乳岩宁联合依西美坦对乳腺癌裸鼠的抑瘤作用及其机理的实验研究
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摘要
目的：观察中药复方乳岩宁联合依西美坦对MCF-7乳腺癌荷瘤裸鼠一般状态、自主活动能力及脏器系数的影响，了解中药复方乳岩宁联合依西美坦是否有协同增效的作用；观察中药复方乳岩宁联合依西美坦对MCF-7乳腺癌细胞的病理形态学影响及其凋亡的影响，探讨其诱导细胞凋亡的作用机制；观察中药复方乳岩宁联合依西美坦对MCF-7乳腺癌细胞周期分布、ERα、p-AKT蛋白表达，了解中药复方乳岩宁联合依西美坦抑制MCF-7乳腺癌细胞增殖的信号转导通路；通过观察中药复方乳岩宁联合依西美坦对MCF-7乳腺癌细胞凋亡相关基因Bcl-2、Bax蛋白及p53、Survivin mRNA的表达的影响，了解其抑制乳腺癌细胞增殖的机理及作用机制。
材料与方法：
1.双侧卵巢切除（去势）模型的制备：
10%水合氯醛，100mg/ml，按4ml/kg体质量腹腔注射麻醉，将裸鼠俯卧于手术台上，四肢固定，背部正中（肋弓下缘）处备皮，络合碘消毒，切开皮肤，于脊柱旁开0.5cm，距肋弓下缘0.5cm，依次切开肌肉，剪开腹膜进入腹腔，可见乳白色组织。轻轻将之提出腹腔，即可见被包裹的粉红色米粒大小“桑椹样”卵巢，分离后钳夹、结扎，剪去卵巢，检查有无渗血，分层缝合皮肤，络合碘切口消毒。同样方法切除对侧卵巢，保温20℃左右待麻醉后苏醒。术后肌注青霉素生理盐水（6万U/ml）抗炎，每天注射0.25ml，并用碘伏切口消毒，连续3d。自由摄水和进食，标准SPF饲料，饲养30d，造模成功。
2. MCF-7荷瘤裸鼠移植模型的建立:
将进入对数生长期的MCF-7细胞用胰酶/EDTA消化后， PBS液清洗2次，加入细胞培养液，直接吹打均匀，0.4%台盼蓝染色，记录活细胞数（＞95%），细胞计数板计数，调节活细胞浓度为1×107/ml，裸鼠右侧胸壁碘伏消毒，使用1ml注射器于2只裸鼠右侧胸壁第二乳垫下接种细胞液0.2ml/只，，接种后10天左右，在接种部位乳垫处出现肿瘤结节，质地较硬，而且逐渐增大，越长越快。当原代肿瘤长至0.8cm3大小时，手术取下肿瘤，使用骨髓穿刺针分别移植至余40只裸小鼠乳垫下：10%水合氯醛（0.02ml/10g体重）腹腔注射麻醉荷瘤裸鼠，无菌条件下取出乳腺癌组织立即放入DMEM培养液中，剪切成1mm3左右的小块,置入骨髓穿刺针中备用。接种前，用碘伏消毒健康裸鼠的右侧胸壁，于右侧胸壁第二乳垫旁开1cm平刺进针，进至乳垫下时插入针芯，将瘤组织留在乳垫下，皮肤消毒，不需要缝合伤口，腹腔一次性注射青霉素1.5万U，裸鼠继续饲养于SPF级箱中。移植后第4-5d后乳垫处即可见肿瘤生长，成瘤率100%，而且形状、大小较整齐，在移植后第15天左右肿瘤可长至9±2mm，经HE染色病理诊断为浸润性导管癌，免疫组化检测ER（+）。
3.实验分组及给药：
将40只造模成功的裸鼠随机分为4组：①模型对照组②西药组（依西美坦）③乳岩宁组（乳岩宁）④结合组（乳岩宁+依西美坦）根据人和鼠给药剂量换算公式计算各组给药的剂量。①模型对照组：0.2ml只-1.d生理盐水灌胃②西药组（依西美坦）：依西美坦按4mg.kg-1体重给药(相当于临床人用量的10倍)，0.2ml只-1.d-1灌胃③乳岩宁组：乳岩宁为33.3g.kg-1体重(相当于临床人用量的12倍)，0.2ml.只-1.d-1灌胃④结合组（乳岩宁+依西美坦），乳岩宁33.3g.kg-1体重+依西美坦4mg.kg-1体重，0.2ml只-1.d-1灌胃，连续给药21天。
4.实验取材
实验结束，各组裸鼠分别进行取材，脱颈处死裸鼠置于冰盒上，完整剥离出瘤体，称重后置于冰盒上，肉眼观察瘤组织后，切取肿瘤全层组织，同时多点取材避免肿瘤坏死组织，共取材5份：（1）细胞周期标本：取下一块组织放入2mlPBS液中。（2）电镜标本：将组织切成大小约为1mm3，立即投入0.25%的戊二醛固定液中，置于冰箱中4℃冷藏备用；（3）RT-PCR标本：2-3个大小约8mm3组织块，置于1mlTrizol中，记号笔标记后，立即置于-80℃冰箱中冻存备用；（4）Western Bloting标本：组织块直接置于EP管中，-80℃冰箱中冻存备用；（5）用于HE、TUNEL检测细胞凋亡及免疫组化检测，标本取材后，置于4%多聚甲醛固定液中4℃冷藏备用。
5.检测指标：
(1)每天观察裸鼠饮食、活动、皮毛色泽、二便及死亡情况，隔日测量绝经后裸鼠体重，绘画体重变化曲线,；分别于用药前、实验第10天、21天用小鼠自主活动测试仪测小鼠5分钟内自主活动次数以判断其活动能力。处死裸鼠摘取肝、肾、脾脏称重并计算脏器指数。脏器指数=脏器重量(mg)/体重(g)。
(2)用药21天后脱颈处死裸鼠，完整剥离肿瘤称重，计算抑瘤率，抑瘤率按照公式=(模型组瘤重一实验组瘤重)/模型组瘤重×100%计算；肿瘤组织经4%多聚甲醛固定，梯度酒精脱水，二甲苯透明，浸蜡与包埋，用石蜡切片机切片，组织切片厚度为5μm，苏木素、伊红染色后光镜观察病理形态学变化；采用TUNNEL法检测肿瘤细胞凋亡率，肿瘤组织，常规石蜡包埋、切片，其余方法步骤按试剂盒说明书进行。细胞核内出现绿色荧光者为阳性细胞，荧光显微镜下观察，每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（Apoptosis Index，AI）。AI=阳性细胞数/计数的细胞个数×100%；进一步以超薄切片术快速冷冻肿瘤组织，采用戊二醛和四氧化锇的双固定法将其固定，脱水、浸透、包埋、切片、透射电镜(TEM)进行超微结构水平上的观测。(3) Annexin V(膜联蛋白V)－PI双染法检测细胞凋亡：乳腺癌组织块置于4℃的磷酸盐缓冲溶液中，机械法剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，调整待测细胞的浓度为5×105-1×106个/ml，取1ml细胞，1000rpm，4℃离心10分钟，弃上清，将细胞重悬于200ul Binding Buffer，加入10ul AnnexinV-FITC和5ul PI，轻轻混匀，避光室温反应15分钟或4℃反应30分钟，加入300ulBinding Buffer后上流式细胞仪检测。结果判定：左上象限：细胞碎片；右上象限：凋亡晚期细胞和死亡细胞；左下象限：活细胞；右下象限：凋亡早期细胞。(4)用药21天后完整剥离出瘤体，称重后置于冰盒上，切开标本取材，置于EP管中，-80℃冰箱中冻存用于Western blot蛋白测定。乳腺癌组织块剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，75％冷乙醇（－20℃预冷）固定细胞12h以上，加入PI（终浓度50ug／ml）和无DNA酶污染的RNA酶（终浓度50ug／ml）1ml染色30min－1h后上流式细胞仪检测细胞周期；应用Western blot蛋白免疫印迹法检测凋亡调控基因P53、 ERɑ表达，并对结果进行统计分析。
(4)RT-PCR的取材是将组织置于1mlTrizol中，标本作好标记后，立即置于-80℃冰箱中冻存备用。采用免疫组织化学法对凋亡调控基因Bcl-2、Bax蛋白的表达进行定性分析； RT-PCR法检测乳腺癌组织中p53、Survivin基因mRNA的表达。
结果：
(1)体重：各组荷瘤裸鼠体重变化比较：给药前各组裸鼠体质量无显著性差异（P=0.790）。给药后一周除了对照组体重持续上升外，其余各组裸鼠体重出现不同程度下降，乳岩宁组(0.63±0.028)，西药组(0.62±0.019)，结合组下降最少(0.20±0.017)，明显低于乳岩宁组及西药组（P﹤0.05），但是这两组比较无显著差异(P>0.05)；后结合组裸鼠体重也呈缓慢上升趋势，平均体重一直高于乳岩宁组及西药组，直至实验的第15天，体重开始缓慢下降。西药组体重一直高于乳岩宁组，直至实验第19天。第21天实验结束时各组体重：西药组最轻，对照组最重，对照组明显高于西药组、乳岩宁组及结合组（P<0.05），但是后三者之间体重比较无显著差别。除外对照组，各组体重下降比较：西药组(1.61±0.12)，乳岩宁组(1.11±0.0.16)，结合组下降最少(0.93±0.06)，各组间比较有统计学差异（P<0.05）；自主活动次数：实验开始时各组裸鼠自主活动次数均在52次/5分钟（简称52次）左右，各组之间比较无统计学差异（p﹥0.05）。在实验第10天，除外对照组，仪器检测各组裸鼠的自主活动次数下降，西药组组下降最明显，低于其余各组（p <0.01）而乳岩宁组与结合组比较无统计学差异（p﹥0.05），在实验的第21天，仍然保持这种下降趋势，由于肿瘤负荷的增加，各组裸鼠的自主活动次数均呈下降趋势，西药依西美坦组下降明显，低于其余各组（p <0.01），即西药组和结合组均施加了依西美坦的因素，可能与依西美坦能够影响荷瘤裸鼠的消化道不良反应而导致体力下降有关，而乳岩宁方能够对这种损伤有修复的作用。各组裸鼠肝、肾、脾脏器指数变化情况：结果显示结合组肝、肾脏器指数最高，都高于对照组，但无显著性差异(p＞0.05)。西药组的肝、肾、脾的脏器指数都高于乳岩宁组，其中肝、脾的脏器指数与乳岩宁组对比有统计学差异（p﹤0.05）。乳岩宁组的肝、肾、脾的脏器指数与结合组比较均有有统计学差异（p﹤0.05）；瘤重：西药组、乳岩宁组、结合组的肿瘤重量均低于对照组，与对照组比较有非常显著差异（p=0.000﹤0.01），其中结合组的瘤重最轻，明显低于单独用药组（p﹤0.01），西药组的瘤重组低于乳岩宁组，有统计学差异（p=0.041﹤0.05）。西药组、乳岩宁组、结合组的抑瘤率分别为34.2%、25.2%、46.3%，说明乳岩宁方对乳腺肿瘤的生长有抑制作用，并与西药依西美坦有协同作用。
