辛伐他汀誘導(dǎo)脈絡(luò)膜黑色素瘤細(xì)胞凋亡和自噬的機(jī)制研究
發(fā)布時(shí)間：2018-08-20 09:21
【摘要】：背景： 脈絡(luò)膜黑色素細(xì)胞瘤是成年人最常見(jiàn)的眼內(nèi)惡性腫瘤，其致死率高。脈絡(luò)膜黑色素細(xì)胞瘤易發(fā)于藍(lán)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(luò)膜黑色素細(xì)胞瘤對(duì)放療化療均不敏感。腫瘤體積的大小與其預(yù)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(lái)治療脈絡(luò)膜黑色素細(xì)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(yīng)用于臨床來(lái)治療高膽固醇血癥，動(dòng)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(yù)防和治療癌癥的作用。但是，辛伐他汀對(duì)脈絡(luò)膜黑色素細(xì)胞瘤的作用研究尚未見(jiàn)報(bào)道。本論文將研究辛伐他汀對(duì)脈絡(luò)膜黑色素細(xì)胞瘤是否具有抗癌作用及可能的作用機(jī)制。 目的： 1．檢測(cè)辛伐他汀在體外對(duì)脈絡(luò)膜黑色素瘤細(xì)胞（OCM-1）增殖的影響。 2．探討辛伐他汀在體外誘導(dǎo)脈絡(luò)膜黑色素瘤細(xì)胞周期阻滯的分子機(jī)制 3．探討辛伐他汀在體外誘導(dǎo)脈絡(luò)膜黑色素瘤細(xì)胞凋亡和自噬的分子機(jī)制。 方法： 1．采用四甲基偶氮唑鹽(MTT法)法，檢測(cè)辛伐他汀在不同時(shí)間和不同濃度下在體外對(duì)OCM-1細(xì)胞增殖的影響。 2.采用流式細(xì)胞儀，RT-PCR技術(shù)和Western-Blot方法探討辛伐他汀在體外誘導(dǎo)脈絡(luò)膜黑色素瘤細(xì)胞周期阻滯的分子機(jī)制。 3.采用細(xì)胞染色方法，流式細(xì)胞儀和Western-Blot方法探討辛伐他汀在體外誘導(dǎo)脈絡(luò)膜黑色素瘤細(xì)胞凋亡和自噬的分子機(jī)制。 結(jié)果： 1.MTT實(shí)驗(yàn)結(jié)果顯示，隨著辛伐他汀濃度（2-10μM）的增加，對(duì)OCM-1細(xì)胞增殖抑制率明顯增加，差異有統(tǒng)計(jì)學(xué)意義(P0.01）；隨著辛伐他汀作用時(shí)間（24h-72h)的延長(zhǎng)，對(duì)OCM-1細(xì)胞增殖抑制率明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.01）。 2.流式細(xì)胞儀結(jié)果顯示，辛伐他�。�4μM，24h-48h）能夠使OCM-1細(xì)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(xì)胞中細(xì)胞周期蛋白cyclinD1,cyclinE的mRNA表達(dá)減少,細(xì)胞周期蛋白依賴性激酶CDK2的mRNA表達(dá)減少，細(xì)胞周期蛋白依賴性激酶抑制因子P21的mRNA表達(dá)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(dá)減少，P21蛋白表達(dá)增加； 3.用Hoe33342對(duì)辛伐他汀處理后的OCM-1細(xì)胞進(jìn)行染色，結(jié)果顯示，辛伐他汀可以誘導(dǎo)OCM-1細(xì)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(shí)間的延長(zhǎng)，細(xì)胞凋亡現(xiàn)象更加明顯。流式細(xì)胞儀結(jié)果顯示，辛伐他汀（4μM，24h-48h）處理的OCM-1細(xì)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(dá)增加，凋亡抑制蛋白Bcl-2，iASPP蛋白表達(dá)減少； 4.MDC染色結(jié)果顯示，辛伐他汀處理后的OCM-1細(xì)胞顯示有更多的MDC著染的自噬泡，說(shuō)明辛伐他汀可以誘導(dǎo)OCM-1細(xì)胞發(fā)生自噬。Western-Blot結(jié)果顯示，辛伐他汀（4μM，24h-48h）處理的OCM-1細(xì)胞中自噬相關(guān)蛋白LC-3表達(dá)增加。 結(jié)論： 1.隨著辛伐他汀作用時(shí)間和濃度的增加，OCM-1細(xì)胞增殖抑制率增加。 2.辛伐他汀可以誘導(dǎo)OCM-1細(xì)胞發(fā)生Gl期阻滯。 3.辛伐他汀可以誘導(dǎo)OCM-1細(xì)胞發(fā)生凋亡。 4.辛伐他汀可以誘導(dǎo)OCM-1細(xì)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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.7
【共引文獻(xiàn)】
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