过大。对面积稍大的静脉畸形,一处进针药液不能浸润到的部位,可采用多点注射。每次注射的药液体积是1.5-6.0ml。治疗后局部压迫穿刺点3min以防药液外溢。根据病变情况决定治疗的次数,2次治疗间隔15d。每次治疗后留观0.5h。三组治疗操作均为一人独立完成。
统计学方法三组性别比较采用卡方检验,年龄、病变面积和治疗次数比较采用完全随机设计资料的方差分析,三组间的疗效比较采用Kruskal-Wallis H秩和检验,三组间的不良反应比较采用卡方检验,P<0.05为差别有统计学意义。所有数据用SPSS16.0分析。
结果三组患者性别、年龄、病变面积差异均无统计学意义(P>0.05)。2mg/ml平阳霉素联合1%利多卡因治疗组的平均治疗次数有降低趋势,但三组治疗次数差异无统计学意义(P=0.586)。经过注射治疗后,随访12-16个月,均未见有肺纤维化及肝肾功能损害现象,三组间的疗效差异无统计学意义(P=0.904)。三组患者治疗后病变部位均有不同程度的肿涨和疼痛,在临床观察中2mg/ml平阳霉素组疼痛表现程度远大于另外两组。各组中均有发热、胃肠道反应及局部水疱或皮肤溃疡、坏死发生。三组不良反应差异无统计学意义(P=0.875)。
结论利多卡因对平阳霉素治疗海绵状静脉畸形未表现出协同作用。
第二节不同浓度平阳霉素治疗海绵状浅表静脉畸形的对照研究
目的比较不同浓度平阳霉素治疗海绵状浅表静脉畸形的临床疗效。
方法自2008年2月-2010年2月,我们采用低浓度平阳霉素(0.5mg/ml)及常规浓度平阳霉素(2mg/ml)分别治疗体表单发、局限性海绵状静脉畸形各32例。所有患者知情同意。建立患者标准化数据收集表格,包括患者年龄、性别、病变部位、大小、病史专科检查、注射药量,治疗次数、临床反应、副作用以及随访。常规消毒后,4号半针头从距病变边缘0.5cm处正常皮肤粘膜处进针,回抽出血确定注射器刺入的静脉区域,缓慢注射至局部肿胀,稍显苍白。对面积稍大的静脉畸形,一处进针药液不能浸润到的部位,可采用多点注射。每次注射的药液体积是0.7-6.0ml。治疗后局部压迫穿刺点3min以防药液外溢。根据病变情况决定治疗的次数,2次治疗间隔15d。每次治疗后留观0.5h。两组治疗操作均为一人独立完成。
统计学方法两组性别比较采用卡方检验,年龄和病变面积比较采用t检验,两组间的疗效比较采用Mann-Whitney U秩和检验,两组间的不良发应采用卡方检验,P<0.05为差别有统计学意义,所有数据用SPSS16.0分析。
结果两组患者性别、年龄、病变面积差异均无统计学意义(P>0.05)。经过1-6次注射治疗后,随访12-16个月,均未见有肺纤维化及肝肾功能损害现象,两组间的疗效差异无统计学意义(P=0.306)。两组患者治疗后病变部位均有不同程度的肿胀和疼痛,在临床观察中低浓度组表现程度远小于正常浓度组。两组不良反应差异无统计学意义(P=0.198)。
结论低浓度平阳霉素硬化治疗重要功能器官的单发、局限性浅表静脉畸形,局部肿胀轻、并发症少,安全有效,患者耐受性好,是一种较好的治疗方式。
第三节低浓度平阳霉素联合地塞米松硬化治疗唇部浅表静脉畸形
目的观察低浓度平阳霉素加地塞米松硬化治疗唇部浅表静脉畸形的临床疗效。
方法2007年1月-2011年1月,应用低浓度平阳霉素(0.5mg/ml)联合地塞米松(1mg/ml)硬化治疗唇部浅表静脉畸形患者68例,其中男29例,女39例,年龄13～69岁；病变范围最小0.5×0.8cm,最大3×2.4cm。根据病变情况决定治疗的次数,2次治疗间隔15d。
结果68例患者最少注射2次,最多注射8次,治疗后随访12～32个月,平均随访21个月。患者外观满意,没有复发倾向。根据疗效评价标准,66例患者治愈,2例患者有效。
结论低浓度平阳霉素联合地塞米松硬化治疗唇部浅表静脉畸形是安全有效的治疗方法。
第四节低浓度平阳霉素注射治疗龟头部静脉畸形
目的探讨龟头部静脉畸形的治疗方法。
方法对2002年1月～2010年10月收治29例龟头部静脉畸形患者,采用低浓度平阳霉素(0.5mg/ml)经皮穿刺注射治疗病变区域。年龄16～33岁,龟头部病变为蓝色、突起的、不规则肿物,指压后可缩小,彩色多普勒超声检查证实为血管样病变,与尿道海绵体或阴茎海绵体之间有清楚的界限。每次治疗注射的药液体积是1～3ml,并在阴茎体部病变区近心端置于止血带,自注射时到注射后5分钟阻断龟头部的血液回流,以保持静脉畸形体内的药物浓度及含量。治疗后外用抗生素软膏,纱布适度加压包扎。根据病变情况决定重复治疗的次数,2次治疗间隔15天。
结果16例患者治疗3次,8例4次,5例6次。治疗后随访12～38个月,平均随访时间为18个月。26名患者治愈,3名患者有效,均没有复发倾向。患者均具有正常的阴茎感觉、勃起和排尿功能,外观满意。
结论低浓度平阳霉素注射治疗是龟头部静脉畸形安全有效的首选的治疗方法。
Venous malformation(VM) results from an error in vascular morphogenesis; veins are dilated with thin walls and abnormal smooth muscle. Consequently, lesions expand, flow stagnates, and clotting occurs. Lesions are blue, soft, and compressible; hard calcified phleboliths may be palpable. VMs may range from small localized skin lesions to diffuse malformations involving multiple tissue planes and vital structures. A VM is especially problematic because it is progressive; it enlarges over time, particularly during adolescence; and often reexpands after treatment.Consequently, most patients who present with congenital lesions will ultimately require intervention.