(2)病理形态学观察：肉眼观察剥离的移植瘤呈圆形、椭圆形或分叶状，肿块质地中等，剖面为苍白色、半透明、质实的实性区，肿瘤大者中心呈灰白色、有坏死现象。HE染色后光镜观察：肿瘤细胞排列成索状或者簇状，间质少，在肿瘤细胞团中可见伴有中央腔隙的小管结构，病理提示为浸润性导管癌生长旺盛，核大，核仁清晰，呈圆形或椭圆形，核分裂相明显，可见散在坏死灶。对照组可见肿瘤细胞密集，排列紊乱，呈团索样结构，肿瘤细胞大，胞质丰富，核大深染，核浆比例失调，病理性核分裂象广泛存在，异型性明显，可见境界清楚的癌巢；而各用药组肿瘤细胞略增大，可见瘤组织结构破坏，大量出血及坏死细胞，异型细胞明显减少，部分细胞固缩成为孤立的细胞，还可见纤维组织。坏死的肿瘤细胞周围有大量的淋巴细胞和白细胞浸润，显示出肿瘤细胞的生长受到抑制。电镜下观察：对照组：乳腺癌细胞生长代谢旺盛，核浆比例失调，核膜完整，可见双层核膜，胞质内大量线粒体，散在核糖体，细胞器完整；西药组：可见大量坏死细胞，细胞膜破裂，胞质内大量空泡，染色质于边积核膜下，线粒体呈髓样改变；乳岩宁组：细胞以凋亡早期为主要改变，细胞体积明显缩小或增大，胞膜可见泡状膨出，核内异染色质增多、边集，浓缩成团块状，分布在核的周边，有的可见不典型的凋亡小体；结合组：出现凋亡中晚期改变，细胞膜破裂，胞质溢出呈空泡样改变，核裂解明显，可见典型的凋亡小体，其由膜包绕，小体内可见核的残片及细胞器。
（3）TUNEL结果：细胞核染成绿色者为阳性细胞，即细胞发生凋亡。每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（AI）。细胞凋亡指数（AI）=凋亡细胞/总细胞数×100%。各用药组与对照组相比，其细胞凋亡指数比较有统计学差异(P=0.000<0.01)；其中结合用药组具有最高的细胞凋亡率，西药组次之，与对照组、单独用药组相比差异均有统计学意义(P=0.000<0.01)。Annexin V(膜联蛋白V)－PI双染法检测细胞凋亡：对照组的细胞存活率最高，明显高于各用药组，差异有统计学差异（P<0.05），细胞凋亡率以结合组（39.25±8.34）最高，明显高于西药组及乳岩宁组（P<0.05），西药组的细胞凋亡率（31.21±7.53）高于乳岩宁组(26.13±6.76)，相比有显著差异性（P<0.05）。(3)流式细胞仪检测细胞周期结果显示：各用药组G0/G1期细胞的比例增多，S期比例明显减少，与模型对照组比较差异显著（P<0.01），结合组的G0/G1期细胞比例最多，S期细胞比例最少，与单纯用药组比较有统计学差异（P<0.05）。但是乳岩宁组与西药组分别对比G0/G1期、S期的细胞比例均无统计学差异（P﹥0.05）；采用Western blot蛋白免疫印迹法，检测各组裸鼠瘤组织中ERα、p-AKT和管家基因β-actin的表达结果：对照组中ERα及p-AKT蛋白高表达，与三个治疗组相比有显著性差异（p﹤0.01）；三个处理组均可下调ERα及p-AKT蛋白的表达，其中以结合组的作用更为显著，与乳岩宁组、西药组相比较有显著性差异（p﹤0.01）。
(4)免疫组化法测定肿瘤组织中Bcl-2、Bax的蛋白表达结果：Bcl-2及Bax蛋白表达定位：主要位于细胞浆中，少数在胞膜。表达阳性的细胞可见黄色、棕黄色的颗粒，尤以核周、胞膜明显，阴性细胞胞浆着色明显减弱，甚至呈阴性。对照组中Bcl-2蛋白存在高表达，Bax低表达，Bcl-2/Bax的比值显著升高，而三个处理组Bcl-2蛋白低表达，Bax蛋白高表达，与对照组相比有显著性差异（p﹤0.01）；西药组、乳岩宁组和结合组，都可不同程度的降低Bcl-2蛋白的表达，提高Bax的表达，并使Bcl-2/Bax的比值降低，其中结合组的作用更为显著，与西药组及乳岩宁组相比较有显著性差异（p﹤0.01）。说明模型对照组中存在乳腺癌细胞凋亡过程受阻，生长迅速，失去控制。各用药组均可诱导MCF-7乳腺癌细胞凋亡，可能与下调Bcl-2蛋白的表达，上调Bax蛋白的表达，使Bcl-2/Bax的比值降低有关。
（5）RT-PCR法检测乳腺癌组织中p53、SurvivinmRNA表达的结果：各用药组都可不同程度的降低p53、Survivin基因mRNA的表达，明显低于模型对照组（p﹤0.05）；其中以结合组的作用最为显著，与单独用药组相比较有显著性差异（p﹤0.01）。
结论:
1.中药乳岩宁能改善荷瘤裸鼠一般状态，减轻依西美坦带来的裸鼠体重下降，改善裸鼠活动能力。
2.中药乳岩宁联合依西美坦具有抑制乳腺癌生长的作用，可能与阻滞细胞周期进程及调节凋亡相关基因Bcl-2、Bax的表达来诱导细胞凋亡有关。
3.中药乳岩宁联合依西美坦下调通过癌组织中p-AKT、ERɑ蛋白的表达来抑制PI3K-AKT信号通路的活性，下调P53、Survivin基因mRNA的表达来抑制乳腺癌细胞增殖的。
Purpose:To observe the influence of Traditional Chinese medicine-Ruyanningand exemestane to the general states, the ability of independent activities andorgan coefficient of nude mouse with MCF-7breast cancer, and to find out ifare there synergistic roles of Chinese medicine-Ruyanning and exemestane. Toobserve the influence of Traditional Chinese medicine-Ruyanning and exemestaneto the pathomorphology and apoptosis of MCF-7breast cancer cell, and to findout the mechanisms of that inducing the apoptosis. To observe the influence ofTraditional Chinese medicine-Ruyanning and exemestane to the cell cycledistribution, and the protein expression of ERα and p-AKT of MCF-7breastcancer cell, and to find out the signal transduction pathway of TraditionalChinese medicine-Ruyanning and exemestane controlling the proliferation ofMCF-7breast cancer cell. To observe the influence of Traditional Chinesemedicine-Ruyanning and exemestane to the expression of the gene of Bcl-2, theprotein of Bax, p53and Survivin mRNA, which are related to the apoptosis ofMCF-7breast cancer cell, and to find out the mechanisms of that controllingthe the proliferation of MCF-7breast cancer cell.
