辛伐他汀誘導脈絡膜黑色素瘤細胞凋亡和自噬的機制研究
發(fā)布時間：2018-08-20 09:21
【摘要】：背景： 脈絡膜黑色素細胞瘤是成年人最常見的眼內(nèi)惡性腫瘤，其致死率高。脈絡膜黑色素細胞瘤易發(fā)于藍眼睛，棕頭發(fā)的白人。其發(fā)病原因不明。目前研究表明，脈絡膜黑色素細胞瘤對放療化療均不敏感。腫瘤體積的大小與其預后和致死率密切相關(guān)。即使手術(shù)切除腫瘤也有約一半的病人因繼發(fā)性轉(zhuǎn)移性并發(fā)癥而死亡。所以，我們有必要尋找一種更有效的方法來治療脈絡膜黑色素細胞瘤。 辛伐他汀屬于羥甲基戊二酸單酰輔酶A(HMG-coA)還原酶抑制劑，目前廣泛應用于臨床來治療高膽固醇血癥，動脈硬化，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，還具有潛在的預防和治療癌癥的作用。但是，辛伐他汀對脈絡膜黑色素細胞瘤的作用研究尚未見報道。本論文將研究辛伐他汀對脈絡膜黑色素細胞瘤是否具有抗癌作用及可能的作用機制。 目的： 1．檢測辛伐他汀在體外對脈絡膜黑色素瘤細胞（OCM-1）增殖的影響。 2．探討辛伐他汀在體外誘導脈絡膜黑色素瘤細胞周期阻滯的分子機制 3．探討辛伐他汀在體外誘導脈絡膜黑色素瘤細胞凋亡和自噬的分子機制。 方法： 1．采用四甲基偶氮唑鹽(MTT法)法，檢測辛伐他汀在不同時間和不同濃度下在體外對OCM-1細胞增殖的影響。 2.采用流式細胞儀，RT-PCR技術(shù)和Western-Blot方法探討辛伐他汀在體外誘導脈絡膜黑色素瘤細胞周期阻滯的分子機制。 3.采用細胞染色方法，流式細胞儀和Western-Blot方法探討辛伐他汀在體外誘導脈絡膜黑色素瘤細胞凋亡和自噬的分子機制。 結(jié)果： 1.MTT實驗結(jié)果顯示，隨著辛伐他汀濃度（2-10μM）的增加，對OCM-1細胞增殖抑制率明顯增加，差異有統(tǒng)計學意義(P0.01）；隨著辛伐他汀作用時間（24h-72h)的延長，對OCM-1細胞增殖抑制率明顯升高,差異有統(tǒng)計學意義(P0.01）。 2.流式細胞儀結(jié)果顯示，辛伐他�。�4μM，24h-48h）能夠使OCM-1細胞發(fā)生Gl期阻滯。RT-PCR結(jié)果顯示辛伐他�。�4μM，24h-48h）處理后的OCM-1細胞中細胞周期蛋白cyclinD1,cyclinE的mRNA表達減少,細胞周期蛋白依賴性激酶CDK2的mRNA表達減少，細胞周期蛋白依賴性激酶抑制因子P21的mRNA表達增加；Western-Blot結(jié)果與RT-PCR結(jié)果一致，，顯示為cyclinD1,cyclinE, CDK2蛋白表達減少，P21蛋白表達增加； 3.用Hoe33342對辛伐他汀處理后的OCM-1細胞進行染色，結(jié)果顯示，辛伐他汀可以誘導OCM-1細胞發(fā)生染色質(zhì)濃縮，凋亡小體形成，而且隨著辛伐他汀作用時間的延長，細胞凋亡現(xiàn)象更加明顯。流式細胞儀結(jié)果顯示，辛伐他�。�4μM，24h-48h）處理的OCM-1細胞中活性氧（ROS）增加， Western-Blot結(jié)果顯示，凋亡相關(guān)蛋白cleaved-caspase-3，cleaved-caspase-9,Bax，P53蛋白表達增加，凋亡抑制蛋白Bcl-2，iASPP蛋白表達減少； 4.MDC染色結(jié)果顯示，辛伐他汀處理后的OCM-1細胞顯示有更多的MDC著染的自噬泡，說明辛伐他汀可以誘導OCM-1細胞發(fā)生自噬。Western-Blot結(jié)果顯示，辛伐他�。�4μM，24h-48h）處理的OCM-1細胞中自噬相關(guān)蛋白LC-3表達增加。 結(jié)論： 1.隨著辛伐他汀作用時間和濃度的增加，OCM-1細胞增殖抑制率增加。 2.辛伐他汀可以誘導OCM-1細胞發(fā)生Gl期阻滯。 3.辛伐他汀可以誘導OCM-1細胞發(fā)生凋亡。 4.辛伐他汀可以誘導OCM-1細胞發(fā)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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R739.7
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R739.7
【共引文獻】
相關(guān)期刊論文 前10條
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