芪丹通脉片干预VEGF的内皮双向作用的机制研究
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摘要
一、研究背景与目的
血管内皮生长因子(VEGF,VEGFA)是促血管新生的关键而有力的调节因子之一。作为一种旁分泌蛋白质,VEGF具有抗内皮细胞凋亡、促内皮细胞有丝分裂和提高血管通透性等作用,是影响血管内环境稳态的重要分子。目前,人们对VEGF生物学作用的双面之争已成热点。一方面,无论在体内还是在体外,VEGF都是内皮细胞的一个促存活因子。缺血、缺氧均可诱导VEGF的表达和分泌。低氧可提高局部组织旁分泌VEGF,并作用在内皮细胞的VEGF受体上,刺激新血管的形成,以适应性调节局部血氧的供应。但是在另一方面,在冠心病、肿瘤、中风、糖尿病等许多疾病状态下,这一适应性调节会发生紊乱,VEGF的“保护性角色”反而转变成主要的致病性病理性因素。在针对这一争论的讨论中,人们逐渐发现,越来越多的依据提示VEGF量的改变与其不同的生物学效应关系密切。已知,血管内皮生长因子受体2(VEGFR2,Flk-1/KDR)是VEGF发挥内皮相关生物学效应的主要受体之一,VEGF激活作用下的KDR表达和胞内转运,调控着内皮细胞对新生血管信号的敏感性,以及下游分子的信号传导。
传统中医药具有趋利避害性调节和维护机体的稳态平衡的优势。研究发现,益气活血类中药对VEGF表达和分泌有双面干预作用。一方面,益气活血中药通过下调VEGF分泌和表达而抑制肿瘤血管生成及血管内皮细胞的增生,减少组织的血供,抑制肿瘤的生长、转移及对机体有害的病理发展;另一方面,益气活血中药通过上调VEGF分泌和表达促进血管新生及血管内皮细胞的修复,在治疗缺血类疾病中独具特色。
既往实验发现,芪丹通脉片(QDTMT)具有内皮细胞保护作用,可提高心肌缺血大鼠心室内VEGF表达,增加缺血区的微血管密度(MVD)。但是,在缺氧状态下,QDTMT是否对内源性VEGF分泌和表达水平存在双向调节,是否对其受体KDR也有调节作用尚属未知。
以往关于活血化瘀中药影响VEGF的实验研究报道,基本上都设计在急性缺氧期,而有关VEGF影响血管增生的实验研究大多设计在疾病的病理改变形成后,后者需要的实验观察时间较长。因此,要进一步明确益气活血化瘀复方中药制剂干预VEGF不同生物效应的具体作用机制,还需要对疾病不同时段进行动态观察。
为此,本研究在动物实验部分选用了便于动态观察的低压氧舱性大鼠缺氧模型。经QDTMT药物干预后,我们选择不同时间点动态测量了大鼠血清中的VEGF浓度变化,并相应观察了与本实验相关性较强的大鼠心、肝、肺多个组织内血管的病理形态改变。
因为接近药物在体内作用的真实过程,血清药理学研究方法近些年发展迅速,本实验在设计中对此部分内容也有所借鉴。在制备了QDTMT含药血清后,为进一步探讨QDTMT的KDR相关分子机制,本实验还加入了体外细胞实验内容。通过培养并鉴定脐静脉内皮细胞(HUVECs),观察了QDTMT含药血清对低氧条件下的内皮细胞一般形态,超微结构和细胞活性的影响,并对细胞内KDR的表达变化进行了一定的观察。该实验从一定程度上证实了VEGF量的变化与其不同生物效应间的相关性,为QDTMT血管保护作用的分子机制提供了实验依据,丰富了中医传统理论中益气活血法有关的现代医学的科学内涵。
二、芪丹通脉片对缺氧大鼠血管内皮生长因子的双向调节作用
1研究方法和内容
1.1动物模型的建立与评估健康雄性SD大鼠18只,置于全自动调节低压低氧舱,6h/d进行缺氧造模。除观察动物一般情况变化外,另取肺组织标本,HE染色观察肺组织内血管增生情况,对模型进行评估。
1.2 QDTMT对缺氧大鼠VEGF的双向调节及血管保护作用健康雄性SD大鼠(n=30)随机分为正常对照组、低氧+QDTMT组(QDTMT组)、低氧+生理盐水组(生理盐水对照组)。动物造模方法同上,并分别在7d、14d两个时间点进行以下内容实验观察。
1)动物麻醉后,从腹主动脉取血后分离血清,800rpm离心25min,分离血清,-70℃存放。使用前经56℃、30min灭活,0.22μm滤膜灭菌,-20℃保存备用。按ELISA试剂盒说明进行血清VEGF测定。
2)取大鼠肺、心、肝组织,经4%多聚甲醛固定24h后,梯度乙醇脱水,石蜡包埋,HE染色,光镜下观察组织病理学改变。另取材3mm3大小肝组织块,经戊二醛固定后,透射电镜观察其超微结构改变。
3) VEGF免疫荧光染色无菌条件下取胸主动脉组织,经4%多聚甲醛固定后,常规石蜡制片。按常规操作顺序进行免疫荧光染色。
2实验结果
2.1 ELISA结果:缺氧7d大鼠血清中VEGF浓度较缺氧前增加,缺氧14d大鼠血清中VEGF浓度进一步急剧增加,组间比较有统计学意义。缺氧7d,QDTMT组大鼠血清中的VEGF含量明显高于正常对照组和生理盐水对照组;而缺氧14d后,QDTMT组大鼠血清中VEGF含量低于生理盐水对照组,组间比较有统计学差异(P<0.05)。
2.2形态学观察结果:光镜下可见,缺氧7d大鼠肺内血管结构改变不明显;缺氧14d大鼠肺组织血管增生明显。QDTMT组大鼠肺、心、肝组织血管增生性改变较生理盐水对照组轻。肝组织电镜结果提示,QDTMT组超微结构损伤较生理盐水对照组轻。而且,在QDTMT组大鼠肝组织内观察到VEGFR2相关的内体样超微结构。
2.3 VEGF免疫荧光染色结果提示,缺氧7d,三组大鼠主动脉内层VEGF表达变化不大;缺氧14d,生理盐水对照组大鼠主动脉内观察到大量VEGF阳性表达,与ELISA结果相一致。
3结论与提示
3.1低压氧舱性缺氧模型下,大鼠血清中的VEGF浓度在不同病理阶段有所变化。