Material and method:
1. Preparation of the model bilateral ovariectony:
10%choral hydrate (100mg/ml) is injected in the intraperitoneal of nude mouseaccording to the standard of4ml/kg. The nude mouse is prone position on theoperating table with the fixed limbs. The skin preparation is on the middle ofthe back with the lodine disinfection. Then we slit the skin on the spine adjacentto open0.5cm, away from the costal arch the lower edge of0.5cm, Cut theperitoneum into the abdominal cavity, and dish the milky organizations from theabdominal cavity. We see the pink mulberry-like ovarian. We separate it, thenclamp, ligature, cut ovarian, check for bleeding, suture the skin and disinfect. The same method is for the contralateral ovarian. The mouse is wake up afteranesthesia in20℃. The mouse is injected in0.25ml penicillin(60000U/ml) afteroperation and disinfect with3days. The mouse is intake water and SPF fodderwith30days. Then the model is success.
2. The creation of model of nude mouse with MCF-7cancer:
MCF-7cell in the phase of logarithmic growth is digested with trypsin/EDTA,then is washed by PBS solution twice. That is added to the cell culture medium,directly evenly pipetted, and stained with0.4%tyrpan blue. We record the numberof viable cells (>95%) and the number of cell counting boards, then adjustingthe viable cell concentration of1×107/ml. The right chest wall of nude mouseis disinfected. We vaccinate two nude mice in right chest wall, under the secondbreast pads with cell liquid0.2ml for every mouse. After10days the tumor nudesare appear in the breast pads, which are hard, gradually increased. When theprimary tumor is0.8cm3,the tumor is removed and transplanted to40nude micewith bone marrow biopsy needle.10%choral hydrate (0.02ml/10g) is injected inthe intraperitoneal of nude mouse with tumor. Breast cancer tissue is removedunder sterile conditions, immediately placed in DMEM, cut into pieces of about1mm3into the bone marrow biopsy needle. Before vaccination the right chest wallof healthy nude mouse is disinfected. We level pierce the needle into the rightchest wall next to the second breast pads1cm, then insert the needle core. Thetumor tissue is left in the breast pads. The skin is disinfected, no need tosuture the wounds. The penicillin1.5million U is injected in theintraperitoneal once. The nude mice are continued to raise in the SPF box. After4-5days the tumor is appear. The rate of tumor formation is100%. And the shapesand sizes are more equal. After15days the tumor can grow to9±2mm. Thepathological diagnosis is invasive ductal carcinoma by HE staining, withimmunohistochemical detection of ER (+).
3.Experimental groups and administration:
The40successful modeling nude mice were randomly divided into four groups:①model controlling group;②group of western medicine (exemestane);③group of Ruyanning;④combined group(exemestane and Ruyanning). Each groupadministered according to the human and mouse dose conversing formula.①modelcontrolling group:0.2ml saline is gavaged for every mouse everyday.②group ofwestern medicine (exemestane): Exemestane is administration according to4mg.kgfor every mouse(equivalent to10times the amount of clinical).0.2ml is gavagedfor every mouse everyday.③group of Ruyanning: Ruyanning is according to33g.kg(equivalent to12times the amount of clinical).0.2ml is gavaged for everymouse everyday.④combined group(exemestane and Ruyanning): Exemestane isaccording to4mg.kg and Ruyanning is according to33g.kg.0.2ml is gavaged forevery mouse everyday. All are administered continuously for21days.
4. Experimental drawing:
After experiment every group is drawn respectively. The nude mice is executedby cervical and placed in the ice box. The tumor is stripped out completely,and placed in the ice box after weighting. After the tumor tissue is observedby eyes, the full-thickness tissue is cut, and the same time it is drawnedmulti-size avoiding necrosis. The materials are5parts.(1)cellcycle specimens: Removing a piece of tissue placed in the PBS liquid of2ml.(2)electron microscopy specimens: Tissue is cut into about1mm3,thenimmediately put into0.25%glutaraldehyde, placed in a refrigerator at4℃spare.(3)RT-PCR specimens:2-3tissue blocks about8mm3is placed in the1ml Trizol. After labeled by permanent marker they are placed immediately in-80℃refrigerator.(4)Western Bloting specimens: Tissue blocks are placeddirectly in the EP tube, frozen in-80°C refrigerator.(5)For HE,TUNEL todetect apoptosis and immune: After drawning the specimens placedin4%paraformaldehyde with4℃.
5.Detection indicators:
(1)To observe the diet, activity, fur color stool,urine and death of nudemice everyday. To measure the weight every other day and paint the weight changecurve. To determine the ability of activity of mice by measuring the frequencyin5minutes with Spontaneous activity tester before treatment, on the10th day of the experiment,21days. The nude mice is executed. The liver, kidney andspleen are weighted and calculated organ index. The organ index=organ weight(mg)/body weight (g).
(2) The nude mice is executed by cervical after21days with medicine. Thetumor is stripped out completely and weighted. The tumor inhibition rate iscalculated. The tumor inhibition rate=(the tumor weigh of model controllinggroup-the tumor weigh of group of experiment)/the tumor weigh of modelcontrolling group×100%. Tumor tissue is fixed by4%paraformaldehyde,dehydrated by alcohol, opened by xylene, embedded in paraffin and sliced bymicrotome. The slice is5μm. Morphological changes are observed by lightmicroscopy after hematoxylin and eosin staining.The rate of apoptosis of tumorcells is detected by TUNEL assay. Tumor tissue is embedded in paraffin andsliced. The rest of the method steps are according to kit instructions. The cellswith appeared green fluorescence in the nucleus are positive cells. Observedby fluorescence microscopy, each stained slice select five400×typical highpower field randomly.100cells are observed per field.500cells are calculated.The average number of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. Further the tumor tissue is quick-frozen byultramicrotomy, fixed by glutaraldehyde and osmium tetroxide fixation,dehydrated, soaked embedded, sliced,observed by transmission electronmicroscopy (TEM)on the ultrastructual level.
(3) Detecting apoptosis by annexin V (annexin V)-PI double staining: Breastcancer tissue blocks are put in4°C phosphate buffer solution. That aredigested by0.25%trypsin30min－1h after mechanically cutting into pieces.Single cell suspension can be get over the200-400mesh filter cells. Themeasuring concentration of the cells is adjusting5×105-1×106cells/ml.1ml of cells is centrifuged1000rpm,4°C for10minutes. The cells wereresuspended in200ul Binding Buffer after Supernatant discarded. That is addedto0ul Annexin V-FITC and5ul PI and mixed gently. The time of reaction is15 minutes in Dark room temperature or30minutes in4℃. It is detected in flowcytometry adding300ul Binding Buffe. The judgment of results: the upper leftquadrant: cell debris; the upper right quadrant: apoptosis late cells and deadcells; the lower left quadrant: living cells: the lower right quadrant: apoptosisearly cells.
(4) The tumor is stripped out completely after21days with medicine. It isweighed and placed in the ice box. The cut specimens are placed in the EP tube,frozen in-80℃refrigerator for Western blot proteindetermination. The tissueof breast cancer after cutting into pieces is digested by0.25%trypsin30min－1h. Single cell suspension can be get over the200-400mesh filter cells. Thecells are fixed more than12hours with75％cold ethanol (-20℃precooling).It is added to1ml of PI(final concentration:50ug／ml) and RNA enzyme withenzyme-free DNA (final concentration:50ug／ml). After strained30minutes itis put into flow cytometry. The expression of apoptosis regulating gene P53,ERɑ is detected by Western blot. And the results are statistical analyze.
