【作者】 周琳；

【导师】 罗志勇；

【作者基本信息】 中南大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 宫颈癌、肺癌是人类常见的一种恶性肿瘤,严重威胁着人类身体的健康。手术、放疗、化疗是目前肿瘤治疗的主要手段。随着现代细胞生物学和分子生物学的快速发展,诱导肿瘤细胞凋亡的凋亡疗法被认为是一种新的有效治疗方法,诱导肿瘤细胞凋亡的天然药物研究已成为近几年的抗肿瘤药物研究热点之一。人参皂苷G-Rh2便是治疗肿瘤药物中天然凋亡诱导剂的一种。G-Rh2是人参中提取的达玛烷二醇型皂苷单体,为人参抗肿瘤效应的主要有效成分。尽管大量研究表明G-Rh2能够诱导多种肿瘤细胞凋亡发挥其体内外广谱抗肿瘤活性,但是G-Rh2诱导肿瘤细胞凋亡的信号转导分子机制,特别是非器官特异性抗肿瘤作用机制还不清楚。本研究以人类宫颈癌HeLa细胞系和肺癌A549细胞系为实验对象,研究G-Rh2诱导HeLa细胞和A549细胞凋亡过程中细胞周期和相关蛋白表达的变化,探讨G-Rh2抗肿瘤作用的分子机制,期望找到各肿瘤细胞共同的关键信号靶蛋白。本实验主要运用MTT法检测0-60μMG-Rh2处理HeLa细胞和A549细胞48h后的增殖情况并分别确定其半数抑制率(IC50),并运用流式细胞术检测0-60μM G-Rh2处理HeLa和A549细胞12、24、48、60和72h后细胞周期改变情况,最后从蛋白水平用Western blotting方法验证相关凋亡信号分子ERα、ERβ、TNFα蛋白表达水平的变化。MTT实验结果显示,0-60μM的G-Rh2处理HeLa和A549细胞48h后,两种细胞的存活率均随着G-Rh2浓度的增加而明显下降,即G-Rh2对HeLa和A549细胞生长的抑制作用具有浓度依赖性效应。G-Rh2对HeLa和A549细胞的IC50值分别为45μM、48μM。流式细胞术分析发现G-Rh2引发肿瘤细胞周期于G1期阻滞,均呈现浓度和时间依赖效应。Western blotting结果表明,两种细胞随着药物浓度的逐渐增大、时间的逐渐延长,ERβ均表达上调,ERα则表达下调。然而HeLa细胞中TNFα表达上调,A549细胞中TNFα则表达不变。G-Rh2与ER拮抗剂MPP、PHTPP、ICI182,780联合处理组中,ERα、ERβ、TNFα表达量不受MPP的影响,而受PHTPP和ICI182,780影响较大。G-Rh2+PHTPP和G-Rh2+ICI182,780组中,ERα、ERβ表达量均降低,HeLa细胞中TNFα表达下调,A549细胞中TNFα表达量不变。综上所述,G-Rh2通过引发HeLa细胞周期G1期阻滞并下调ERa、上调ERβ蛋白质水平的表达,进而上调TNFα蛋白质水平的表达,而诱导HeLa细胞凋亡。G-Rh2通过引发A549细胞周期G1期阻滞并下调ERa蛋白质水平的表达和上调ERβ蛋白质水平的表达,而诱导A549细胞凋亡。ERα或(和)ERβ可能作为了G-Rh2诱导各肿瘤细胞凋亡的共同关键信号蛋白。更多还原

【Abstract】 Uterine cervix cancer and lung cancer are common malignant tumors all over the world. The two both threaten the human health. Surgery, radiotherapy, and chemotherapy are the three major cancer therapeutic strategies. With the development of modern cellular biology and molecular biology, apoptosis therapy by inducing apoptosis of tumor cells is considered to be a new and effective treatment. Exploration of efficient and low toxicity apoptosis inducers may be the most promising direction for antitumor drugs, natural medicine research on inducing apoptosis of tumor cells has become one of the hotspots of anticancer drug research in recent years.Ginsenoside Rh2 is one of the natural inducers of apoptosis for cancer treatment. It is a dammarane saponin derived from Panax ginseng, has been identified as the major component responsible for ginseng’s antitumor action. Although accumulating evidences showed that G-Rh2 can induce apoptosis of various tumors and play broad-spectrum anti-tumor activity in vivo and in vitro, the cell signal transduction molecular mechanism of inducing apoptosis of tumor cells by G-Rh2, especially its non-organic specific antitumor effects, has not been fully elucidated. In this study we used human cervical cancer cell line HeLa and lung cancer cell line A549 as experimental subjects to study the cell cycle and related protein expression change during the apoptosis process of these cells induced by G-Rh2 and explore the molecular mechanism of G-Rh2’s antitumor activity.We used MTT way to detect proliferation of HeLa cells and A549 cells treated by G-Rh2 of 0-60μM-dose for 48 hours and separately calculate the IC50 of G-Rh2. Flow cytometry (FCM) was used to detect the change of cell cycle about these two cells treated with G-Rh2 of 0-60μM-dose for 12,24,48,60 and 72 hours. Finally, Western blotting was used to analyze the expression of correlative protein ERa, ERP and TNFa. The results of MTT showed that the survival rate of HeLa and A549 cells were in a dose-dependent manner. The IC50 of G-Rh2 when treated HeLa is 45μM and A549 is 48μM. FCM analysis found that G-Rh2 blocked the cycle within G1 phase of these two cells in a dose-and time-dependent manner. The results of Western blotting showed that the expression of ERa was down-regulated and ERβwas up-regulated, in a dose-and time-dependent manner. However, the expression of TNFa was up-regulated in HeLa cells, but didn’t change in A549 cells. While treating tumor cells with G-Rh2 respectively combined with ER antagonist MPP, PHTPP and ICI 182,780, compared to the control group, the expression of ERa, ERβ, TNFa were not affected by MPP,but affected by PHTPP and ICI182,780 larger. The expression of ERa and ERβin the G-Rh2 combined with PHTPP and ICI182,780 treatment group were lower, and the expression of TNFa in HeLa cells was reduced, but the expression of TNFa did not change in A549 cells.Conclusively, G-Rh2 can induce apoptosis of HeLa cells through the G1 cell cycle arrest and regulating the expression level of estrogen receptor ERα, ERβand TNFα.And it also can induce apoptosis of A549 cells through the G1 cell cycle arrest and regulating the expression level of ERa and ERβto inhibit the proliferation of tumor cells. ERαor/and ERβmay be as the common and critical signal protein of G-Rh2-induced-apoptosis in all the tumor cells.更多还原