The first-line treatment of problematic VMs is sclerotherapy, which is generally safer and more effective than resection. Sclerotherapy involves the injection of a sclerosant into the malformation, which then cause cellular destruction, thrombosis, and intense inflammation. Scarring leads to shrinkage of the lesion. Good to excellent results are obtained in75%to90%of patients, including reduction in the size of the malformation and alleviation of the symptoms. Often, multiple treatments are required.
In our country, Pingyangmycin sclerotherapy as a conventional treatment method for the treatment of venous malformation is observed to have exact curative effect. In order to guide application, therapeutic method of venous malformation with Pingyangmycin and lidocaine was proposed by committee of Oral and Maxillofacial Surgery, Branch Association of stomatology, China medical Association in2011.On one hand, Lidocaine as amide local anaesthetics can be used at different sites to block sensory nerve and lead to anesthesia, with strong penetrability and diffusivity. During the treatment of venous malformation, it can help to alleviate reflective vasospasm caused by the pain due to embolisation with Pingyangmycin and promote the medicine to fill up the nidus more completely. On the other hand, Lidocaine as calmodulin inhibitor has sensitivity-increasing effect for antineoplastic drug.
This subject adopts the spleen of Kunming mouse as the animal model for human cavernous venous malformation to observe toxic effects of Lidocaine of different concentrations and Pingyangmycin of different concentrations to splenic vascular endothelial cells at different time, and toxic effects of combined medicine of Pingyangmycin and Lidocaine of different concentrations to vascular endothelial cells at different time, and to explore whether Lidocaine has synergic action to Pingyangmycin for the treatment of cavernous venous malformation by means of paraffin section optical microscopic observation, ultrathin section electron microscopic observation, labeling apoptotic cells in terminal transferase mediated nick end labeling method (TUNEL method), comparing apoptosis rate according to Image-Pro Plus6.0pathologic cells'image analysis system, and detecting expression of cysteinyl aspartate specific protease-3(Caspase-3) of apoptosis factor by real-time quantitative PCR technology. Retrospectively analyze treating cavernous venous malformation with Pingyangmycin and Lidocaine of different concentrations to explore the influence of Lidocaine for curative effect in aspect of clinic medication proportion; retrospectively analyze treating superficial cavernous venous malformation with Pingyangmycin of different concentrations to explore the influence of Pingyangmycin concentration for therapeutic effect and guide clinic medication; retrospectively analyze treating superficial venous malformation with Pingyangmycin of low concentration to conclude therapeutic effect.