(5)The drawning of RT-PCR is that the tissue is put in1ml Trizol, and afterremarked put in-80℃refrigerator. The expression of apoptosis regulating geneBcl-2, Bax protein are qualitatively analyze by immunohistochemical method. Theexpression of p53、Survivin gene mRNA in breast cancer tissue by RT-PCR method.
Results:
(1)weight: the change of nude mice weight of each group: The nude mice weightof each group is no significant difference (P=0.790) before the administration.After a week the weight of the rest of the group is decrease in addition thatthe weight of controlling group is continued to rise. The group of Ruyanningis0.63±0.028. The group of western medicine is0.62±0.019. The combined group(0.20±0.017)is lighter than the group of Ruyanning and western medicine(P﹤0.05).But there are no significant difference between them(P>0.05). Theweight of combined group is gradually upward and the average weight is higherthan the group of Ruyanning and western medicine. After15days the weight isdecrease gradually. The weight of the group of western medicine is higher than the group of Ruyanning until19days. After21days the weights: The weight ofthe group of western medicine is lightest. The controlling group is heaviest.The controlling group is higher than the group of combined, Ruyanning andwestern medicine(P<0.05). But there are no significant difference among them.In addition to the controlling group the decreasing change of other group: Thegroup of western medicine is1.61±0.12. are no difference is1.11±0.0.16.Thecombined is0.93±0.06. There are differences among them(P<0.05).The timesof autonomic activities: At the beginning the times of each group is up about52/5minutes (abbreviation52). There are no difference among them(P>0.05).After10days in addition to the controlling group the times of each group isdecreased. The group of western medicine is lower than other group(p <0.01).Butthere are no difference between the group of Ruyanning and combined(P>0.05).After21days the decreasing trend is still. The times of autonomicactivities of each group are decrease because of the tumor. The group of westernmedicine is lower than other group(p <0.01).Because of exemestane in the groupof western medicine and combined, which can influence the gastrointestinaladverse reactions leading to physical falling. And the Ruyanning can repair thedamage. The change of liver, spleen and kidney of each group: The index of liverand kidney of combined group are higher than the controlling,but there is nodifference between them(P>0.05). The index of liver,spleen and kidney of thegroup of western medicine is higher than the Ruyanning. And there is differenceof index of liver and spleen between the group of western medicine and theRuyanning(P<0.05). There are differences between the index of liver, spleenand kidney of the Ruyanning and the combined(P<0.05).The weight of tumor: Theweight of tumor of western medicine, Ruyanning and the combined are all lowerthan the controlling. There are differences with the controlling(P﹤0.01).The lightest is combined group, which is lower than the group with single drug(p﹤0.01). The weight of tumor of western medicine is lower than the Ruyanning.There are differences(P﹤0.05).The rate of inhibition to tumor of westernmedicine, Ruyanning and the combined is respectively34.2%、25.2%、46.3%. The Ruyanning can inhibit the growth of breast cancer and has synergy withexemestane.
(2) pathomorphological observation: The tumor is circular, oval or lobulatedby eyes. The texture is middle. The section is pale, translucent and realqualities area. The pale necrosis is appeared in the larger tumor. Observationby light microscopy after HE staining: Tumor cells are arranged in cords orclusterswith less interstitial. There are central lacunar small pipe structurein the mass of tumor cells. The pathology is that invasive ductal carcinoma isvigorous growth, large nucleus, clear rounded or oval nucleolus, obvious mitoticand scattered necrotic foci. The controlling group is that the tumor cells aredensity, disorganized, cord-like. The tumor cells are large with abundantcytoplasm, large nucleus deeply stained, imbalance of nucleus and plasma. Thepathological mitotic is appear with significant atypia and clear cancer nests.And other groups are that tumor cells are slightly increased. The structure ofthe tumor tissue is destructed with bleeding and necrotic cells, atypical cellsreducing, some cells becoming isolating cells and fibrous tissue. There are alarge number of lymphocytes and leukocyte around the necrotic tumor cells. Thatmeans the growth of tumor cells is inhibited. Observation by electron microscopy:the controlling group: The breast cancer cell growth and metabolism are vigorous.The nucleus and cytoplasm is imbalance. The nuclear membrane is integrity withdouble nuclear membrane. There are a large number of mitochondria in thecytoplasm, scattered ribosomes and complete organs. The group of westernmedicine: There are much necrotic cells, rupturing membrane, the large numberof vacuoles in the cytoplasm, chromatin in the edge area of the nuclear membrane,mitochondria with medullary changes. The group of Ruyanning: The cells are earlyapoptotic. The Cell volume is significantly reduced or increased. Thebubble-like swelling is appear in the membrane. The nuclear heterochromatin isincrease, distributed in the periphery of the nucleus, and some with typicalapoptotic bodies. The combined group: The cells are late apoptotic. The cellmembrane is rupture. Cytoplasmic overflow is vacuolar changes, with the typical apoptotic bodies by the surrounding with membrane. There are nuclear fragmentsand organelles in small body.
(3)The results of TUNEL: The cells with appeared green fluorescence in thenucleus are positive cells. That means apoptosis. Observed by fluorescencemicroscopy, each stained slice select five400×typical high power fieldrandomly.100cells are observed per field.500cells are calculated. The averagenumber of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. There are differences in AI of the controlling groupand other groups (P=0.000<0.01). The rate of apoptosis of the combined groupis highest, then the western medicine. There are differences in them and thecontrolling and single drugs(P=0.000<0.01). Detecting apoptosis by annexin V(annexin V)-PI double staining: The cell viability of controlling is higher thanother groups. There are differences in them(P<0.05).The apoptosis rate ofcombined(39.25±8.34)is higher than the western medicine and Ruyaning(P<0.05).The apoptosis rate of western medicine (31.21±7.53)is higher than the Ruyaning(26.13±6.76). There are differences(P<0.05).
(4)The results of flow cytometry cell cycle: The proportion of cells inG0/G1phase of each treating group is increased, and S phase is significantlyreduced. The difference was significantly (P <0.01) comparing with the modelgroup. The proportion of cells in G0/G1phase of compared group is the most,and S phase is the least. There is difference comparing with the group of singledrug(P<0.05).But there is no difference between the group of Ruyanning andwestern medicine in the proportion of cells in G0/G1and S phase(P﹥0.05).The results of the expression of ERα、p-AKT and geneβ-actin in the tissue ofnude mouse by Western blot: The expression of ERαand p-AKT in controlling groupis high. The difference was significantly (P <0.01) comparing with other threegroups. Other three groups all can make down the expression of ERαand p-AKT.The most significant is combined group. The difference was significantly (P<0.01) comparing with the group of Ruyanning and western medicine.
(5)The results of the expression of Bcl-2、Bax in tumor tissue byimmunohistochemistry: The position of the expression of Bcl-2、Bax is the mostin cytoplasm, some in membrane. The positive expression of cell is yellow, brownparticles, especially in perinuclear and membrane. The negative cell iscytoplasmic colored significantly diminishing, or even negative. In controllinggroup the expression of Bcl-2is high, Bax is low, and the ratio of Bcl-2/Baxis significantly increased. In other three groups the expression of Bcl-2islow, Bax is high, and the difference is significant comparing with thecontrolling group(p﹤0.01).The group of western medicine, Ruyanning andcombined can reduce the expression of Bcl-2, increase Bax, and reduce the ratioof Bcl-2/Bax. The combined group has more role, and the difference issignificant comparing with the group of western medicine, Ruyanning(p﹤0.01).Wecan see the process of apoptosis is blocked in the controlling group, with rapidgrowth and loss of control. Other groups can induce the apoptosis of MCF-7breastcancer cell. That maybe related to reduce the expression of Bcl-2, increaseBax, and reduce the ratio of Bcl-2/Bax.
(6)The results of the expression of p53、SurvivinmRNA in tumor tissue byRT-PCR: Each group with drug all can low the expression of p53、SurvivinmRNA,lower than the controlling group(P<0.05).The compared group is most significant,having difference with the group with single drug(p﹤0.01).Conclusions:
1. The Traditional Chinese medicine-Ruyanning can improve the general statesand the ability of independent activities of nude mouse with MCF-7breast cancer,and reduce the side effect of exemestane decreasing the weight of mouse.
2. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit thegrowth of breast cancer, which maybe related to block the procession of cellcycle and regulate the expression of gene Bcl-2、Bax to induce apoptosis.
3. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit theactivity of PI3K-AKT signal pathway by reducing the expression of p-AKT、ERɑin cancer tissue, and inhibit the proliferation of breast cancer cell by reducing the expression of P53、SurvivingenemRNA.
材料与方法：
1.双侧卵巢切除（去势）模型的制备：
10%水合氯醛，100mg/ml，按4ml/kg体质量腹腔注射麻醉，将裸鼠俯卧于手术台上，四肢固定，背部正中（肋弓下缘）处备皮，络合碘消毒，切开皮肤，于脊柱旁开0.5cm，距肋弓下缘0.5cm，依次切开肌肉，剪开腹膜进入腹腔，可见乳白色组织。轻轻将之提出腹腔，即可见被包裹的粉红色米粒大小“桑椹样”卵巢，分离后钳夹、结扎，剪去卵巢，检查有无渗血，分层缝合皮肤，络合碘切口消毒。同样方法切除对侧卵巢，保温20℃左右待麻醉后苏醒。术后肌注青霉素生理盐水（6万U/ml）抗炎，每天注射0.25ml，并用碘伏切口消毒，连续3d。自由摄水和进食，标准SPF饲料，饲养30d，造模成功。
2. MCF-7荷瘤裸鼠移植模型的建立:
将进入对数生长期的MCF-7细胞用胰酶/EDTA消化后， PBS液清洗2次，加入细胞培养液，直接吹打均匀，0.4%台盼蓝染色，记录活细胞数（＞95%），细胞计数板计数，调节活细胞浓度为1×107/ml，裸鼠右侧胸壁碘伏消毒，使用1ml注射器于2只裸鼠右侧胸壁第二乳垫下接种细胞液0.2ml/只，，接种后10天左右，在接种部位乳垫处出现肿瘤结节，质地较硬，而且逐渐增大，越长越快。当原代肿瘤长至0.8cm3大小时，手术取下肿瘤，使用骨髓穿刺针分别移植至余40只裸小鼠乳垫下：10%水合氯醛（0.02ml/10g体重）腹腔注射麻醉荷瘤裸鼠，无菌条件下取出乳腺癌组织立即放入DMEM培养液中，剪切成1mm3左右的小块,置入骨髓穿刺针中备用。接种前，用碘伏消毒健康裸鼠的右侧胸壁，于右侧胸壁第二乳垫旁开1cm平刺进针，进至乳垫下时插入针芯，将瘤组织留在乳垫下，皮肤消毒，不需要缝合伤口，腹腔一次性注射青霉素1.5万U，裸鼠继续饲养于SPF级箱中。移植后第4-5d后乳垫处即可见肿瘤生长，成瘤率100%，而且形状、大小较整齐，在移植后第15天左右肿瘤可长至9±2mm，经HE染色病理诊断为浸润性导管癌，免疫组化检测ER（+）。
3.实验分组及给药：
将40只造模成功的裸鼠随机分为4组：①模型对照组②西药组（依西美坦）③乳岩宁组（乳岩宁）④结合组（乳岩宁+依西美坦）根据人和鼠给药剂量换算公式计算各组给药的剂量。①模型对照组：0.2ml只-1.d生理盐水灌胃②西药组（依西美坦）：依西美坦按4mg.kg-1体重给药(相当于临床人用量的10倍)，0.2ml只-1.d-1灌胃③乳岩宁组：乳岩宁为33.3g.kg-1体重(相当于临床人用量的12倍)，0.2ml.只-1.d-1灌胃④结合组（乳岩宁+依西美坦），乳岩宁33.3g.kg-1体重+依西美坦4mg.kg-1体重，0.2ml只-1.d-1灌胃，连续给药21天。
4.实验取材
实验结束，各组裸鼠分别进行取材，脱颈处死裸鼠置于冰盒上，完整剥离出瘤体，称重后置于冰盒上，肉眼观察瘤组织后，切取肿瘤全层组织，同时多点取材避免肿瘤坏死组织，共取材5份：（1）细胞周期标本：取下一块组织放入2mlPBS液中。（2）电镜标本：将组织切成大小约为1mm3，立即投入0.25%的戊二醛固定液中，置于冰箱中4℃冷藏备用；（3）RT-PCR标本：2-3个大小约8mm3组织块，置于1mlTrizol中，记号笔标记后，立即置于-80℃冰箱中冻存备用；（4）Western Bloting标本：组织块直接置于EP管中，-80℃冰箱中冻存备用；（5）用于HE、TUNEL检测细胞凋亡及免疫组化检测，标本取材后，置于4%多聚甲醛固定液中4℃冷藏备用。
5.检测指标：
(1)每天观察裸鼠饮食、活动、皮毛色泽、二便及死亡情况，隔日测量绝经后裸鼠体重，绘画体重变化曲线,；分别于用药前、实验第10天、21天用小鼠自主活动测试仪测小鼠5分钟内自主活动次数以判断其活动能力。处死裸鼠摘取肝、肾、脾脏称重并计算脏器指数。脏器指数=脏器重量(mg)/体重(g)。
(2)用药21天后脱颈处死裸鼠，完整剥离肿瘤称重，计算抑瘤率，抑瘤率按照公式=(模型组瘤重一实验组瘤重)/模型组瘤重×100%计算；肿瘤组织经4%多聚甲醛固定，梯度酒精脱水，二甲苯透明，浸蜡与包埋，用石蜡切片机切片，组织切片厚度为5μm，苏木素、伊红染色后光镜观察病理形态学变化；采用TUNNEL法检测肿瘤细胞凋亡率，肿瘤组织，常规石蜡包埋、切片，其余方法步骤按试剂盒说明书进行。细胞核内出现绿色荧光者为阳性细胞，荧光显微镜下观察，每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（Apoptosis Index，AI）。AI=阳性细胞数/计数的细胞个数×100%；进一步以超薄切片术快速冷冻肿瘤组织，采用戊二醛和四氧化锇的双固定法将其固定，脱水、浸透、包埋、切片、透射电镜(TEM)进行超微结构水平上的观测。(3) Annexin V(膜联蛋白V)－PI双染法检测细胞凋亡：乳腺癌组织块置于4℃的磷酸盐缓冲溶液中，机械法剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，调整待测细胞的浓度为5×105-1×106个/ml，取1ml细胞，1000rpm，4℃离心10分钟，弃上清，将细胞重悬于200ul Binding Buffer，加入10ul AnnexinV-FITC和5ul PI，轻轻混匀，避光室温反应15分钟或4℃反应30分钟，加入300ulBinding Buffer后上流式细胞仪检测。结果判定：左上象限：细胞碎片；右上象限：凋亡晚期细胞和死亡细胞；左下象限：活细胞；右下象限：凋亡早期细胞。(4)用药21天后完整剥离出瘤体，称重后置于冰盒上，切开标本取材，置于EP管中，-80℃冰箱中冻存用于Western blot蛋白测定。乳腺癌组织块剪碎后用0.25%胰酶消化30min－1h，过200－400目筛网过滤细胞，获得单细胞悬液，75％冷乙醇（－20℃预冷）固定细胞12h以上，加入PI（终浓度50ug／ml）和无DNA酶污染的RNA酶（终浓度50ug／ml）1ml染色30min－1h后上流式细胞仪检测细胞周期；应用Western blot蛋白免疫印迹法检测凋亡调控基因P53、 ERɑ表达，并对结果进行统计分析。
(4)RT-PCR的取材是将组织置于1mlTrizol中，标本作好标记后，立即置于-80℃冰箱中冻存备用。采用免疫组织化学法对凋亡调控基因Bcl-2、Bax蛋白的表达进行定性分析； RT-PCR法检测乳腺癌组织中p53、Survivin基因mRNA的表达。
结果：
(1)体重：各组荷瘤裸鼠体重变化比较：给药前各组裸鼠体质量无显著性差异（P=0.790）。给药后一周除了对照组体重持续上升外，其余各组裸鼠体重出现不同程度下降，乳岩宁组(0.63±0.028)，西药组(0.62±0.019)，结合组下降最少(0.20±0.017)，明显低于乳岩宁组及西药组（P﹤0.05），但是这两组比较无显著差异(P>0.05)；后结合组裸鼠体重也呈缓慢上升趋势，平均体重一直高于乳岩宁组及西药组，直至实验的第15天，体重开始缓慢下降。西药组体重一直高于乳岩宁组，直至实验第19天。第21天实验结束时各组体重：西药组最轻，对照组最重，对照组明显高于西药组、乳岩宁组及结合组（P<0.05），但是后三者之间体重比较无显著差别。除外对照组，各组体重下降比较：西药组(1.61±0.12)，乳岩宁组(1.11±0.0.16)，结合组下降最少(0.93±0.06)，各组间比较有统计学差异（P<0.05）；自主活动次数：实验开始时各组裸鼠自主活动次数均在52次/5分钟（简称52次）左右，各组之间比较无统计学差异（p﹥0.05）。在实验第10天，除外对照组，仪器检测各组裸鼠的自主活动次数下降，西药组组下降最明显，低于其余各组（p <0.01）而乳岩宁组与结合组比较无统计学差异（p﹥0.05），在实验的第21天，仍然保持这种下降趋势，由于肿瘤负荷的增加，各组裸鼠的自主活动次数均呈下降趋势，西药依西美坦组下降明显，低于其余各组（p <0.01），即西药组和结合组均施加了依西美坦的因素，可能与依西美坦能够影响荷瘤裸鼠的消化道不良反应而导致体力下降有关，而乳岩宁方能够对这种损伤有修复的作用。各组裸鼠肝、肾、脾脏器指数变化情况：结果显示结合组肝、肾脏器指数最高，都高于对照组，但无显著性差异(p＞0.05)。西药组的肝、肾、脾的脏器指数都高于乳岩宁组，其中肝、脾的脏器指数与乳岩宁组对比有统计学差异（p﹤0.05）。乳岩宁组的肝、肾、脾的脏器指数与结合组比较均有有统计学差异（p﹤0.05）；瘤重：西药组、乳岩宁组、结合组的肿瘤重量均低于对照组，与对照组比较有非常显著差异（p=0.000﹤0.01），其中结合组的瘤重最轻，明显低于单独用药组（p﹤0.01），西药组的瘤重组低于乳岩宁组，有统计学差异（p=0.041﹤0.05）。西药组、乳岩宁组、结合组的抑瘤率分别为34.2%、25.2%、46.3%，说明乳岩宁方对乳腺肿瘤的生长有抑制作用，并与西药依西美坦有协同作用。