3.2 QDTMT对VEGF分泌呈双向动态调节作用,可能是其血管保护作用主要机制之一。
三、芪丹通脉片对缺氧脐静脉血管内皮细胞保护作用研究
1研究方法和内容
1.1体外培养人脐静脉血管内皮细胞(HUVECs)及鉴定
参照文献记载的细胞培养方法,从人脐静脉分离内皮细胞进行原代及传代培养。利用显微镜和免疫荧光染色技术,从形态学和Ⅷ因子膜抗原鉴定培养的内皮细胞。
1.2 QDTMT含药血清对缺氧HUVECs增殖、凋亡的影响
1.2.1制备QDTMT含药血清
成年雄性SD大鼠18只,分组及处理同动物实验部分内容。大鼠缺氧造模后,经腹主动脉取血并分离血清,存放于-70℃备用。参照文献及预实验比较,确定最佳含药血清浓度为10%后,用含100ml/L胎牛血清的M199培养基将3种血清的浓度均配制为10%。
1.2.2 MTT法检测细胞活性
选择生长状态良好的3-5代HUVECs,用含100ml/L胎牛血清的M199培养24h,再用无血清M199继续培养6h。将细胞分为A、B、C3份:(1)A份,加入10%空白对照组大鼠血清;(2)B份,加入10%生理盐水对照组大鼠血清;(3)C份,加入10%QDTMT组大鼠血清。除A份细胞在CO2培养箱正常培养24h外,其余两份细胞放入三相气体培养箱中,在37℃、5%CO2以及2%O2浓度条件下培养24h。MTT法常规操作,检测三份细胞的活性。
1.2.3流式细胞术检测细胞凋亡细胞分组及处理同上,流式细胞仪常规操作测量细胞凋亡率。
1.3 VEGFR2免疫荧光染色同上处理细胞后,使用常规免疫荧光技术,观察三份细胞中VEGFR2的表达情况。
1.4细胞超微结构观察细胞同上处理,经离心、收集后,戊二醛固定,制备电镜标本。透射电镜下观察三份细胞超微结构。
2实验结果
2.1细胞鉴定及超微结构观察:在倒置显微镜下可见,培养的内皮细胞呈典型铺路石子状排列生长。免疫荧光鉴定结果提示,培养的内皮细胞胞浆呈Ⅷ因子阳性表达。透射电镜观察其超微结构,可见内皮细胞特有的短棒状细胞器Weible-Palade小体。
2.2 QDTMT含药血清对缺氧HUVECs的影响
1) MTT结果提示,与A份细胞相比,缺氧使B份和C份细胞增殖受到抑制;与B份相比,QDTMT含药血清干预后的C份内皮细胞增殖明显,组间比较有统计学差异(P<0.05)。
2)流式细胞术检测结果提示,缺氧使B、C两份细胞的凋亡率较A份细胞凋亡率增高;QDTMT含药血清干预后的C份内皮细胞凋亡率低于B份细胞,组间比较有统计学差异(P<0.05)。
2.3免疫荧光结果提示,C份内皮细胞上表达的VEGFR2阳性信号强于B份细胞。
2.4电镜结果提示,在C份细胞的超微结构中发现与VEGFR2转运有关的内涵体结构。
3结论与提示
在低氧状态下,体外培养的脐静脉内皮细胞凋亡率增加、增殖减少,QDTMT含药血清干预后的HUVECs则表现出了细胞增殖提高、凋亡率降低。QDTMT对缺氧HUVECs的保护作用可能和它促进内皮细胞VEGFR2表达有关。
一、Research background and aim
The role of vascular endothelial growth factor (VEGF, also referred to as VEGFA) in the regulation of angiogenesis is the object of intense investigation now. As the most critical and potent proangiogenic regulator, VEGF is a protein with antiapoptotic, mitogenic, and permeability -increasing activities specific for vascular endothelium. For one hand, VEGF is a survival factor for ECs, both in vitro and in vivo. But for the other hand, VEGF has been implicated in pathological angiogenesis associated with tumors, stroke,diabetes, coronary artery disease. The expression and secretion of VEGF is in ischemia and oxygen deficiency. There is much evidence that diverse biological effects of VEGF on ECs have much to do with its concentration. It has been an agreement that VEGF stimulates vascular endothelial cells mainly through VEGF receptor 2 ( VEGFR2,Flk-1/KDR ). VEGF-stimulated recycling of KDR regulates the sensitivity of endothelial cells to proangiogenic signals.