Part I Experimental Study on Influence of Lidocaine for the Treatment of Venous Malformation by Pingyangmycin
Experiment1Influence of Lidocaine of Different Concentrations for Mouse Splenic Vascular Endothelial Cells
Objective:To explore cytotoxicity of lidocaine to vascular endothelial cells.
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.1%Lidocaine group,0.2%Lidocaine group, and0.4%Lidocaine group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Mice was narcotized with10%chloral hydrate (3ml/kg) by intraperitoneal injection and disinfected. Surgical incision into the abdominal wall, turn up the stomach to reveal the spleen, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to contrast group and inject0.3ml Lidocaine Hydrochloride of different concentrations to experimental group. Withdraw injector at uniform speed while injecting, slightly press the pinhole of injector to stop bleeding, sew by layers, and continue to feed. Process4mice in control group on the2nd,5th,8th, and14th day after surgery, and6mice in experimental group B, C, and D. Open the peritoneal cavity along original cut after anesthesia, dissociate and cut off blood vessel and ligate mesentery around spleen, fetch the spleen, remove its head and tail, divided the middle section of the spleen into two parts., put into10%neutral formalin or2.5%glutaraldehyde solution respectively. Observe specimen changes at different concentrations and different times after HE staining; detect the apoptosis in TUNEL method by the following steps:prepare2sections for each specimen and analyze TUNEL labeling section of every group by Image-Pro Plus6.0pathologic cells' image analysis system. Randomly select6images under optical microscope at400times in every section.count TUNEL positive cell ratio (=6positive cell counts at high magnification/total cell counts×100%) in each section and calculate its mean value. Observe the changes of vascular endothelial cells under electron microscope.
Statistical Method:Normal distributed measurement data is described by mean±standard deviation (χ±s).Factor analysis was used to test the effect of interaction between group and time. If the effect of interaction was statistically significant then analyzed the effect alone. If the data are normal distribution and equal variance, then analyze by one-way ANOVA; if the data are abnormal distribution and (or) heterogeneity of variance, then adopt Kruskal-Wallis H Rank sum test; and if there is statistical significance, further make multiple comparison in SNK method, in which, if P﹤0.05, the disparity has statistical significance. The data were analyzed by SPSS software16.0.
Result:The appearances of physiological saline group have no significant changes on the2nd,5th,8th, and14th day, through general observation, manifesting that the capsulae is smooth and complete, the edge is tidy and without swelling. There is no obvious difference between every experimental group and control group.
Under microscope, physiological saline group shows normal spleen performances at every time point:the spleen surface is with a thin layer of fibrous tissue, the outer layer are monolayer mesothelial cells; under which the red and white pulps are clear, blood sinus is not dilated, the splenic sinus is lined with monolayer endothelial cells which are complete, the blood sinus is filled with appropriate amount of erythrocyte, there are no inflammatory cells infiltrated in mesenchyme, and the histiocyte has no hyperplasia; the splenic corpuscle has clear structure and the germinal center is not dilated.0.1%Lidocaine group:there is no obvious difference compared with contraol group at every time point.0.2%Lidocaine group:in2d day group, it shows a normal spleen performance; in5th day group, the splenic sinus bleeds occasionally; in8th day group, there are occasionally inflammatory cells infiltrated in mesenchyme and there appears local mononuclear or polynuclear macrophage occasionally; in14th day group, the infiltrated inflammatory cells are more than that in8th day group, there are fibrous protein exuding occasionally, and there appears polynuclear macrophages that swallow cell debris occasionally.0.4%Lidocaine group:in2nd day group, the splenic sinus is occasionally dilated and hyperemic, the red and white pulps are clear, and there are occasionally inflammatory cells infiltrated in mesenchyme; in5th day group, splenic sinus is dilated and obviously hyperemic, and inflammatory cells infiltrated in mesenchyme is increased; in8th day group, there exists interstitial cell infiltration, there is a small quantity of fibrous protein exuding, partial spleen trabecular structure is slightly obscure, and there appears polynuclear macrophages that swallow cell debris; in14th day group, the interstitial cell infiltration and fibrous protein exudation are increased, there appears fewer polynuclear macrophages that swallow cell debris locally, and the structures of partial red and white pulps are obscure.