(2)病理形态学观察：肉眼观察剥离的移植瘤呈圆形、椭圆形或分叶状，肿块质地中等，剖面为苍白色、半透明、质实的实性区，肿瘤大者中心呈灰白色、有坏死现象。HE染色后光镜观察：肿瘤细胞排列成索状或者簇状，间质少，在肿瘤细胞团中可见伴有中央腔隙的小管结构，病理提示为浸润性导管癌生长旺盛，核大，核仁清晰，呈圆形或椭圆形，核分裂相明显，可见散在坏死灶。对照组可见肿瘤细胞密集，排列紊乱，呈团索样结构，肿瘤细胞大，胞质丰富，核大深染，核浆比例失调，病理性核分裂象广泛存在，异型性明显，可见境界清楚的癌巢；而各用药组肿瘤细胞略增大，可见瘤组织结构破坏，大量出血及坏死细胞，异型细胞明显减少，部分细胞固缩成为孤立的细胞，还可见纤维组织。坏死的肿瘤细胞周围有大量的淋巴细胞和白细胞浸润，显示出肿瘤细胞的生长受到抑制。电镜下观察：对照组：乳腺癌细胞生长代谢旺盛，核浆比例失调，核膜完整，可见双层核膜，胞质内大量线粒体，散在核糖体，细胞器完整；西药组：可见大量坏死细胞，细胞膜破裂，胞质内大量空泡，染色质于边积核膜下，线粒体呈髓样改变；乳岩宁组：细胞以凋亡早期为主要改变，细胞体积明显缩小或增大，胞膜可见泡状膨出，核内异染色质增多、边集，浓缩成团块状，分布在核的周边，有的可见不典型的凋亡小体；结合组：出现凋亡中晚期改变，细胞膜破裂，胞质溢出呈空泡样改变，核裂解明显，可见典型的凋亡小体，其由膜包绕，小体内可见核的残片及细胞器。
（3）TUNEL结果：细胞核染成绿色者为阳性细胞，即细胞发生凋亡。每张染色后的切片随机选择5个400×典型的高倍视野，每个视野分别观察100个细胞，共500个细胞，计算出每100个细胞内的阳性细胞平均数作为凋亡指数（AI）。细胞凋亡指数（AI）=凋亡细胞/总细胞数×100%。各用药组与对照组相比，其细胞凋亡指数比较有统计学差异(P=0.000<0.01)；其中结合用药组具有最高的细胞凋亡率，西药组次之，与对照组、单独用药组相比差异均有统计学意义(P=0.000<0.01)。Annexin V(膜联蛋白V)－PI双染法检测细胞凋亡：对照组的细胞存活率最高，明显高于各用药组，差异有统计学差异（P<0.05），细胞凋亡率以结合组（39.25±8.34）最高，明显高于西药组及乳岩宁组（P<0.05），西药组的细胞凋亡率（31.21±7.53）高于乳岩宁组(26.13±6.76)，相比有显著差异性（P<0.05）。(3)流式细胞仪检测细胞周期结果显示：各用药组G0/G1期细胞的比例增多，S期比例明显减少，与模型对照组比较差异显著（P<0.01），结合组的G0/G1期细胞比例最多，S期细胞比例最少，与单纯用药组比较有统计学差异（P<0.05）。但是乳岩宁组与西药组分别对比G0/G1期、S期的细胞比例均无统计学差异（P﹥0.05）；采用Western blot蛋白免疫印迹法，检测各组裸鼠瘤组织中ERα、p-AKT和管家基因β-actin的表达结果：对照组中ERα及p-AKT蛋白高表达，与三个治疗组相比有显著性差异（p﹤0.01）；三个处理组均可下调ERα及p-AKT蛋白的表达，其中以结合组的作用更为显著，与乳岩宁组、西药组相比较有显著性差异（p﹤0.01）。
(4)免疫组化法测定肿瘤组织中Bcl-2、Bax的蛋白表达结果：Bcl-2及Bax蛋白表达定位：主要位于细胞浆中，少数在胞膜。表达阳性的细胞可见黄色、棕黄色的颗粒，尤以核周、胞膜明显，阴性细胞胞浆着色明显减弱，甚至呈阴性。对照组中Bcl-2蛋白存在高表达，Bax低表达，Bcl-2/Bax的比值显著升高，而三个处理组Bcl-2蛋白低表达，Bax蛋白高表达，与对照组相比有显著性差异（p﹤0.01）；西药组、乳岩宁组和结合组，都可不同程度的降低Bcl-2蛋白的表达，提高Bax的表达，并使Bcl-2/Bax的比值降低，其中结合组的作用更为显著，与西药组及乳岩宁组相比较有显著性差异（p﹤0.01）。说明模型对照组中存在乳腺癌细胞凋亡过程受阻，生长迅速，失去控制。各用药组均可诱导MCF-7乳腺癌细胞凋亡，可能与下调Bcl-2蛋白的表达，上调Bax蛋白的表达，使Bcl-2/Bax的比值降低有关。
（5）RT-PCR法检测乳腺癌组织中p53、SurvivinmRNA表达的结果：各用药组都可不同程度的降低p53、Survivin基因mRNA的表达，明显低于模型对照组（p﹤0.05）；其中以结合组的作用最为显著，与单独用药组相比较有显著性差异（p﹤0.01）。
结论:
1.中药乳岩宁能改善荷瘤裸鼠一般状态，减轻依西美坦带来的裸鼠体重下降，改善裸鼠活动能力。
2.中药乳岩宁联合依西美坦具有抑制乳腺癌生长的作用，可能与阻滞细胞周期进程及调节凋亡相关基因Bcl-2、Bax的表达来诱导细胞凋亡有关。
3.中药乳岩宁联合依西美坦下调通过癌组织中p-AKT、ERɑ蛋白的表达来抑制PI3K-AKT信号通路的活性，下调P53、Survivin基因mRNA的表达来抑制乳腺癌细胞增殖的。
Purpose:To observe the influence of Traditional Chinese medicine-Ruyanningand exemestane to the general states, the ability of independent activities andorgan coefficient of nude mouse with MCF-7breast cancer, and to find out ifare there synergistic roles of Chinese medicine-Ruyanning and exemestane. Toobserve the influence of Traditional Chinese medicine-Ruyanning and exemestaneto the pathomorphology and apoptosis of MCF-7breast cancer cell, and to findout the mechanisms of that inducing the apoptosis. To observe the influence ofTraditional Chinese medicine-Ruyanning and exemestane to the cell cycledistribution, and the protein expression of ERα and p-AKT of MCF-7breastcancer cell, and to find out the signal transduction pathway of TraditionalChinese medicine-Ruyanning and exemestane controlling the proliferation ofMCF-7breast cancer cell. To observe the influence of Traditional Chinesemedicine-Ruyanning and exemestane to the expression of the gene of Bcl-2, theprotein of Bax, p53and Survivin mRNA, which are related to the apoptosis ofMCF-7breast cancer cell, and to find out the mechanisms of that controllingthe the proliferation of MCF-7breast cancer cell.
