辛伐他汀诱导脉络膜黑色素瘤细胞凋亡和自噬的机制研究

发布时间：2018-08-20 09:21

【摘要】：背景：脉络膜黑色素细胞瘤是成年人最常见的眼内恶性肿瘤，其致死率高。脉络膜黑色素细胞瘤易发于蓝眼睛，棕头发的白人。其发病原因不明。目前研究表明，脉络膜黑色素细胞瘤对放疗化疗均不敏感。肿瘤体积的大小与其预后和致死率密切相关。即使手术切除肿瘤也有约一半的病人因继发性转移性并发症而死亡。所以，我们有必要寻找一种更有效的方法来治疗脉络膜黑色素细胞瘤。辛伐他汀属于羟甲基戊二酸单酰辅酶A(HMG-coA)还原酶抑制剂，目前广泛应用于临床来治疗高胆固醇血症，动脉硬化，冠心病，其性能安全，耐受性好。辛伐他汀除了具有降脂作用外，还具有潜在的预防和治疗癌症的作用。但是，辛伐他汀对脉络膜黑色素细胞瘤的作用研究尚未见报道。本论文将研究辛伐他汀对脉络膜黑色素细胞瘤是否具有抗癌作用及可能的作用机制。目的： 1．检测辛伐他汀在体外对脉络膜黑色素瘤细胞（OCM-1）增殖的影响。 2．探讨辛伐他汀在体外诱导脉络膜黑色素瘤细胞周期阻滞的分子机制 3．探讨辛伐他汀在体外诱导脉络膜黑色素瘤细胞凋亡和自噬的分子机制。方法： 1．采用四甲基偶氮唑盐(MTT法)法，检测辛伐他汀在不同时间和不同浓度下在体外对OCM-1细胞增殖的影响。 2.采用流式细胞仪，RT-PCR技术和Western-Blot方法探讨辛伐他汀在体外诱导脉络膜黑色素瘤细胞周期阻滞的分子机制。 3.采用细胞染色方法，流式细胞仪和Western-Blot方法探讨辛伐他汀在体外诱导脉络膜黑色素瘤细胞凋亡和自噬的分子机制。结果： 1.MTT实验结果显示，随着辛伐他汀浓度（2-10μM）的增加，对OCM-1细胞增殖抑制率明显增加，差异有统计学意义(P0.01）；随着辛伐他汀作用时间（24h-72h)的延长，对OCM-1细胞增殖抑制率明显升高,差异有统计学意义(P0.01）。 2.流式细胞仪结果显示，辛伐他汀（4μM，24h-48h）能够使OCM-1细胞发生Gl期阻滞。RT-PCR结果显示辛伐他汀（4μM，24h-48h）处理后的OCM-1细胞中细胞周期蛋白cyclinD1,cyclinE的mRNA表达减少,细胞周期蛋白依赖性激酶CDK2的mRNA表达减少，细胞周期蛋白依赖性激酶抑制因子P21的mRNA表达增加；Western-Blot结果与RT-PCR结果一致，，显示为cyclinD1,cyclinE, CDK2蛋白表达减少，P21蛋白表达增加； 3.用Hoe33342对辛伐他汀处理后的OCM-1细胞进行染色，结果显示，辛伐他汀可以诱导OCM-1细胞发生染色质浓缩，凋亡小体形成，而且随着辛伐他汀作用时间的延长，细胞凋亡现象更加明显。流式细胞仪结果显示，辛伐他汀（4μM，24h-48h）处理的OCM-1细胞中活性氧（ROS）增加， Western-Blot结果显示，凋亡相关蛋白cleaved-caspase-3，cleaved-caspase-9,Bax，P53蛋白表达增加，凋亡抑制蛋白Bcl-2，iASPP蛋白表达减少； 4.MDC染色结果显示，辛伐他汀处理后的OCM-1细胞显示有更多的MDC着染的自噬泡，说明辛伐他汀可以诱导OCM-1细胞发生自噬。Western-Blot结果显示，辛伐他汀（4μM，24h-48h）处理的OCM-1细胞中自噬相关蛋白LC-3表达增加。结论： 1.随着辛伐他汀作用时间和浓度的增加，OCM-1细胞增殖抑制率增加。 2.辛伐他汀可以诱导OCM-1细胞发生Gl期阻滞。 3.辛伐他汀可以诱导OCM-1细胞发生凋亡。 4.辛伐他汀可以诱导OCM-1细胞发生自噬。
[Abstract]:Background:
Choroidal melanocytoma is the most common intraocular malignancy in adults with a high mortality rate. Choroidal melanocytoma is prone to occur in white people with blue eyes and brown hair. Even if the tumor is removed surgically, about half of the patients die of secondary metastatic complications. Therefore, it is necessary to find a more effective treatment for choroidal melanoma.
Simvastatin is a kind of HMG-coA reductase inhibitor. It is widely used in clinical treatment of hypercholesterolemia, atherosclerosis, coronary heart disease. It is safe and well tolerated. Simvastatin has not only lipid-lowering effect, but also potential role in the prevention and treatment of cancer. The effect of simvastatin on choroidal melanoma has not been reported yet. This study will investigate whether simvastatin has anticancer effect on choroidal melanoma and its possible mechanism.
Objective:
1. to detect the effect of simvastatin on the proliferation of choroidal melanoma cells (OCM-1) in vitro.
2. to explore the molecular mechanism of cell cycle arrest induced by simvastatin in vitro.
