细胞色素P4501A1在白藜芦醇抑制人肝癌细胞增殖及促进其凋亡中的作用

发布时间：2018-03-09 05:17

  本文选题：白藜芦醇　切入点：人肝癌细胞SMMC-7721　出处：《四川医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:原发性肝癌是常见的消化系统恶性肿瘤之一,恶性程度较高,病灶容易转移,治疗效果差。白藜芦醇(Resveratrol)是从虎杖、葡萄等植物中提取的一种多酚类物质,近年来研究显示,白藜芦醇具有抗肿瘤、保护血管神经、抗炎、影响骨钙水平等作用,是治疗脂类代谢紊乱、炎症和心脏疾病的有效成分。据文献报道,白藜芦醇具有抗肝癌的作用,但抗肝癌作用具体机制尚不清楚。诱导肿瘤细胞凋亡、抑制肿瘤细胞增殖是研究抗肿瘤药物的常规途径,本研究拟从体外角度,研究白藜芦醇对人肝癌细胞SMMC-7721增殖及凋亡的影响,并初步探讨其可能的作用机制。通过CCK-8法检测白藜芦醇影响人肝癌细胞SMMC-7721增殖的时效及量效关系,以确定白藜芦醇发挥最大作用的浓度及时间范围。应用Hoechst-33258染色及双染流式细胞术(Annexin V/P)检测凋亡细胞形态学及量的变化,Real time-PCR技术检测白藜芦醇干预前后人肝癌细胞SMMC-7721表达细胞色素P4501A1 m RNA(CYP1A1)的变化。蛋白印记分析(Western blot analysis)法检测白藜芦醇干预前后人肝癌细胞SMMC-7721表达细胞色素P4501A1蛋白的变化。方法:分别用浓度25μmol/L、50μmol/L、100μmol/L、200μmol/L的白藜芦醇作用人肝癌细胞24h、48h、72h,CCK-8法检测白藜芦醇对人肝癌细胞SMMC-7721的增殖抑制作用。分别以无处理的人肝癌细胞SMMC-7721及相同剂量的DMSO为空白和随机对照组,Annexin V/PI流式细胞术观察100μmol/L的白藜芦醇作用72h后对人肝癌细胞SMMC-7721凋亡率的影响,Hoechst-33258观察100μmol/L白藜芦醇作用72h后人肝癌细胞SMMC-7721中凋亡细胞形态学的变化。Real-time PCR分析100μmol/L的白藜芦醇作用前后对人肝癌细胞SMMC-7721表达CYP1A1m RNA的影响。蛋白印记分析(Western blot analysis)法检测100μmol/L的白藜芦醇作用前后人肝癌细胞SMMC-7721中CYP1A1蛋白表达情况。结果:CCK-8检测结果显示,在体外培养状态下,白藜芦醇能显著抑制人肝癌细胞SMMC-7721的增殖,在本实验的浓度剂量范围内,随着白藜芦醇浓度的不断增大,抑制增殖的效果明显增强,呈浓度依赖关系。同一浓度随着作用时间的不断延长,抑制增殖的效果明显增强,呈时间依赖性。不同浓度白藜芦醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌细胞24h后增殖抑制率分别为(8.38±1.75%、15.40±3.32%、22.35±2.43%、15.57±1.73%),不同浓度白藜芦醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌细胞48h后增殖抑制率分别为(12.60±1.51%、20.70±3.21%、40.60±2.33%、51.67±3.70%),不同浓度白藜芦醇(25μmol/L、50μmol/L、100μmol/L、200μmol/L)作用人肝癌细胞72h后增殖抑制率分别为(15.42±3.54%、24.57±3.81%、60.51±3.50%、65.51±2.41%),经统计分析得出,不同浓度白藜芦醇对人肝癌细胞SMMC-7721增殖抑制率不同(F=14.03,P=0.03),相同浓度白藜芦醇在不同作用时间对人肝癌细胞SMMC-7721增殖抑制率也有明显差异(F=15.04,P0.05),所以一定浓度白藜芦醇作用一定时间范围内,其对人肝癌细胞SMMC-7721的增殖抑制作用存在浓度及时间依赖性。从人肝癌细胞SMMC-7721在白藜芦醇作用下的量效曲线上可以看出:100μmol/L白藜芦醇作用72h后随药物浓度的升高抑制率斜率开始下降,说明单位时间内抑制效率开始下降,因此认为100μmol/L白藜芦醇作用72h对人肝癌细胞SMMC-7721的抑制作用最为明显。后续实验以此作为作用截止点。Hoechst-33258染色结果显示:100μmol/L的白藜芦醇作用72h后人肝癌细胞SMMC-7721细胞核呈现明显凋亡形态,随机对照组及空白组人肝癌细胞SMMC-7721细胞核无明显变化。双染色流式细胞仪检测细胞凋亡率结果显示:空白对照组、随机对照组、100μmol/L白藜芦醇实验组的凋亡率分别为10.27%、12.31%、38.54%。RT-PCR结果:100μmol/L白藜芦醇作用72h后细胞CYP1A1m RNA的表达出现显著下降。进一步检测发现CYP1A1蛋白的表达也呈现下降趋势。结论:体外培养状态下,白藜芦醇能抑制人肝癌细胞SMMC-7721的增殖、促进其凋亡,其机制可能与白藜芦醇在转录水平下调CYP1A1m RNA的表达、从而在翻译水平减少CYP1A1的蛋白表达有关。
[Abstract]:Objective: primary hepatocellular carcinoma is one of the most common malignant tumor of digestive system, the higher the degree of malignancy, the lesion is easy to transfer and poor treatment effect. Resveratrol (Resveratrol) from Polygonum cuspidatum, an extraction of polyphenols from grape plants, recent studies have shown that resveratrol has anti-tumor anti-inflammatory, protect blood vessels and nerves. The effect of bone calcium levels, etc., is the treatment