Transmission Electron Microscope Observation:It was revealed that three groups (A, B and C group)showed the similar represent at the different time:red pulp in large quantities,integrate structure of blood sinus, good cell junction, many red blood cell in blood sinus, lymphocytes dispersed well and integrate organelles. No apoptotic bodies was observed under electronic microscope. In the D group, there was no obvious histology change of spleen at the2nd and5th day,but failure of blood sinus and apoptotic bodies can be observed at8th and14th day.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance (F组间=107.676, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=4.725, P时间=0.005) and the effect of interaction between group and time was statistically significant.(F组间×时间=13.682, P组间×时间=0.000)
The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828). Both B (χ2=3.687, P=0.297)and C (χ2=4.167, P=0.244)group showed the same result as A in the tests. The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group D (x2=19.747, P=0.000).14th day group>8th day group>5th day group, and14th day group>2nd day group.
Apoptosis rate comparison between all groups at different time points:in2nd day group, the comparison differences between every group have no statistical significance (F=1.386, P=0.279); in5th day group,8th group, and14group, the apoptosis rates of0.4%Lidocaine group are rising compared with other groups, D group> A group (P<0.05), D group>B group (P<0.05) and D group>C group (P <0.05).
Conclusion:When Lidocaine is of the concentrations of0.1%and0.2%, mice splenic vascular endothelial cells has no histological changes and all physiological functions work well, which cannot lead cell apoptosis. But as the intervention concentration of Lidocaine is increased and the action time is prolonged, the Lidocaine will have cytotoxicity and lead to cell apoptosis. Lidocaine inducing mice splenic vascular endothelial cells apoptosis has a time and concentration dependence.
Experiment2Influence of Pingyangmycin of Different Concentrations for
Mouse Splenic Vascular Endothelial Cells
Objective:Explore Cytotoxicity of Pingyangmycin to Vascular Endothelial Cells.
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.5mg/ml Pingyangmycin group,1mg/ml Pingyangmycin group, and2mg/ml Pingyangmycin group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Find mouse spleen according to the method of experiment1, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to control group and inject0.3ml Pingyangmycin of different concentrations to experimental group. Treatment after surgery is the same as experiment1and the statistical method is the same as experiment1.
Result:The appearances of physiological saline group have no significant changes.0.5mg/ml Pingyangmycin group:in2nd day group, the splenic tissue is dull-red and bright, without obvious change; in5th day group, individual specimen has slightly dark color, and the capsulae is a little strained, in8th day group, the margin of partial specimen is slightly blunt and with mild swelling; in14th day group, most specimens become dark, slightly sunk, and the turgidity of splenic capsula disappears; lmg/ml Pingyangmycin group:in2nd day group, partial capsula serosa lienis is slightly strained and slightly swelling; in5th day group, capsula serosa lienis become dark, the margin is slightly blunt and with mild swelling; in8th day group, the turgidity of capsula serosa lienis disappears, and a slight irregularity in the surface of the partial area.; in14th day group, part of specimen atrophy, the margin is uneven and has incisure, there is white stripe cicatrix on surface, and adheres to the surrounding tissues.2mg/ml Pingyangmycin:in2nd day group, the capsula serosa lienis is strained, the margin is slightly blunt and with swelling of different degrees; in5th day group, the turgidity of capsula serosa lienis disappears, the color becomes dark, and a slight irregularity in the surface of the partial area; in8th day group, the spleen atrophy at different degrees, partial specimen has margin incisure, the capsula has concave-convex, there is white stripe cicatrix on surface, and adheres to the surrounding tissues; in14th day group, most specimens have hard and dull-red spleens, the capsulas are concave-convex and atrophy, there is white cicatrix on surface, spleen margins are sharp and have irregular incisures, and adhere to the surrounding tissues and are remarkable macroscopic shrink.