3. to explore the molecular mechanism of simvastatin inducing apoptosis and autophagy in choroidal melanoma cells in vitro.
Method:
1. The effect of simvastatin on the proliferation of OCM-1 cells in vitro was detected by MTT assay.
2. To investigate the molecular mechanism of simvastatin-induced choroidal melanoma cell cycle arrest in vitro, flow cytometry, RT-PCR and Western-Blot methods were used.
3. To investigate the molecular mechanism of simvastatin-induced apoptosis and autophagy in choroidal melanoma cells in vitro by cell staining, flow cytometry and Western-Blot assay.
Result:
1. MTT assay showed that with the increase of simvastatin concentration (2-10 mu M), the inhibition rate of OCM-1 cell proliferation increased significantly (P 0.01); with the extension of simvastatin action time (24-72 h), the inhibition rate of OCM-1 cell proliferation increased significantly (P 0.01).
2. The results of flow cytometry showed that simvastatin (4 mu M, 24 h-48 h) could induce Gl phase arrest in OCM-1 cells. RT-PCR showed that the expression of cyclin D1 and cyclin E mRNA, the expression of cyclin-dependent kinase CDK 2 mRNA and cyclin-dependent protein were decreased in the OCM-1 cells treated with simvastatin (4 mu, 24 h-48 h). The expression of sex kinase inhibitor P21 mRNA increased, Western-Blot results were consistent with RT-PCR results, showing that cyclin D1, cyclin E, CDK2 protein expression decreased, P21 protein expression increased.
3. OCM-1 cells treated with simvastatin were stained with Hoe33342. The results showed that simvastatin could induce chromatin condensation and apoptotic corpuscle formation in OCM-1 cells, and the phenomenon of apoptosis was more obvious with the extension of simvastatin treatment time. Flow cytometry showed that simvastatin (4 mu M, 24 h-48 h) treated OCM-1 cells. Western-Blot analysis showed that the expression of cleaved-caspase-3, cleaved-caspase-9, Bax and P53 protein increased, while the expression of Bcl-2 and iASPP protein decreased.
4. The results of MDC staining showed that there were more MDC-stained autophagic vesicles in the OCM-1 cells treated with simvastatin, suggesting that simvastatin could induce autophagy in OCM-1 cells.
Conclusion:
1. with the increase of the time and concentration of simvastatin, the inhibition rate of OCM-1 cell proliferation increased.
2. simvastatin can induce Gl arrest in OCM-1 cells.
3. simvastatin can induce apoptosis in OCM-1 cells.
4. simvastatin can induce autophagy in OCM-1 cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.7

【共引文献】