TM6SF2 E167K多态性在非酒精性脂肪性肝病中的作用研究

发布时间：2018-05-22 11:35

本文选题：基因多态性 + 非酒精性脂肪肝　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:探讨TM6SF2 E167K多态性在体外培养肝细胞HEPA1-6中的作用机制。方法:(1)利用10%胎牛血清、1%抗生素的低糖DMEM培养液,在37℃、5%CO2、饱和湿度的培养箱内培养肝癌细胞HEPA1-6。(2)构建稳定表达TM6SF2基因E167K野生型、突变型以及空病毒载体的肝癌细胞HEPA1-6细胞株。(3)流式细胞仪检测细胞周期。(4)油红0染色显示各组细胞甘油三酯含量变化,全自动生物化学分析仪测定各组细胞内甘油三酯和总胆固醇的含量。(5)Western blot检测细胞周期调节因子Cyclin D1、p53、P16、P27、P21和Rb,细胞炎症因子TNF-β、IL-2、IL-6和IL-8,胆固醇调节元件结合蛋白(SREBP-1c)和脂肪酸合成酶(FASN)的蛋白表达情况。荧光定量PCR检测m RNA的表达情况。两组间比较采用独立样本t检验。结果:(1)突变组与野生组相比,G1期细胞比例减少(突变型G1期36.26±0.31%VS野生型G1期57.63±0.28%,t值为88.607,P0.05),S期与G2/M期细胞比例相应增加(突变型S期28.41±0.31%VS野生型S期19.54±0.25%,突变型G2/M期35.23±0.14%VS野生型G2/M期25.77±0.51%,t值分别为38.577,9.982,P0.05),突变组与对照组相比,G1期细胞比例减少(对照组G1期58.36±0.21%,t值为97.595,P0.05),S期与G2/M期细胞比例增加(对照组S期19.31±0.25%,G2/M期25.61±0.36%,t值分别为39.577,29.201,P0.05),野生型与对照组相比不存在统计学差异(P0.05)。与野生型相比,突变组Cyclin D1、p53和Rbm RNA相对表达量明显增加(2.03±0.01VS1.00±0.00,1.88±0.05VS1.48±0.09,1.29±0.06VS1.18±0.01,t值为178.532,6.729,3.132,P0.05),P27m RNA相对表达量明显降低(0.56±0.02VS0.82±0.05,t值为8.363,P0.05)。与对照组相比,突变组Cyclin D1、p53、Rb m RNA相对表达量明显增加(对照组1.04±0.06,1.37±0.03,1.15±0.03,t值为28.190,15.152,3.615,P0.05),P27m RNA相对表达量明显降低(对照组0.85±0.05,t值为9.328,P0.05)。野生型和对照组相比不存在统计学差异(P0.05)。P16和P21m RNA的表达在三组中均无统计学差异(P0.05)。(2)与对照组相比,突变组炎症因子IL-2、IL-6 m RNA的表达明显增高(1.24±0.07VS 1.00±0.04,1.24±0.10 VS 1.01±0.04,t值分别为5.161,3.698,P值均0.05),与野生组相比,突变组炎症因子IL-2、IL-6 m RNA的表达明显增高(野生组IL-2:1.00±0.05,野生组IL-6:1.00±0.09,t值分别为4.829,3.089,P值均0.05)。突变组炎症因子TNF-βm RNA的表达与对照组相比不存在统计学差异(1.04±0.05 VS 1.03±0.03,t值为0.297,P0.05)。突变组炎症因子IL-8 m RNA的表达与野生组相比不存在统计学差异(1.17±0.02 VS 1.13±0.04,t值为1.550,P0.05)。(3)油红O染色结果显示突变组甘油三酯含量较野生组与对照组明显增加。突变组的甘油三酯含量较野生组和对照组明显升高(0.54±0.02 VS 0.20±0.02 VS 0.24±0.02,t值分别为20.859,18.405,P值均0.01)。与野生型和对照组相比,突变组SREBP-lc、FASN m RNA表达量明显增加(P值均0.01),突变组SREBP-lc、FASN蛋白表达较野生组与对照组明显增加(P值均0.01)。突变组SREBP-lc与甘油三酯水平均呈正相关(r=0.913,P0.01)。结论:(1)TM6SF2基因E167K多态性通过上调Cyclin D1、p53、Rb的表达和下调P27的表达引起HEPA1-6细胞周期紊乱。(2)TM6SF2基因E167K多态性可促进炎症因子IL-2、IL-6的表达,可促进肝损伤的进展。(3)TM6SF2基因E167K多态性可促进脂肪合成增加,甘油三酯的合成增加可能通过脂质代谢密切相关基因SREBP-lc、FASN起作用,此研究为探讨非酒精性脂肪性肝病的发病机制提供了新线索。
[Abstract]:Objective: To investigate the mechanism of TM6SF2 E167K polymorphism in the culture of hepatocyte HEPA1-6 in vitro. Methods: (1) using 10% fetal bovine serum, 1% antibiotic low glucose DMEM culture, incubating hepatoma cells HEPA1-6. (2) in a incubator of 37, 5%CO2 and saturated humidity (2) to construct a stable expression of TM6SF2 gene E167K wild-type, mutant and empty virus vector HEPA1-6 cell line of hepatoma cell. (3) flow cytometry was used to detect cell cycle. (4) oil and red 0 staining showed the change of triglyceride content in each group. The content of triglyceride and total cholesterol in each cell was measured by automatic biochemical analyzer. (5) Western blot was used to detect cell cycle regulator Cyclin D1, p53, P16, P27, P21 and Rb, and cytis The expression of TNF- beta, IL-2, IL-6 and IL-8, the protein expression of cholesterol regulator element binding protein (SREBP-1c) and fatty acid synthetase (FASN). The fluorescent quantitative PCR was used to detect the expression of M RNA. The two groups were compared with independent sample t test. Results: (1) the proportion of G1 phase cells was decreased compared with that of the wild group (36.26 of the mutant G1). The wild type G1 period was 57.63 + 0.28%, the T value was 88.607, P0.05), the proportion of the S phase and the G2/M stage increased correspondingly (19.54 + 0.25% in the 28.41 + 0.31%VS wild type S phase of the mutant S phase, the mutant G2/M 35.23 + 0.14%VS wild type G2/M period 25.77 + 0.51%, the T value respectively). 