It is well known that traditional Chinese medicine (TCM) is potential to the regulation of homeostasis. Recent studies have demonstrated that TCM of reinforcing Qi and promoting blood circulation exert two-ways regulation of VEGF expression and secretion. Some researchers have showed that TCM can upregulate the level of VEGF as an attractive approach in therapeutic angiogenesis. Conversely, a variety of studies suggest that TCM play an important role in antiangiogenic therapies.
Previously, we have described Qidantongmai tablet (QDTMT) protect vascular endothelial cell (VEC), increase the VEGF expression in ventricles of myocardial ischemia rats and improve the microvessel density(MVD) in the ischemia zones. However, in hypoxic condition, whether QDTMT has regulation effects on VEGF and KDR is not known. What’more, a long time investigation is required of the complex pathophysiological process in animal experiments.
In this study, the hypobaria hypoxia chamber is used for the rat hypoxic models. After QDTMT treatment, the concentrations of VEGF in rat serum is detected. The pathological changes of rat tissues and the expression of VEGF in blood vessel are observed in this study. In vitro, we have cultured and subcultured human umbilical endothelial cells(HUVECs), and have observed effects of serum containing QDTMT on endothelial proliferation and apoptosis in hypoxia. In addition, the expression of KDR is also investigated in HUVECs. We use this research to improve understanding of the balance between VEGF level and its biological activies,and of the relationship between the arterioprotection of endothelium and YiqiHuoxue principle in TCM.
二、Regulation effect of QDTMT on VEGF expression in rats with hypoxic model
1 Methods
1.1 The establishment of animal model
Male Sprague-Dawley rats (n=18) were exposed to low pressure and low oxygen conditions in hypobaric and hypoxic chamber ,6 h /d, for the estab lishment of animal model. After observation of the general state of rats, the lung tissues of rats were collected with Hemotoxylin and Eosin dye, and investigated the proliferation of vascular.
1.2 Effect of QDTMT on rats with hypoxic model Male Sprague-Dawley rats (n=30) were randomly divided into 3 groups: normal control, hypoxia+ QDTMT (QDTMT), hypoxia + normal saline(hypoxic control). With the animal model of hypobaric hypoxia (HBH) for 7d and 14d,we investigated the pathological changes. Then , as following:
1) After hypoxia, the serum level of VEGF in rats were detected by ELISA kit.
