内质网应激通路在姜黄素对人肝癌SMMC-7721细胞增殖抑制和凋亡诱导中的作用研究

发布时间：2018-03-12 18:07

本文选题：姜黄素　切入点：内质网应激　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:姜黄素(Curcumin,Cur),作为一种多酚类化学提取物,是从姜科类植物等中药的根或者茎中提炼的,颜色黄、性味苦,为结晶粉末,很难溶于水,易溶于有机溶剂,为常用的食物添加剂和染色剂,具有抗氧化、抗炎、抗动脉粥样硬化、抗微生物、抗突变等作用。1995年Menon等首次提出姜黄素具有抗癌等药理活性的可能后,大量临床前及临床实验研究已证实其抗癌作用。发现姜黄素对宫颈癌、前列腺癌等恶性肿瘤的生长和转移均有良好的抑制作用。鉴于肝癌恶性程度高,早期发现困难,中晚期肝癌手术价值低,且化疗效果差、毒副作用大等现状,众多学者对姜黄素的药理作用,及治疗肿瘤的作用机制(其中包括对肝癌的治疗)等进行了大量的实验研究,但其内在的确切机制还不清楚。本实验旨在探索姜黄素治疗肝癌的机制,故做如下探究:研究对象选用的是人肝癌SMMC-7721细胞:(1)姜黄素在处理肝癌细胞之后,细胞增殖、凋亡发生了怎样的变化,以及这些变化所潜在的机制;(2)姜黄素在处理肝癌细胞之后内质网应激发生了怎样的变化,且主要通过其中哪条信号通路发挥作用;(3)内质网应激在姜黄素处理肝癌细胞后,对增殖、凋亡产生了怎样的影响。以上研究目的是为肝癌的治疗寻找天然、无毒或低毒、有效的治疗药物,同时也进一步探寻姜黄素在肿瘤治疗中的机理,以及在肝癌治疗研究领域的潜在疗效。研究方法:1、采用不同浓度的姜黄素(control、2.5μM、5μM、10μM、20μ、50μM)分别处理人肝癌SMMC-7721细胞12h,24h,48h后,各浓度组肝癌细胞活力以MTT法检测;姜黄素处理肝癌细胞12h后的凋亡率以细胞流式技术检测。最后与对照组进行分析比较。以此探究姜黄素对肝癌细胞增殖的影响以及对肝癌细胞凋亡的影响。2、采用不同浓度的姜黄素(control、2.5μM、5μM、10μM、20μM、50μM)处理人肝癌SMMC-7721细胞12h后,裂解细胞,并提取各组细胞蛋白。Western Blot检测内质网应激发生的标志性蛋白GRP78,通路蛋白p-e IF2α、IRE1、CHOP的表达情况。以此探究姜黄素作用肝癌细胞后,对内质网应激反应信号通路的影响。3、通过药物干预,首先以内质网应激反应抑制剂sal预处理人肝癌SMMC-7721细胞12h,之后再应用不同浓度姜黄素(control、2.5μM、5μM、10μM、20μ、50μM)处理人肝癌SMMC-7721细胞12h,随后,肝癌细胞活力采用MTT法检测;肝癌细胞凋亡情况采用流式细胞技术检测;内质网应激反应有关蛋白的表达情况采用Western Blot的法进行检测。并且和姜黄素单独处理组相比较进行统计学分析。以此探究姜黄素作用人肝癌SMMC-7721细胞是否通过诱导内质网应激反应起作用。研究结果:1、在姜黄素药物剂量增加的同时,并且延长其作用时间,MTT检测发现人肝癌SMMC-7721细胞光吸收值显著降低,活力细胞显著减少,进一步表明姜黄素对人肝癌SMMC-7721细胞的具有增殖抑制作用,并与对照组相比较,具有统计学意义(均p0.05)。2、不同浓度姜黄素作用肝癌SMMC-7721细胞12h后,流式细胞技术检测细胞凋亡发现,肝癌细胞的凋亡率随姜黄素药物浓度的增加而显著上升,并与对照组相比较,具有统计学意义(均p0.05)。3、Western Blot结果显示姜黄素能够诱导人肝癌SMMC-7721细胞内质网应激反应有关蛋白GRP78、p-e IF2α、IRE1以及CHOP等的表达。并随姜黄素药物剂量的上升,蛋白表达量渐趋升高,具有剂量依赖性关系,相应姜黄素浓度组与对照组进行比较p0.05。4、应用内质网应激抑制剂sal,预处理人肝癌SMMC-7721细胞12h,随后再应用姜黄素作用该细胞,MTT和流式细胞凋亡结果分别显示:姜黄素单独处理组的人肝癌SMMC-7721细胞较sal预处理后再以姜黄素处理的肝癌细胞增殖抑制率更加显著(p0.05);细胞凋亡率也升高。5、内质网应激抑制剂sal预处理人肝癌SMMC-7721细胞12h,随后再应用姜黄素作用该细胞,Western Blot结果示:内质网应激有关蛋白的表达被sal显著的下调。姜黄素单独处理组的肝癌细胞内质网应激有关蛋白的表达量较sal预处理后再以姜黄素处理的肝癌细胞内质网应激相关蛋白的表达量明显升高,并具有统计学意义(p0.05)。研究结论:姜黄素能够显著抑制人肝癌SMMC-7721细胞增殖,并通过内质网应激信号通路对肝癌细胞发挥促凋亡作用;应用内质网应激抑制剂sal,预处理人肝癌SMMC-7721细胞后,进一步证实了姜黄素通过激活肝癌细胞内质网应激反应来诱导细胞凋亡的。上述结论对姜黄素的临床实验研究、肝癌治疗方法的探索均具有重大意义。
[Abstract]:Objective: curcumin (Curcumin, Cur), as a kind of polyphenolic chemical extracts, derived from Zingiberaceae plants such as Chinese medicine roots or stems, color yellow, bitter, crystalline powder, very soluble in water, soluble in organic solvents, used as food additives and dyeing agent, antioxidant, anti-inflammatory, anti atherosclerosis, anti microbial, anti mutation effect of.1995 Menon first proposed curcumin has anticancer activity of May, a large number of preclinical and clinical experimental studies showed that the anticancer effect of cervical cancer. Jiang Huang found that good inhibition of growth and metastasis of malignant tumor prostate cancer. Given the high degree of malignancy, early found difficulties in advanced liver cancer surgery and chemotherapy with low value, poor effect, side effects and so on situation, many scholars on the pharmacological effects of curcumin, and its anti-tumor effect The mechanism (including the treatment of liver cancer) were a large number of experimental studies, but the exact mechanism is not clear. To explore the mechanism of curcumin in the treatment of hepatocellular carcinoma in this study, so do the following research: the research object is the selection of human hepatocellular carcinoma SMMC-7721 cells: (1) curcumin after treatment of hepatocellular carcinoma cells what happens to the proliferation, apoptosis, and the mechanism of these changes have the potential; (2) after the treatment of curcumin in hepatocellular carcinoma cell endoplasmic reticulum stress what changes have taken place, and which play a role mainly through which signal pathways; (3) the endoplasmic reticulum stress in cells treated with curcumin, on the proliferation, the how the influence of apoptosis. The purpose of the study is to find more natural for the treatment of liver cancer, non-toxic or low toxic and effective drug treatment, but also to further explore the mechanism of curcumin in the treatment of cancer, as well as in the treatment of liver cancer The potential clinical research field. Methods: 1, using different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20, 50 M) were treated with human hepatocellular carcinoma cell line SMMC-7721 12h, 24h, 48h, each group was detected by MTT cell activity of