Under microscope, physiological saline group shows normal spleen performances at every time point.0.5mg/ml Pingyangmycin:in2nd day group, individual sinus lienis is dilated and hyperemic, and there exists little inflammatory cell infiltration; in5th day group, there exists interstitial inflammatory cell infiltration, sinusoidal endothelial cells are swelling, individuals are degeneration and with little fibrous protein exudation, in8th day group, splenic sinuses are dilated and obviously hyperemic, sinusoidal endothelial cells are swelling and degeneration, part of splenic cords are obscure, and there exists more inflammatory cell infiltration, fibrous protein exudation, and histiocytosis; in14th day group, there exist fibrous protein exudation, degeneration, and fibroplastic proliferation, erythrocytes diffuse, part of splenic sinuses are sunk and destroyed, and splenic corpuscle atropies and has uneven forms, lmg/ml Pingyangmycin:in2nd day group, splenic sinus is hyperemic and dilated, there exists little inflammatory cell infiltration, and there appear polynuclear macrophages occasionally; in5th day group, splenic tissue is obviously hyperemic, and has inflammatory cell infiltration, fibrous protein deposits, endothelial cells swell and degeneration, and polynuclear macrophages increase; in8th day group, splenic cords are obscure, fibrous protein deposits with hyperplasia of fibrous tissue, and it is clear to see fresh bleeding; in14th day group, splenic sinuses are degeneration and destroyed, splenic corpuscle atrophise and has uneven forms, there exist fibrous protein exudation, degeneration, and more mononuclear or polynuclear macrophages gather.2mg/ml Pingyangmycin:in2nd day group, the splenic sinus is dilated and hyperemic, sinusoidal endothelial cells swell, individual sinusoidal endothelial cell and splenic cord fibrocyte degenerates, or cell nucleus concentrates, crushes, the splenic cord structure is obscure, and there exists little inflammatory cell infiltration and histiocyte hyperplasia; in5th day group, the splenic tissue is hyperemic obviously, more sinusoidal endothelial cells and splenic cord fibrocyte degenerate, a large number of cell nucleuses concentrate, crush, and disintegrate, much more mononuclear or polynuclear macrophages gather, the splenic cord structure is obscure, and there exists more inflammatory cell infiltration and histiocyte hyperplasia; in8th day group, splenic sinuses are destroyed, endothelial cell nuclei dissolves and shatters, and a large number of mononuclear or polynuclear macrophages gather, there exist fibrous protein exudation, degeneration, fibrous tissue proliferates, erythrocyte diffuse, part of splenic corpuscles shrink and with uneven forms, and the margin is hyperemic and bleeding; in14th day group, splenic sinus endothelial cell apoptosis increases, splenic corpuscle collapses and shrink, fibrous tissue proliferates, and the capsula is thicken.
Under Electron Microscope, It was revealed that groups A showed the represent as experiment1.Failure of blood sinus, apoptotic bodies, expanded Endoplasmic Reticulum and structural change of mitochondrion can be observed in varying degrees in group B,C and D at the different times.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance ((F组间=9549.392, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=2499.782, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=279.087, P组间×时间=0.000). The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828) The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group B (F=1144.825, P=0.000),C (F=1494.281, P=0.000)and D(F=963.028, P=0.000),14th day group>8thday group>5th day group,>2nd day group. Apoptosis rate comparison between all groups at different time points:in2nd (F=1232.831, P=0.000),5th (F=1201.614, P=0.000),8th (F=2134.600, P=0.000) and14th (F=14886.604, P=0.000) day group, the comparison differences between each group have statistical significance. D group>C group>B group> A group.