Material and method:
1. Preparation of the model bilateral ovariectony:
10%choral hydrate (100mg/ml) is injected in the intraperitoneal of nude mouseaccording to the standard of4ml/kg. The nude mouse is prone position on theoperating table with the fixed limbs. The skin preparation is on the middle ofthe back with the lodine disinfection. Then we slit the skin on the spine adjacentto open0.5cm, away from the costal arch the lower edge of0.5cm, Cut theperitoneum into the abdominal cavity, and dish the milky organizations from theabdominal cavity. We see the pink mulberry-like ovarian. We separate it, thenclamp, ligature, cut ovarian, check for bleeding, suture the skin and disinfect. The same method is for the contralateral ovarian. The mouse is wake up afteranesthesia in20℃. The mouse is injected in0.25ml penicillin(60000U/ml) afteroperation and disinfect with3days. The mouse is intake water and SPF fodderwith30days. Then the model is success.
2. The creation of model of nude mouse with MCF-7cancer:
MCF-7cell in the phase of logarithmic growth is digested with trypsin/EDTA,then is washed by PBS solution twice. That is added to the cell culture medium,directly evenly pipetted, and stained with0.4%tyrpan blue. We record the numberof viable cells (>95%) and the number of cell counting boards, then adjustingthe viable cell concentration of1×107/ml. The right chest wall of nude mouseis disinfected. We vaccinate two nude mice in right chest wall, under the secondbreast pads with cell liquid0.2ml for every mouse. After10days the tumor nudesare appear in the breast pads, which are hard, gradually increased. When theprimary tumor is0.8cm3,the tumor is removed and transplanted to40nude micewith bone marrow biopsy needle.10%choral hydrate (0.02ml/10g) is injected inthe intraperitoneal of nude mouse with tumor. Breast cancer tissue is removedunder sterile conditions, immediately placed in DMEM, cut into pieces of about1mm3into the bone marrow biopsy needle. Before vaccination the right chest wallof healthy nude mouse is disinfected. We level pierce the needle into the rightchest wall next to the second breast pads1cm, then insert the needle core. Thetumor tissue is left in the breast pads. The skin is disinfected, no need tosuture the wounds. The penicillin1.5million U is injected in theintraperitoneal once. The nude mice are continued to raise in the SPF box. After4-5days the tumor is appear. The rate of tumor formation is100%. And the shapesand sizes are more equal. After15days the tumor can grow to9±2mm. Thepathological diagnosis is invasive ductal carcinoma by HE staining, withimmunohistochemical detection of ER (+).
3.Experimental groups and administration:
The40successful modeling nude mice were randomly divided into four groups:①model controlling group;②group of western medicine (exemestane);③group of Ruyanning;④combined group(exemestane and Ruyanning). Each groupadministered according to the human and mouse dose conversing formula.①modelcontrolling group:0.2ml saline is gavaged for every mouse everyday.②group ofwestern medicine (exemestane): Exemestane is administration according to4mg.kgfor every mouse(equivalent to10times the amount of clinical).0.2ml is gavagedfor every mouse everyday.③group of Ruyanning: Ruyanning is according to33g.kg(equivalent to12times the amount of clinical).0.2ml is gavaged for everymouse everyday.④combined group(exemestane and Ruyanning): Exemestane isaccording to4mg.kg and Ruyanning is according to33g.kg.0.2ml is gavaged forevery mouse everyday. All are administered continuously for21days.
4. Experimental drawing:
After experiment every group is drawn respectively. The nude mice is executedby cervical and placed in the ice box. The tumor is stripped out completely,and placed in the ice box after weighting. After the tumor tissue is observedby eyes, the full-thickness tissue is cut, and the same time it is drawnedmulti-size avoiding necrosis. The materials are5parts.(1)cellcycle specimens: Removing a piece of tissue placed in the PBS liquid of2ml.(2)electron microscopy specimens: Tissue is cut into about1mm3,thenimmediately put into0.25%glutaraldehyde, placed in a refrigerator at4℃spare.(3)RT-PCR specimens:2-3tissue blocks about8mm3is placed in the1ml Trizol. After labeled by permanent marker they are placed immediately in-80℃refrigerator.(4)Western Bloting specimens: Tissue blocks are placeddirectly in the EP tube, frozen in-80°C refrigerator.(5)For HE,TUNEL todetect apoptosis and immune: After drawning the specimens placedin4%paraformaldehyde with4℃.
5.Detection indicators:
(1)To observe the diet, activity, fur color stool,urine and death of nudemice everyday. To measure the weight every other day and paint the weight changecurve. To determine the ability of activity of mice by measuring the frequencyin5minutes with Spontaneous activity tester before treatment, on the10th day of the experiment,21days. The nude mice is executed. The liver, kidney andspleen are weighted and calculated organ index. The organ index=organ weight(mg)/body weight (g).
(2) The nude mice is executed by cervical after21days with medicine. Thetumor is stripped out completely and weighted. The tumor inhibition rate iscalculated. The tumor inhibition rate=(the tumor weigh of model controllinggroup-the tumor weigh of group of experiment)/the tumor weigh of modelcontrolling group×100%. Tumor tissue is fixed by4%paraformaldehyde,dehydrated by alcohol, opened by xylene, embedded in paraffin and sliced bymicrotome. The slice is5μm. Morphological changes are observed by lightmicroscopy after hematoxylin and eosin staining.The rate of apoptosis of tumorcells is detected by TUNEL assay. Tumor tissue is embedded in paraffin andsliced. The rest of the method steps are according to kit instructions. The cellswith appeared green fluorescence in the nucleus are positive cells. Observedby fluorescence microscopy, each stained slice select five400×typical highpower field randomly.100cells are observed per field.500cells are calculated.The average number of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. Further the tumor tissue is quick-frozen byultramicrotomy, fixed by glutaraldehyde and osmium tetroxide fixation,dehydrated, soaked embedded, sliced,observed by transmission electronmicroscopy (TEM)on the ultrastructual level.
(3) Detecting apoptosis by annexin V (annexin V)-PI double staining: Breastcancer tissue blocks are put in4°C phosphate buffer solution. That aredigested by0.25%trypsin30min－1h after mechanically cutting into pieces.Single cell suspension can be get over the200-400mesh filter cells. Themeasuring concentration of the cells is adjusting5×105-1×106cells/ml.1ml of cells is centrifuged1000rpm,4°C for10minutes. The cells wereresuspended in200ul Binding Buffer after Supernatant discarded. That is addedto0ul Annexin V-FITC and5ul PI and mixed gently. The time of reaction is15 minutes in Dark room temperature or30minutes in4℃. It is detected in flowcytometry adding300ul Binding Buffe. The judgment of results: the upper leftquadrant: cell debris; the upper right quadrant: apoptosis late cells and deadcells; the lower left quadrant: living cells: the lower right quadrant: apoptosisearly cells.
(4) The tumor is stripped out completely after21days with medicine. It isweighed and placed in the ice box. The cut specimens are placed in the EP tube,frozen in-80℃refrigerator for Western blot proteindetermination. The tissueof breast cancer after cutting into pieces is digested by0.25%trypsin30min－1h. Single cell suspension can be get over the200-400mesh filter cells. Thecells are fixed more than12hours with75％cold ethanol (-20℃precooling).It is added to1ml of PI(final concentration:50ug／ml) and RNA enzyme withenzyme-free DNA (final concentration:50ug／ml). After strained30minutes itis put into flow cytometry. The expression of apoptosis regulating gene P53,ERɑ is detected by Western blot. And the results are statistical analyze.
(5)The drawning of RT-PCR is that the tissue is put in1ml Trizol, and afterremarked put in-80℃refrigerator. The expression of apoptosis regulating geneBcl-2, Bax protein are qualitatively analyze by immunohistochemical method. Theexpression of p53、Survivin gene mRNA in breast cancer tissue by RT-PCR method.