2) After hypoxia , lung tissue,heart tissue and liver tissue were fixed with 4% paraffin for about 24 h, and embedded, treated with Hemotoxylin and Eosin dye ,and observed with light microscope. The ultrastructure of liver tissue was observed with electron microscope too.
3) VEGF expression Open the chest under the sterile condition and the thoracic aorta tissues were fixed with 4% paraform for 8 h .The VEGF positive cells were detected by immunofluorescence in aorta.
2 Results
2.1 ELISA After 7d of hypoxia, we found that the concentration of VEGF in serum of QDTMT group was higher than that of normal control and hypoxic control (P < 0.05); after 14d of hypoxia, the VEGF level of hypoxic control was higher than that of QDTMT group (P< 0.05).
2.2 Pathological changes results Afer 7d of hypoxia, pathological changes of rats showed that blood vessel structural changes were slight in all the groups; afer 14d of hypoxia, pathological changes of vascullar structural were clear in hypoxic control under microscope. Pathological changes were decreased in QDTMT group. In addition , we found the endosome structure in QDTMT group with electron microscope.
2.3 Immunofluorescence results After 14d of hypoxia, a lot of VEGF positive cells were seen in aorta of hypoxic control group.
3 Conclusions
3.1 The level of VEGF in rat serum changed in different pathological stages in this HBH hypoxia model.
3.2 Two-ways regulation effect on VEGF level may be one of the mechanism for QDTMT to protect the endothelial tissue in hypoxia condition.
三、Study on protective effects of QDTMT to anoxic HUVECs
1 Methods
1.1 Cell cultivation and identification Human umbilical vein endothelial cells(HUVECs) were got from infant umbilical cord for primary cultivation and subcultivation as previously described. HUVECs were identified by morphologic character and membrane antigenⅧfactor. HUVECs were subcultured for use at passage 3-5.
1.2 Effects of serum containing QDTMT on HUVECs activities in hypoxia.
1.2.1 preparation for serum containing QDTMT According to different treatment factors , male Sprague-Dawley rats (n=18) were randomly divided into three groups. After hypoxic treatment, serum containing QDTMT was collected and stored in -70℃(refer to study in vivo) .It was showed that the best dilution of serum containing QDTMT was 10%. After diluted in M199 media, the serum concentration of all groups was 10%.
1.2.2 MTT for HUVECs cytoactive
HUVECs between passage 3 and 5 were cultured with M199 media for 24h, and were free of M199 media for 6h. Then cells were divided into three groups: (1)A, treated with 10% serum from normoxia control ; (2)B, treated with 10% serum from hypoxic control; (3) C, treated with 10% serum from QDTMT group. Except for normoxia control,cells in the other two groups were exposed in hypoxia condition for 24 h. The proliferation of HUVECs was observed in MTT.
1.2.3 The apoptosis ratewas tested with FCM in all groups.
1.3 Using immunofluorescence microscope, we examined the expression of VEGFR2 in HUVECs of all groups.
1.4 After fixed with glutaraldehyde the ultrastructure of HUVECs was observed with electron microscope.
2 Results
2.1 The morphology of cultured cells was obviously changed in the shape of cobble-stone under microscope. We also found the characteristic Weible-Palade bodies in cultured HUVECs. In addition , cells were seen stained with factorⅧby immunofluorescence microscope.
2.2 The effect of serum containing QDTMT on hypoxic HUVECs.
1) The MTT showed that the cell cytoactive of B ans C was lower than that of A. QDTMT promoted the proliferation of HUVECs in hypoxic condition, and the proliferation level was higher in C group treated with serum containing QDTMT than that of B group (P < 0.05).
2) The FCM assay showed that the apoptosis rate was lower in C group than that of B group (P < 0.05).
2.3 Result of immunofluorescence The VEGFR2 expression level of HUVECs was higher in C group than that of B.
2.4 With electron microscope, we fund the endosome in C group. Endosome is an important intracellular pool for VEGFR2/KDR mobilization within endothelial cells.
3 Conclusions
The results demonstrated that serum containing QDTMT improved the activities of antiapoptosis and proliferation of cultured HUVECs .To enhance the expression of VEGFR2 may be one of the endothelial protective effects of QDTMT.