Conclusion:Pingyangmycin can lead to obvious vascular endothelial cell apoptosis at the concentration of0.5mg/ml. Its cytotoxicity to vascular endothelial cell shows obvious time and concentration dependence.
Experiment3Influence of Pingyangmycin and Lidocaine of Different Concentrations for Mouse Splenic Vascular Endothelial Cells
Objective:To Explore Whether Lidocaine Has synergistic effect on Pingyangmycin for Treatment of Cavernous Venous Malformation
Method:Separate88female Kunming mice into4groups at random according to randomized block design principle, in which16mice in group A are physiological saline control group;72mice in experimental group are divided into three groups B, C and D,24in each group, which are separately0.5mg/ml Pingyangmycin group,0.5mg/ml Pingyangmycin+0.1%Lidocaine group, and0.5mg/ml Pingyangmycin +0.2%Lidocaine group. Every group is divided into4subgroups according to schedule (2,5,8and14d group). The control group has4in every schedule subgroup, and the experimental group has6in every schedule subgroup. Find mouse spleen according to the method of experiment1, puncture in long axis direction of the spleen with lml injector. Inject0.3ml physiological saline to control group and inject0.5mg/ml Pingyangmycin solution or mixed solution of Pingyangmycin and Lidocaine0.3ml. Fetch mouse spleen at every time point according to the method of experiment1. Remove its head and tail, divide the middle section of the spleen into two parts in group A, then put into10%neutral formalin and2.5%glutaraldehyde solution respectively; divide the middle section of the spleen into three parts in experimental group, place one part is preserved in liquid nitrogen; put the other two parts into10%neutral formalin and2.5%glutaraldehyde solution respectively. The liquid nitrogen frozen tissue is used for real-time quantitative (RT-PCR) check of proapoptotic protein Caspase-3. The statistical method is the same as that in experiment1.
Result:The appearances of group A have no significant changes on the2nd,5th,8th, and14th day. Group B:appearances at every timing point are the same as the performances of Pingyangmycin group of the same concentration in experiment2. No significant changes were observed in group C compared with the group B. group D: in2nd day group, the splenic tissue is dull-red and bright, partial capsula serosa lienis is slightly strained and slightly swelling; in5th day group, spleen become dark and the margin is slightly blunt; in8th day group, the turgidity of capsula serosa lienis disappears, the color becomes dark, and slight irregularity in the surface of the partial area.; in14th day group, part of specimen the margin is uneven and has incisure.
Under microscope, group A shows normal spleen performances at every timing point. Group B, the performances at every time point are the same as the performances of Pingyangmycin group of the same concentration in experiment2. group C:The performances at every timing point have no obvious changes compared with that of group B. group D:in2nd day group, partial splenic sinus is dilated and hyperemic, there exists little inflammatory cell infiltration, and there appear polynuclear macrophages occasionally; in5th day group, splenic tissue is obviously hyperemic, splenic sinus has inflammatory cell infiltration, little fibrous protein deposits, endothelial cells swell and denaturize, and polynuclear macrophages increase; in8th day group, splenic cords are obscure, fibrous protein deposits with hyperplasia of fibrous tissue, and it is clear to see fresh bleeding; in14th day group, part of splenic sinuses are destroyed, partial splenic corpuscle shrinks and has uneven forms, there exists fibrous protein exudation, degeration, and proliferation, and more mononuclear or polynuclear macrophages gather.
Under Electron Microscope, It was revealed that groups A showed the represent as experiment1.Failure of blood sinus, apoptotic bodies, expanded Endoplasmic Reticulum and structural change of mitochondrion can be observed in varying degrees in group B, C and D at the different times.