of lipid metabolism disorder, effective components of inflammation and heart disease. It is reported that resveratrol has anti hepatoma effect, but the anti cancer effects of the specific mechanism is still unclear. Induction of tumor cell apoptosis, inhibit the proliferation of tumor cells is the study of conventional ways of anticancer drugs, the in vitro study from the angle of effect of resveratrol on proliferation and apoptosis of human hepatocellular carcinoma cell line SMMC-7721, and to explore its possible mechanism. Through the detection of resveratrol in human hepatocellular carcinoma fine CCK-8 method. Time effect and dose effect relationship of SMMC-7721 cell proliferation, to determine resveratrol concentration and time play the biggest role. The scope of application of Hoechst-33258 staining and double staining flow cytometry (Annexin V/P) and the amount of morphological changes of apoptotic cells were detected, Real time-PCR detection of resveratrol in human hepatocellular carcinoma cells before intervention SMMC-7721 expression of cytochrome P4501A1 m RNA (CYP1A1). The changes of the Western blot analysis (Western blot analysis) method for the detection of resveratrol intervention before human hepatocellular carcinoma cell SMMC-7721 expression changes of cytochrome P4501A1 protein. Methods: with concentration of 25 mol/L, 50 mol/L, 100 mol/L, 200 mol/L of resveratrol in human hepatocellular carcinoma cells 24h 48h, 72h, and to investigate the inhibitory effect of resveratrol on proliferation of CCK-8 human hepatocellular carcinoma cell line SMMC-7721. In human hepatoma SMMC-7721 cells without treatment and the same dose of DMSO is empty and random on white Control group, resveratrol effect 72h Annexin V/PI flow cytometry in 100 mol/L on human hepatoma cell apoptosis rate of SMMC-7721, before and after treatment with resveratrol Hoechst-33258 changes of.Real-time PCR apoptosis 100 mol/L resveratrol 72h human hepatocellular carcinoma cells SMMC-7721 in the analysis of 100 mol/L the expression of CYP1A1m in human hepatoma cell line SMMC-7721 RNA. Western blot analysis (Western blot analysis) were detected before and after treatment with resveratrol 100 mol/L CYP1A1 protein expression in human hepatocellular carcinoma cell SMMC-7721. Results: CCK-8 test results showed that in vitro, resveratrol can significantly inhibit human hepatocellular carcinoma cell line SMMC-7721 the proliferation of concentration in the dose range of this experiment, with the increasing concentration of resveratrol, inhibited the proliferation effect significantly enhanced in a concentration dependent manner. With the same concentration 鐫，

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