Results:
(1)weight: the change of nude mice weight of each group: The nude mice weightof each group is no significant difference (P=0.790) before the administration.After a week the weight of the rest of the group is decrease in addition thatthe weight of controlling group is continued to rise. The group of Ruyanningis0.63±0.028. The group of western medicine is0.62±0.019. The combined group(0.20±0.017)is lighter than the group of Ruyanning and western medicine(P﹤0.05).But there are no significant difference between them(P>0.05). Theweight of combined group is gradually upward and the average weight is higherthan the group of Ruyanning and western medicine. After15days the weight isdecrease gradually. The weight of the group of western medicine is higher than the group of Ruyanning until19days. After21days the weights: The weight ofthe group of western medicine is lightest. The controlling group is heaviest.The controlling group is higher than the group of combined, Ruyanning andwestern medicine(P<0.05). But there are no significant difference among them.In addition to the controlling group the decreasing change of other group: Thegroup of western medicine is1.61±0.12. are no difference is1.11±0.0.16.Thecombined is0.93±0.06. There are differences among them(P<0.05).The timesof autonomic activities: At the beginning the times of each group is up about52/5minutes (abbreviation52). There are no difference among them(P>0.05).After10days in addition to the controlling group the times of each group isdecreased. The group of western medicine is lower than other group(p <0.01).Butthere are no difference between the group of Ruyanning and combined(P>0.05).After21days the decreasing trend is still. The times of autonomicactivities of each group are decrease because of the tumor. The group of westernmedicine is lower than other group(p <0.01).Because of exemestane in the groupof western medicine and combined, which can influence the gastrointestinaladverse reactions leading to physical falling. And the Ruyanning can repair thedamage. The change of liver, spleen and kidney of each group: The index of liverand kidney of combined group are higher than the controlling,but there is nodifference between them(P>0.05). The index of liver,spleen and kidney of thegroup of western medicine is higher than the Ruyanning. And there is differenceof index of liver and spleen between the group of western medicine and theRuyanning(P<0.05). There are differences between the index of liver, spleenand kidney of the Ruyanning and the combined(P<0.05).The weight of tumor: Theweight of tumor of western medicine, Ruyanning and the combined are all lowerthan the controlling. There are differences with the controlling(P﹤0.01).The lightest is combined group, which is lower than the group with single drug(p﹤0.01). The weight of tumor of western medicine is lower than the Ruyanning.There are differences(P﹤0.05).The rate of inhibition to tumor of westernmedicine, Ruyanning and the combined is respectively34.2%、25.2%、46.3%. The Ruyanning can inhibit the growth of breast cancer and has synergy withexemestane.
(2) pathomorphological observation: The tumor is circular, oval or lobulatedby eyes. The texture is middle. The section is pale, translucent and realqualities area. The pale necrosis is appeared in the larger tumor. Observationby light microscopy after HE staining: Tumor cells are arranged in cords orclusterswith less interstitial. There are central lacunar small pipe structurein the mass of tumor cells. The pathology is that invasive ductal carcinoma isvigorous growth, large nucleus, clear rounded or oval nucleolus, obvious mitoticand scattered necrotic foci. The controlling group is that the tumor cells aredensity, disorganized, cord-like. The tumor cells are large with abundantcytoplasm, large nucleus deeply stained, imbalance of nucleus and plasma. Thepathological mitotic is appear with significant atypia and clear cancer nests.And other groups are that tumor cells are slightly increased. The structure ofthe tumor tissue is destructed with bleeding and necrotic cells, atypical cellsreducing, some cells becoming isolating cells and fibrous tissue. There are alarge number of lymphocytes and leukocyte around the necrotic tumor cells. Thatmeans the growth of tumor cells is inhibited. Observation by electron microscopy:the controlling group: The breast cancer cell growth and metabolism are vigorous.The nucleus and cytoplasm is imbalance. The nuclear membrane is integrity withdouble nuclear membrane. There are a large number of mitochondria in thecytoplasm, scattered ribosomes and complete organs. The group of westernmedicine: There are much necrotic cells, rupturing membrane, the large numberof vacuoles in the cytoplasm, chromatin in the edge area of the nuclear membrane,mitochondria with medullary changes. The group of Ruyanning: The cells are earlyapoptotic. The Cell volume is significantly reduced or increased. Thebubble-like swelling is appear in the membrane. The nuclear heterochromatin isincrease, distributed in the periphery of the nucleus, and some with typicalapoptotic bodies. The combined group: The cells are late apoptotic. The cellmembrane is rupture. Cytoplasmic overflow is vacuolar changes, with the typical apoptotic bodies by the surrounding with membrane. There are nuclear fragmentsand organelles in small body.
(3)The results of TUNEL: The cells with appeared green fluorescence in thenucleus are positive cells. That means apoptosis. Observed by fluorescencemicroscopy, each stained slice select five400×typical high power fieldrandomly.100cells are observed per field.500cells are calculated. The averagenumber of positive cells per100cells is calculated as apoptoticindex(Apoptosis Index, AI).AI=the number of positive cells/the number ofcalculating cells×100%. There are differences in AI of the controlling groupand other groups (P=0.000<0.01). The rate of apoptosis of the combined groupis highest, then the western medicine. There are differences in them and thecontrolling and single drugs(P=0.000<0.01). Detecting apoptosis by annexin V(annexin V)-PI double staining: The cell viability of controlling is higher thanother groups. There are differences in them(P<0.05).The apoptosis rate ofcombined(39.25±8.34)is higher than the western medicine and Ruyaning(P<0.05).The apoptosis rate of western medicine (31.21±7.53)is higher than the Ruyaning(26.13±6.76). There are differences(P<0.05).
(4)The results of flow cytometry cell cycle: The proportion of cells inG0/G1phase of each treating group is increased, and S phase is significantlyreduced. The difference was significantly (P <0.01) comparing with the modelgroup. The proportion of cells in G0/G1phase of compared group is the most,and S phase is the least. There is difference comparing with the group of singledrug(P<0.05).But there is no difference between the group of Ruyanning andwestern medicine in the proportion of cells in G0/G1and S phase(P﹥0.05).The results of the expression of ERα、p-AKT and geneβ-actin in the tissue ofnude mouse by Western blot: The expression of ERαand p-AKT in controlling groupis high. The difference was significantly (P <0.01) comparing with other threegroups. Other three groups all can make down the expression of ERαand p-AKT.The most significant is combined group. The difference was significantly (P<0.01) comparing with the group of Ruyanning and western medicine.
(5)The results of the expression of Bcl-2、Bax in tumor tissue byimmunohistochemistry: The position of the expression of Bcl-2、Bax is the mostin cytoplasm, some in membrane. The positive expression of cell is yellow, brownparticles, especially in perinuclear and membrane. The negative cell iscytoplasmic colored significantly diminishing, or even negative. In controllinggroup the expression of Bcl-2is high, Bax is low, and the ratio of Bcl-2/Baxis significantly increased. In other three groups the expression of Bcl-2islow, Bax is high, and the difference is significant comparing with thecontrolling group(p﹤0.01).The group of western medicine, Ruyanning andcombined can reduce the expression of Bcl-2, increase Bax, and reduce the ratioof Bcl-2/Bax. The combined group has more role, and the difference issignificant comparing with the group of western medicine, Ruyanning(p﹤0.01).Wecan see the process of apoptosis is blocked in the controlling group, with rapidgrowth and loss of control. Other groups can induce the apoptosis of MCF-7breastcancer cell. That maybe related to reduce the expression of Bcl-2, increaseBax, and reduce the ratio of Bcl-2/Bax.
(6)The results of the expression of p53、SurvivinmRNA in tumor tissue byRT-PCR: Each group with drug all can low the expression of p53、SurvivinmRNA,lower than the controlling group(P<0.05).The compared group is most significant,having difference with the group with single drug(p﹤0.01).Conclusions:
1. The Traditional Chinese medicine-Ruyanning can improve the general statesand the ability of independent activities of nude mouse with MCF-7breast cancer,and reduce the side effect of exemestane decreasing the weight of mouse.
2. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit thegrowth of breast cancer, which maybe related to block the procession of cellcycle and regulate the expression of gene Bcl-2、Bax to induce apoptosis.
3. The Traditional Chinese medicine-Ruyanning and exemestane can inhibit theactivity of PI3K-AKT signal pathway by reducing the expression of p-AKT、ERɑin cancer tissue, and inhibit the proliferation of breast cancer cell by reducing the expression of P53、SurvivingenemRNA.
引文
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[1]王歌，殷东风，高宏．殷东风运用中药饮片治疗晚期乳腺癌的规律研究[J]．辽宁中医杂志，2010,37(11)：2157-2159
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