Factor analysis was used to test the endothelial cell apoptosis rate, the comparisons of four groups have statistical significance (F组间=1510.583, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=917.257, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=88.540, P组间×时间=0.000). The results of analysis show that the difference of apoptosis rate had no statistics significance at four time points in the group A (F=0.296, P=0.828) The results of analysis show that the difference of apoptosis rate had statistics significance at four time points in the group B (χ2=21.600, P=0.000),C (F=876.169, P=0.000) and D (F=21.600, P=0.000),14th day group>8thday group>5th day group,>2nd day group. Apoptosis rate comparison between all groups at different time points:in2nd (χ2=17.360, P=0.0011,5th (χ2=17.202, P=0.001),8th (χ2=17.715, P=0.001) and14th (χ2=17.146, P=0.001) day group, the comparison differences between each group have statistical significance. D group>C group>A group, D group>B group> A group.
Factor analysis was used to test the caspase-3copy numbers, the comparisons of three groups have statistical significance (F组间=9549.392, P组间=0.000),the comparisons at the four time points have statistical significance (F时间=2499.782, P时间=0.000) and the effect of interaction between group and time was statistically significant (F组间×时间=279.087, P组间×时间=0.000).The results of analysis show that the difference of caspase-3copy numbers had statistics significance at four time points in the group B(χ2=21.600, P=0.000),C (χ2=21.600, P=0.000) and D (F=21.600, P=0.000),14th day group>8th day group>5th day group,>2nd day group. Caspase-3copy numbers comparison between all groups at different time points:in2nd day group, the comparison differences between all groups have statistical significance (χ2=15.158, P=0.001), group D>group C>group B. In5th day group (χ2=11.415, P=0.003),8th day group (χ2=11.474, P=0.003), and14th day group (F=127.582, P=0.000), the comparison between all groups, group D>group C; group D> group B.
Conclusion:When applying0.5mg/mI Pingyangmycin and0.1%Lidocaine, the apoptosis rate of splenic endothelial cell has no obvious change compared with only applying Pingyangmycin, and Lidocaine does not have synergistic action for apoptosis induced by Pingyangmycin; when applying0.5mg/ml Pingyangmycin and0.2%Lidocaine, the apoptosis rate of splenic endothelial cell is obviously increased compared with only applying Pingyangmycin and is obviously increased compared with applying0.5mg/ml Pingyangmycin and0.1%Lidocaine, Lidocaine has synergistic action for apoptosis induced by Pingyangmycin and the action improves as time goes on. It implies that Lidocaine's synergistic action to Pingyangmycin has time and concentration dependence.
Part II The Clinical Study on Pingyangmycin Sclerotherapy of Cavernous Venous Malformation
Section I Effect of Lidocaine in Defferent Concentration with Pingyangycin on Cavernous Venous Malformation
Objective To investigate the clinical efficacy of Lidocaine in defferent concentration with Pingyangycin oncavernous venous malformation.
Method Patients with cavernous venous malformation were treated with Lidocaine in defferent concentration with Pingyangycin in our department from June2008to June2010. Patients were divided into three groups:treated with1mg/ml Pingyangmycin (group A), treated with lmg/ml Pingyangmycin with0.5%Lidocaine (group B), treated with lmg/ml Pingyangmycin with1%Lidocaine (group C). All of the patients presented with a measurable lesion. Informed consent was given to the use of drug as injection therapy. A standardised data collection sheet recorded patient details including:age, sex, weight, location of lesion, size, clinical history, special investigations, dosage, clinical response, side effects and follow-up. Colour photographs were taken of every patient before, during, and after completion of treatment. All patients underwent direct intralesional injection of different solution.A needle connected to a syringe containing liquid medicine was inserted into the venous space, and the intraluminal position of its tip was confirmed by blood aspiration. The sclerosant was then injected slowly into the lesion. The volume of the solution varied from1.5to6.0ml per injection.They were observed0.5h and ischarged home. Treatment was performed in several stages. The applications were carried out after intervals of15days. Procedure was done by one operator.
Utilizing Chi-square test, analysis of variance and rank sum texst to analyse the collected data in SPSS16.0software. If P<0.05, the difference has statistical significance.