Curcumin; apoptosis treatment of hepatocellular carcinoma cells after 12h with rate of flow cytometry detection. Finally, compared with control group. In order to study the effect of curcumin on proliferation of hepatocellular carcinoma cells and the effect on apoptosis of hepatocellular carcinoma cells.2, using different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20 M. 50 M) treatment of human hepatocellular carcinoma SMMC-7721 cells after 12h cell lysis, protein extraction and.Western Blot cells were detected in endoplasmic reticulum stress the marker protein of GRP78 pathway protein P-E IF2 alpha, IRE1, CHOP expression. In order to explore the effects of curcumin on liver cancer cells, endoplasmic reticulum stress signaling pathway The effect of.3, through drug intervention, first of all to the endoplasmic reticulum stress reaction inhibitor Sal pretreated human hepatocellular carcinoma cell line SMMC-7721 12h, after the application of different concentrations of curcumin (control, 2.5 M, 5 M, 10 M, 20 mu, 50 mu M) treatment of human hepatocellular carcinoma cell line SMMC-7721 12h, then, cell viability by MTT assay; apoptosis of hepatocellular carcinoma cells by flow cytometry; expression of endoplasmic reticulum stress related protein by Western Blot assay. And curcumin treatment group compared with statistical analysis. To explore whether Jiang Huang effects of human hepatoma SMMC-7721 cells induced by endoplasmic reticulum stress play a role. Results: 1, increase in curcumin dose and at the same time, the role of time, MTT detected in human hepatocellular carcinoma SMMC-7721 cells light absorption value was significantly decreased, cell viability significantly decreased further The curcumin can inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells, and compared with the control group, with statistical significance (.2, P0.05) of different concentrations of curcumin in SMMC-7721 cells after 12h cell apoptosis was evaluated by flow cytometry showed that the apoptosis rate increased with the concentration of curcumin increased significantly, phase and compared with the control group, with statistical significance (P0.05).3, Western Blot results showed that curcumin can induce endoplasmic reticulum stress reaction on hepatocellular carcinoma cells SMMC-7721 protein GRP78, P-E expression of IRE1 and IF2 alpha, CHOP. And with the rise of curcumin dose, the expression gradually increased with dose dependent relationship accordingly, the concentration of curcumin group compared with control group p0.05.4, application of endoplasmic reticulum stress inhibitor Sal, pretreatment of human hepatocellular carcinoma cell line SMMC-7721 12h, then the application of curcumin for With the MTT cells, and apoptosis by flow cytometry results showed that curcumin treatment group were independent of human hepatocellular carcinoma SMMC-7721 cells were pretreated with Sal on proliferation of hepatocellular carcinoma cells treated with curcumin inhibition rate was more significant (P0.05); the apoptosis rate also increased.5, endoplasmic reticulum stress inhibitor Sal pretreated human hepatocellular carcinoma cell line SMMC-7721 12h then, the application of curcumin in Western cells, Blot showed that the expression of endoplasmic reticulum stress related protein Sal was significantly decreased. The expression of hepatocellular carcinoma cells treated with curcumin alone endoplasmic reticulum stress related proteins than Sal after pretreatment with the expression of endoplasmic reticulum stress related protein in hepatocellular carcinoma cells treated with curcumin significantly increased the amount of and, with statistical significance (P0.05). Conclusion: curcumin can inhibit the proliferation of human hepatocellular carcinoma SMMC-7721 cells through endoplasmic reticulum stress signaling pathway on liver cancer cells The essential application of apoptosis; endoplasmic reticulum stress inhibitor Sal, pretreatment of human hepatocellular carcinoma SMMC-7721 cells, further confirmed that curcumin through activation of stress induced apoptosis of hepatoma cells to endoplasmic reticulum. Clinical and experimental study of curcumin in the above conclusions, explore the method of treatment of liver cancer is of great significance.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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