血管内皮生长因子(VEGF,VEGFA)是促血管新生的关键而有力的调节因子之一。作为一种旁分泌蛋白质,VEGF具有抗内皮细胞凋亡、促内皮细胞有丝分裂和提高血管通透性等作用,是影响血管内环境稳态的重要分子。目前,人们对VEGF生物学作用的双面之争已成热点。一方面,无论在体内还是在体外,VEGF都是内皮细胞的一个促存活因子。缺血、缺氧均可诱导VEGF的表达和分泌。低氧可提高局部组织旁分泌VEGF,并作用在内皮细胞的VEGF受体上,刺激新血管的形成,以适应性调节局部血氧的供应。但是在另一方面,在冠心病、肿瘤、中风、糖尿病等许多疾病状态下,这一适应性调节会发生紊乱,VEGF的“保护性角色”反而转变成主要的致病性病理性因素。在针对这一争论的讨论中,人们逐渐发现,越来越多的依据提示VEGF量的改变与其不同的生物学效应关系密切。已知,血管内皮生长因子受体2(VEGFR2,Flk-1/KDR)是VEGF发挥内皮相关生物学效应的主要受体之一,VEGF激活作用下的KDR表达和胞内转运,调控着内皮细胞对新生血管信号的敏感性,以及下游分子的信号传导。
传统中医药具有趋利避害性调节和维护机体的稳态平衡的优势。研究发现,益气活血类中药对VEGF表达和分泌有双面干预作用。一方面,益气活血中药通过下调VEGF分泌和表达而抑制肿瘤血管生成及血管内皮细胞的增生,减少组织的血供,抑制肿瘤的生长、转移及对机体有害的病理发展;另一方面,益气活血中药通过上调VEGF分泌和表达促进血管新生及血管内皮细胞的修复,在治疗缺血类疾病中独具特色。
既往实验发现,芪丹通脉片(QDTMT)具有内皮细胞保护作用,可提高心肌缺血大鼠心室内VEGF表达,增加缺血区的微血管密度(MVD)。但是,在缺氧状态下,QDTMT是否对内源性VEGF分泌和表达水平存在双向调节,是否对其受体KDR也有调节作用尚属未知。
以往关于活血化瘀中药影响VEGF的实验研究报道,基本上都设计在急性缺氧期,而有关VEGF影响血管增生的实验研究大多设计在疾病的病理改变形成后,后者需要的实验观察时间较长。因此,要进一步明确益气活血化瘀复方中药制剂干预VEGF不同生物效应的具体作用机制,还需要对疾病不同时段进行动态观察。
为此,本研究在动物实验部分选用了便于动态观察的低压氧舱性大鼠缺氧模型。经QDTMT药物干预后,我们选择不同时间点动态测量了大鼠血清中的VEGF浓度变化,并相应观察了与本实验相关性较强的大鼠心、肝、肺多个组织内血管的病理形态改变。
因为接近药物在体内作用的真实过程,血清药理学研究方法近些年发展迅速,本实验在设计中对此部分内容也有所借鉴。在制备了QDTMT含药血清后,为进一步探讨QDTMT的KDR相关分子机制,本实验还加入了体外细胞实验内容。通过培养并鉴定脐静脉内皮细胞(HUVECs),观察了QDTMT含药血清对低氧条件下的内皮细胞一般形态,超微结构和细胞活性的影响,并对细胞内KDR的表达变化进行了一定的观察。该实验从一定程度上证实了VEGF量的变化与其不同生物效应间的相关性,为QDTMT血管保护作用的分子机制提供了实验依据,丰富了中医传统理论中益气活血法有关的现代医学的科学内涵。
二、芪丹通脉片对缺氧大鼠血管内皮生长因子的双向调节作用
1研究方法和内容
1.1动物模型的建立与评估健康雄性SD大鼠18只,置于全自动调节低压低氧舱,6h/d进行缺氧造模。除观察动物一般情况变化外,另取肺组织标本,HE染色观察肺组织内血管增生情况,对模型进行评估。
1.2 QDTMT对缺氧大鼠VEGF的双向调节及血管保护作用健康雄性SD大鼠(n=30)随机分为正常对照组、低氧+QDTMT组(QDTMT组)、低氧+生理盐水组(生理盐水对照组)。动物造模方法同上,并分别在7d、14d两个时间点进行以下内容实验观察。
1)动物麻醉后,从腹主动脉取血后分离血清,800rpm离心25min,分离血清,-70℃存放。使用前经56℃、30min灭活,0.22μm滤膜灭菌,-20℃保存备用。按ELISA试剂盒说明进行血清VEGF测定。
2)取大鼠肺、心、肝组织,经4%多聚甲醛固定24h后,梯度乙醇脱水,石蜡包埋,HE染色,光镜下观察组织病理学改变。另取材3mm3大小肝组织块,经戊二醛固定后,透射电镜观察其超微结构改变。
3) VEGF免疫荧光染色无菌条件下取胸主动脉组织,经4%多聚甲醛固定后,常规石蜡制片。按常规操作顺序进行免疫荧光染色。
2实验结果
2.1 ELISA结果:缺氧7d大鼠血清中VEGF浓度较缺氧前增加,缺氧14d大鼠血清中VEGF浓度进一步急剧增加,组间比较有统计学意义。缺氧7d,QDTMT组大鼠血清中的VEGF含量明显高于正常对照组和生理盐水对照组;而缺氧14d后,QDTMT组大鼠血清中VEGF含量低于生理盐水对照组,组间比较有统计学差异(P<0.05)。
2.2形态学观察结果:光镜下可见,缺氧7d大鼠肺内血管结构改变不明显;缺氧14d大鼠肺组织血管增生明显。QDTMT组大鼠肺、心、肝组织血管增生性改变较生理盐水对照组轻。肝组织电镜结果提示,QDTMT组超微结构损伤较生理盐水对照组轻。而且,在QDTMT组大鼠肝组织内观察到VEGFR2相关的内体样超微结构。
2.3 VEGF免疫荧光染色结果提示,缺氧7d,三组大鼠主动脉内层VEGF表达变化不大;缺氧14d,生理盐水对照组大鼠主动脉内观察到大量VEGF阳性表达,与ELISA结果相一致。
3结论与提示
3.1低压氧舱性缺氧模型下,大鼠血清中的VEGF浓度在不同病理阶段有所变化。
3.2 QDTMT对VEGF分泌呈双向动态调节作用,可能是其血管保护作用主要机制之一。
三、芪丹通脉片对缺氧脐静脉血管内皮细胞保护作用研究