Result The ages, sexes and clinical characteristics of patients with cavernous venous malformation were not significantly different (P>0.05). There were decreasing trend of mean therapeutic times in group C, but therapeutic times in three groups had no significant difference (P=0.586). Twelve to16months followed up after termination of the therapy. All patiens had no functional lesion of liver and kidney and pulmonary fibrosis. Curative effect in three groups had no significant difference (P=0.904).All patients showed different degree hypertrophy and pain and the postoperative pain degrees of group A is more than group B and group C by clinical observation. Adverse reatction and complication in patients of the three groups:fever, gastrointestinal reatction, local blister, skin ulcer and local necrosis. The rate of complications and drug adverse reactions were similar in two groups (P=0.875)
Conclusion Lidocaine has no synergistic effect with Pingyangmycin in the process of treating cavernous venous malformation.
Section II Comparison among treatment of cavernous venous malformation with Pingyangmycin of different concentration
Objective To compare the clinical efficacy of cavernous venous malformation treated with Pingyangmycin of different concentration
Method Patients with cavernous venous malformation were treated with Pingyangycin of defferent concentration in our department from February2008to February2010. Patients were divided into two groups:treated with0.5mg/ml Pingyangmycin (group A), treated with2mg/ml Pingyangmycin (group B),32patients in each group. All of the patients presented with a measurable lesion. Informed consent was given to the use of drug as injection therapy. A standardised data collection sheet recorded patient details including:age, sex, weight, location of lesion, size, clinical history, special investigations, dosage, clinical response, side effects and follow-up. Colour photographs were taken of every patient before, during, and after completion of treatment. All patients underwent direct intralesional injection of different solution. A needle connected to a syringe containing liquid medicine was inserted into the venous space, and the intraluminal position of its tip was confirmed by blood aspiration. The sclerosant was then injected slowly into the lesion. The volume of the solution varied from0.7to6.0ml per injection. They were observed0.5h and is charged home. Treatment was performed in several stages. The applications were carried out after intervals of15days. Procedure was done by one operator.
Utilizing Chi-square test, t-test, rank sum test to analyse the collected data in SPSS16.0software. If P<0.05, the difference has statistical significance.
Result The ages, sexes and clinical characteristics of patients with cavernous venous malformation were not significantly different(P>0.05). Cases had treated for1to6times and12to16months followed up after termination of the therapy. All patiens had no functional lesion of liver and kidney and pulmonary fibrosis. All patients showed different degree hypertrophy and pain and that of groupB is more than group A by clinical observation. Curative effect in two groups had no significant difference (P=0.306). The rate of complications and drug adverse reactions were similar in two groups (P=0.198)
Conclusion Sclerotherapy with low concentration Pingyangmycin is proved to be a safe, effective therapy for superficial sporadic, solitary venous malformations of critical organs.
Section III Percutaneous sclerotherapy for superficial venous malformations of the lips with low concentration Pingyangmycin and dexamethasonum
Objective To evaluate the clinical effect of using low concentration Pingyangmycin and dexamethasonum to treat superficial vascular malformation of the lips.
Methods Sixty eight patients with superficial venous malformation of the lips(29males and39females, age range:13-69years)were treated in our department from January2007to January2011.The lesions ranged in size from0.5×0.8cm to3×2.1cm. Pingyangmycin(about0.5mg/ml) and dexamethasonum (about1mg/ml) was injected into the center and periphery of the superficial venous malformation, the injection was repeated every15days when necessary.
Results All patients required multiple therapeutic sessions. At a mean follow-up of21months (range12to32),66patients were cured and2had marked improvement. No patients presented with signs of recurrence.
Conclusion Percutaneous Sclerotherapy with low concentration Pingyangmycin and dexamethasonum is proved to be a safe, effective therapy for superficial venous malformations of the lips.
Section IV Treatment for venous malformations of the glans penis with low concentration Pingyangmycin
Objective To study the therapeutic methods of venous malformations of the glans penis.
Methods From January2002to October2010,29patients, aged16～33years old, were treated with low concentration Pingyangmycin (0.5mg/ml) and the volume of the solution varied from1to3ml per injection.. Physical examination showed light-t
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