【作者】 许景艳；

【导师】 欧阳建；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 [目的]本研究探讨藤黄酸在体外抑制套细胞淋巴瘤细胞株Jeko-1增殖的效应及可能机理。[方法]以不同浓度(0、0.065、0.125、0.25、0.5、1.0、2.0、4.0μg/ml)藤黄酸处理套细胞淋巴瘤Jeko-1细胞株12h-36h,免疫组化法检测细胞Ki-67阳性表达率,细胞毒性检测(?)(CCK-8)法检测藤黄酸对Jeko-1细胞及正常人骨髓单个核细胞的增殖抑制作用：碘化丙啶(PI)染色分析检测细胞周期改变；采用Annexin V/PI双染法观察细胞形态并检测细胞的凋亡,JC-1染色法检测细胞线粒体跨膜电位(△Ψm)水平；分光光度法检测细胞半胱氨酸蛋白酶Caspase-3、Caspase-8、Caspase-9阳性细胞水平；蛋白免疫印迹术检钡(?)Bcl-2、Bax蛋白水平。[结果]1.藤黄酸呈时间和浓度依赖性抑制Jeko-1细胞的增殖,藤黄酸对Jeko-1细胞增殖抑制率在低浓度组(0.065、0.125、0.25μg/ml)与对照组比较差异不显著(P>0.05),较高浓度组(0.5、1.0、2.0、4.0μg/ml)与对照组比较差异显著(P<0.05)：藤黄酸对正常人骨髓单个核细胞加药组与对照组比较,差异均无显著性(P>0.05)。藤黄酸可下调Jeko-1细胞Ki67的表达,且呈药物浓度依赖性。2.藤黄酸作用于Jeko-1细胞株24h后呈典型凋亡形态学改变,各浓度(0、0.5、1.0、2.0、4.0μg/ml藤黄酸对Jeko-1细胞作用24h的细胞周期未见明显影响。3.藤黄酸呈浓度依赖性增高Jeko-1细胞株的损伤线粒体跨膜电位(△ψm)水平。4.藤黄酸作用JeKo-1细胞24小时,用药标本较对照标本的caspase-3、caspase-8和caspase-9活化比例明显升高(p<0.05)。caspase-3、caspase-8、和caspase-9的活化呈现浓度依赖性。5.藤黄酸作用Jeko-1细胞24h后,随着藤黄酸浓度的增高,Bcl-2蛋白表达逐渐下降,而Bax蛋白表达逐渐增强。与对照组相比,在1μg/ml浓度时Bax/Bcl-2蛋白比值明显增高(P<0.05)。[结论]1.藤黄酸呈浓度依赖性抑制JeKo-1细胞的增殖。2.藤黄酸呈浓度依耐性诱导JeKo-1细胞凋亡,机理是通过线粒体通路和膜受体通路诱导Jeko-1细胞凋亡,其诱导凋亡作用并非通过特异的细胞周期阻滞来实现。3.藤黄酸诱导人套细胞淋巴瘤Jeko-1细胞凋亡时,主要通过活化Caspase-3、Caspase-8, Caspase-9和受损△ψm增加途径而实现诱导细胞凋亡,藤黄酸通过减低JeKo-1细胞中Bcl-2蛋白的水平,上调Bax蛋白的水平诱导细胞凋亡。更多还原

【Abstract】 [Objective] To study the mechanisms in gambogic acid (GA)-induced JeKo-1human Mantle Cell Lymphoma cell apoptosis in vitro.[Methods] the proliferation of GA-treated Jeko-1cells was measured by CCK-8assay and ki-67immunocytochemical detection. Appotosis, cell cycle and mitochondrial membrane potential were measured by flow cytometric analysis. Caspase-3,-8and-9were detected by colorimetric assay. Bcl-2and Bax were analyzed by Western blot.[Results]1. GA inhibited Jeko-1cells’ growth in a time-and dose-dependent manner. Lower GA concentration (0.065、0.125、0.25μg/ml) could not inhibit the proliferation of Jeko-1cells(P>0.05). Higher GA(0.5、1.0、2.0、4.0μg/ml) concentrationcould inhibit the proliferation of Jeko-1cells (P<0.05).2. GA did not significantly influence the cell cycle of Jeko-1cell lines at the concentration of0、0.5、1.0、2.0、4.0μg/ml for24h.3. The typical apoptosis morphology change could be visualized after Jeko-1cell lines were incubated with GA. GA induces apoptosis in Jeko-1cells, which was involved in reducing the membrane potential of mitochondria,4. GA increased the apoptosis rate of Jeko-1cell line and increased activated caspase-3, caspase-8, caspase-9positive cell number for different degree in24h.5. There was a significant decrease in Bcl-2expression and increase in Bax expression in a dose-dependent manner after the GA treatment on Jeko-1cell for24h. Compared with the control group, with the concentration of1ug/ml, ratio between Bax and Bcl-2protein increased significantly (P<0.05).[Conclusion]1. GA has inhibitive effect on Jeko-1cell lines’ growth in a concentration dependent manner in vitro.2. GA induces apoptosis on Jeko-1cell lines in a concentration dependent manner. The Mechanism is through the mitochondrial pathway and membrane receptor pathway induce Jeko-1cell apoptosis.The cell cycles are not disturbed obviously in Jeko-1cell lines.3. GA induced apoptosis in human MCL Jeko-1cells by regulating Bcl-2/Bax and activating caspase-3,-8and-9via mitochondrial pathway without affecting cell cycle.更多还原