1研究方法和内容
1.1体外培养人脐静脉血管内皮细胞(HUVECs)及鉴定
参照文献记载的细胞培养方法,从人脐静脉分离内皮细胞进行原代及传代培养。利用显微镜和免疫荧光染色技术,从形态学和Ⅷ因子膜抗原鉴定培养的内皮细胞。
1.2 QDTMT含药血清对缺氧HUVECs增殖、凋亡的影响
1.2.1制备QDTMT含药血清
成年雄性SD大鼠18只,分组及处理同动物实验部分内容。大鼠缺氧造模后,经腹主动脉取血并分离血清,存放于-70℃备用。参照文献及预实验比较,确定最佳含药血清浓度为10%后,用含100ml/L胎牛血清的M199培养基将3种血清的浓度均配制为10%。
1.2.2 MTT法检测细胞活性
选择生长状态良好的3-5代HUVECs,用含100ml/L胎牛血清的M199培养24h,再用无血清M199继续培养6h。将细胞分为A、B、C3份:(1)A份,加入10%空白对照组大鼠血清;(2)B份,加入10%生理盐水对照组大鼠血清;(3)C份,加入10%QDTMT组大鼠血清。除A份细胞在CO2培养箱正常培养24h外,其余两份细胞放入三相气体培养箱中,在37℃、5%CO2以及2%O2浓度条件下培养24h。MTT法常规操作,检测三份细胞的活性。
1.2.3流式细胞术检测细胞凋亡细胞分组及处理同上,流式细胞仪常规操作测量细胞凋亡率。
1.3 VEGFR2免疫荧光染色同上处理细胞后,使用常规免疫荧光技术,观察三份细胞中VEGFR2的表达情况。
1.4细胞超微结构观察细胞同上处理,经离心、收集后,戊二醛固定,制备电镜标本。透射电镜下观察三份细胞超微结构。
2实验结果
2.1细胞鉴定及超微结构观察:在倒置显微镜下可见,培养的内皮细胞呈典型铺路石子状排列生长。免疫荧光鉴定结果提示,培养的内皮细胞胞浆呈Ⅷ因子阳性表达。透射电镜观察其超微结构,可见内皮细胞特有的短棒状细胞器Weible-Palade小体。
2.2 QDTMT含药血清对缺氧HUVECs的影响
1) MTT结果提示,与A份细胞相比,缺氧使B份和C份细胞增殖受到抑制;与B份相比,QDTMT含药血清干预后的C份内皮细胞增殖明显,组间比较有统计学差异(P<0.05)。
2)流式细胞术检测结果提示,缺氧使B、C两份细胞的凋亡率较A份细胞凋亡率增高;QDTMT含药血清干预后的C份内皮细胞凋亡率低于B份细胞,组间比较有统计学差异(P<0.05)。
2.3免疫荧光结果提示,C份内皮细胞上表达的VEGFR2阳性信号强于B份细胞。
2.4电镜结果提示,在C份细胞的超微结构中发现与VEGFR2转运有关的内涵体结构。
3结论与提示
在低氧状态下,体外培养的脐静脉内皮细胞凋亡率增加、增殖减少,QDTMT含药血清干预后的HUVECs则表现出了细胞增殖提高、凋亡率降低。QDTMT对缺氧HUVECs的保护作用可能和它促进内皮细胞VEGFR2表达有关。
一、Research background and aim
The role of vascular endothelial growth factor (VEGF, also referred to as VEGFA) in the regulation of angiogenesis is the object of intense investigation now. As the most critical and potent proangiogenic regulator, VEGF is a protein with antiapoptotic, mitogenic, and permeability -increasing activities specific for vascular endothelium. For one hand, VEGF is a survival factor for ECs, both in vitro and in vivo. But for the other hand, VEGF has been implicated in pathological angiogenesis associated with tumors, stroke,diabetes, coronary artery disease. The expression and secretion of VEGF is in ischemia and oxygen deficiency. There is much evidence that diverse biological effects of VEGF on ECs have much to do with its concentration. It has been an agreement that VEGF stimulates vascular endothelial cells mainly through VEGF receptor 2 ( VEGFR2,Flk-1/KDR ). VEGF-stimulated recycling of KDR regulates the sensitivity of endothelial cells to proangiogenic signals.