8.36 + 0.21%, t value 97.595, P0.05), the proportion of S and G2/M cells increased (19.31 + 0.25%, G2/M period 25.61 + 0.36%, t value 39.577,29.201, P0.05) in the control group, and there was no statistical difference between the wild type and the control group (P0.05). 0 + 0.00,1.88 + 0.05VS1.48 + 0.09,1.29 + 0.06VS1.18 + 0.01, t value 178.532,6.729,3.132, P0.05), the relative expression of P27m RNA decreased significantly (0.56 + 0.02VS0.82 + 0.05, t was 8.363, P0.05). 0,15.152,3.615, P0.05), the relative expression of P27m RNA decreased significantly (0.85 + 0.05 in the control group, t value 9.328, P0.05). There was no statistical difference between the wild type and the control group (P0.05).P16 and P21m RNA expression in the three groups (P0.05). (2) the expression of the inflammatory factor IL-2 was significantly increased compared with the control group. High (1.24 + 0.07VS 1 + 0.04,1.24 + 0.10 VS 1.01 + 0.04, t value respectively 5.161,3.698, P value 0.05). Compared with the wild group, the expression of inflammatory factors IL-2, IL-6 m RNA increased obviously (wild group IL-2:1.00 + 0.05, wild group IL-6:1.00 0.09, respectively 0.05). Compared with the control group, there was no statistical difference (1.04 + 0.05 VS 1.03 + 0.03, t value 0.297, P0.05). The expression of IL-8 m RNA in the mutant group was not statistically different from that in the wild group (1.17 + 0.02 VS 1.13 + 0.04, t value 1.550, P0.05). (3) the oil red O staining results showed that the content of triglyceride in the mutant group was better than that in the wild group and the control group The content of triglyceride in the mutant group was significantly higher than that in the wild group and the control group (0.54 + 0.02 VS 0.20 + 0.02 VS 0.24 + 0.02, t value 20.859,18.405, P value 0.01). Compared with the wild type and the control group, the mutation group SREBP-lc, FASN m RNA expression increased significantly (P value 0.01), the mutant group SREBP-lc, the FASN protein expression was more than the wild group and the wild group. The control group increased significantly (P value was 0.01). SREBP-lc in the mutant group was positively correlated with triglyceride (r=0.913, P0.01). Conclusion: (1) the E167K polymorphism of the TM6SF2 gene can cause the disorder of the HEPA1-6 cell cycle by up regulation of Cyclin D1, p53, Rb expression and down-regulation P27. (2) the polymorphism of the gene can promote the expression of inflammatory factors. It can promote the progress of liver injury. (3) the E167K polymorphism of TM6SF2 gene can promote the increase of fat synthesis, and the synthesis of triglycerides may be mediated by the gene SREBP-lc and FASN, which is closely related to lipid metabolism. This study provides a new clue to explore the pathogenesis of nonalcoholic fatty liver disease.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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