It is well known that traditional Chinese medicine (TCM) is potential to the regulation of homeostasis. Recent studies have demonstrated that TCM of reinforcing Qi and promoting blood circulation exert two-ways regulation of VEGF expression and secretion. Some researchers have showed that TCM can upregulate the level of VEGF as an attractive approach in therapeutic angiogenesis. Conversely, a variety of studies suggest that TCM play an important role in antiangiogenic therapies.
Previously, we have described Qidantongmai tablet (QDTMT) protect vascular endothelial cell (VEC), increase the VEGF expression in ventricles of myocardial ischemia rats and improve the microvessel density(MVD) in the ischemia zones. However, in hypoxic condition, whether QDTMT has regulation effects on VEGF and KDR is not known. What’more, a long time investigation is required of the complex pathophysiological process in animal experiments.
In this study, the hypobaria hypoxia chamber is used for the rat hypoxic models. After QDTMT treatment, the concentrations of VEGF in rat serum is detected. The pathological changes of rat tissues and the expression of VEGF in blood vessel are observed in this study. In vitro, we have cultured and subcultured human umbilical endothelial cells(HUVECs), and have observed effects of serum containing QDTMT on endothelial proliferation and apoptosis in hypoxia. In addition, the expression of KDR is also investigated in HUVECs. We use this research to improve understanding of the balance between VEGF level and its biological activies,and of the relationship between the arterioprotection of endothelium and YiqiHuoxue principle in TCM.
二、Regulation effect of QDTMT on VEGF expression in rats with hypoxic model
1 Methods
1.1 The establishment of animal model
Male Sprague-Dawley rats (n=18) were exposed to low pressure and low oxygen conditions in hypobaric and hypoxic chamber ,6 h /d, for the estab lishment of animal model. After observation of the general state of rats, the lung tissues of rats were collected with Hemotoxylin and Eosin dye, and investigated the proliferation of vascular.
1.2 Effect of QDTMT on rats with hypoxic model Male Sprague-Dawley rats (n=30) were randomly divided into 3 groups: normal control, hypoxia+ QDTMT (QDTMT), hypoxia + normal saline(hypoxic control). With the animal model of hypobaric hypoxia (HBH) for 7d and 14d,we investigated the pathological changes. Then , as following:
1) After hypoxia, the serum level of VEGF in rats were detected by ELISA kit.
2) After hypoxia , lung tissue,heart tissue and liver tissue were fixed with 4% paraffin for about 24 h, and embedded, treated with Hemotoxylin and Eosin dye ,and observed with light microscope. The ultrastructure of liver tissue was observed with electron microscope too.
3) VEGF expression Open the chest under the sterile condition and the thoracic aorta tissues were fixed with 4% paraform for 8 h .The VEGF positive cells were detected by immunofluorescence in aorta.
2 Results
2.1 ELISA After 7d of hypoxia, we found that the concentration of VEGF in serum of QDTMT group was higher than that of normal control and hypoxic control (P < 0.05); after 14d of hypoxia, the VEGF level of hypoxic control was higher than that of QDTMT group (P< 0.05).
2.2 Pathological changes results Afer 7d of hypoxia, pathological changes of rats showed that blood vessel structural changes were slight in all the groups; afer 14d of hypoxia, pathological changes of vascullar structural were clear in hypoxic control under microscope. Pathological changes were decreased in QDTMT group. In addition , we found the endosome structure in QDTMT group with electron microscope.
2.3 Immunofluorescence results After 14d of hypoxia, a lot of VEGF positive cells were seen in aorta of hypoxic control group.
3 Conclusions
3.1 The level of VEGF in rat serum changed in different pathological stages in this HBH hypoxia model.
3.2 Two-ways regulation effect on VEGF level may be one of the mechanism for QDTMT to protect the endothelial tissue in hypoxia condition.
三、Study on protective effects of QDTMT to anoxic HUVECs
1 Methods
1.1 Cell cultivation and identification Human umbilical vein endothelial cells(HUVECs) were got from infant umbilical cord for primary cultivation and subcultivation as previously described. HUVECs were identified by morphologic character and membrane antigenⅧfactor. HUVECs were subcultured for use at passage 3-5.
1.2 Effects of serum containing QDTMT on HUVECs activities in hypoxia.
1.2.1 preparation for serum containing QDTMT According to different treatment factors , male Sprague-Dawley rats (n=18) were randomly divided into three groups. After hypoxic treatment, serum containing QDTMT was collected and stored in -70℃(refer to study in vivo) .It was showed that the best dilution of serum containing QDTMT was 10%. After diluted in M199 media, the serum concentration of all groups was 10%.
1.2.2 MTT for HUVECs cytoactive
HUVECs between passage 3 and 5 were cultured with M199 media for 24h, and were free of M199 media for 6h. Then cells were divided into three groups: (1)A, treated with 10% serum from normoxia control ; (2)B, treated with 10% serum from hypoxic control; (3) C, treated with 10% serum from QDTMT group. Except for normoxia control,cells in the other two groups were exposed in hypoxia condition for 24 h. The proliferation of HUVECs was observed in MTT.
1.2.3 The apoptosis ratewas tested with FCM in all groups.
1.3 Using immunofluorescence microscope, we examined the expression of VEGFR2 in HUVECs of all groups.
1.4 After fixed with glutaraldehyde the ultrastructure of HUVECs was observed with electron microscope.
2 Results
2.1 The morphology of cultured cells was obviously changed in the shape of cobble-stone under microscope. We also found the characteristic Weible-Palade bodies in cultured HUVECs. In addition , cells were seen stained with factorⅧby immunofluorescence microscope.
2.2 The effect of serum containing QDTMT on hypoxic HUVECs.
1) The MTT showed that the cell cytoactive of B ans C was lower than that of A. QDTMT promoted the proliferation of HUVECs in hypoxic condition, and the proliferation level was higher in C group treated with serum containing QDTMT than that of B group (P < 0.05).
2) The FCM assay showed that the apoptosis rate was lower in C group than that of B group (P < 0.05).
2.3 Result of immunofluorescence The VEGFR2 expression level of HUVECs was higher in C group than that of B.
2.4 With electron microscope, we fund the endosome in C group. Endosome is an important intracellular pool for VEGFR2/KDR mobilization within endothelial cells.
3 Conclusions
The results demonstrated that serum containing QDTMT improved the activities of antiapoptosis and proliferation of cultured HUVECs .To enhance the expression of VEGFR2 may be one of the endothelial protective effects of QDTMT.
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