本文選題：基因多態(tài)性 + 非酒精性脂肪肝�。� 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:探討TM6SF2 E167K多態(tài)性在體外培養(yǎng)肝細(xì)胞HEPA1-6中的作用機(jī)制。方法:(1)利用10%胎牛血清、1%抗生素的低糖DMEM培養(yǎng)液,在37℃、5%CO2、飽和濕度的培養(yǎng)箱內(nèi)培養(yǎng)肝癌細(xì)胞HEPA1-6。(2)構(gòu)建穩(wěn)定表達(dá)TM6SF2基因E167K野生型、突變型以及空病毒載體的肝癌細(xì)胞HEPA1-6細(xì)胞株。(3)流式細(xì)胞儀檢測細(xì)胞周期。(4)油紅0染色顯示各組細(xì)胞甘油三酯含量變化,全自動生物化學(xué)分析儀測定各組細(xì)胞內(nèi)甘油三酯和總膽固醇的含量。(5)Western blot檢測細(xì)胞周期調(diào)節(jié)因子Cyclin D1、p53、P16、P27、P21和Rb,細(xì)胞炎癥因子TNF-β、IL-2、IL-6和IL-8,膽固醇調(diào)節(jié)元件結(jié)合蛋白(SREBP-1c)和脂肪酸合成酶(FASN)的蛋白表達(dá)情況。熒光定量PCR檢測m RNA的表達(dá)情況。兩組間比較采用獨(dú)立樣本t檢驗(yàn)。結(jié)果:(1)突變組與野生組相比,G1期細(xì)胞比例減少(突變型G1期36.26±0.31%VS野生型G1期57.63±0.28%,t值為88.607,P0.05),S期與G2/M期細(xì)胞比例相應(yīng)增加(突變型S期28.41±0.31%VS野生型S期19.54±0.25%,突變型G2/M期35.23±0.14%VS野生型G2/M期25.77±0.51%,t值分別為38.577,9.982,P0.05),突變組與對照組相比,G1期細(xì)胞比例減少(對照組G1期58.36±0.21%,t值為97.595,P0.05),S期與G2/M期細(xì)胞比例增加(對照組S期19.31±0.25%,G2/M期25.61±0.36%,t值分別為39.577,29.201,P0.05),野生型與對照組相比不存在統(tǒng)計(jì)學(xué)差異(P0.05)。與野生型相比,突變組Cyclin D1、p53和Rbm RNA相對表達(dá)量明顯增加(2.03±0.01VS1.00±0.00,1.88±0.05VS1.48±0.09,1.29±0.06VS1.18±0.01,t值為178.532,6.729,3.132,P0.05),P27m RNA相對表達(dá)量明顯降低(0.56±0.02VS0.82±0.05,t值為8.363,P0.05)。與對照組相比,突變組Cyclin D1、p53、Rb m RNA相對表達(dá)量明顯增加(對照組1.04±0.06,1.37±0.03,1.15±0.03,t值為28.190,15.152,3.615,P0.05),P27m RNA相對表達(dá)量明顯降低(對照組0.85±0.05,t值為9.328,P0.05)。野生型和對照組相比不存在統(tǒng)計(jì)學(xué)差異(P0.05)。P16和P21m RNA的表達(dá)在三組中均無統(tǒng)計(jì)學(xué)差異(P0.05)。(2)與對照組相比,突變組炎癥因子IL-2、IL-6 m RNA的表達(dá)明顯增高(1.24±0.07VS 1.00±0.04,1.24±0.10 VS 1.01±0.04,t值分別為5.161,3.698,P值均0.05),與野生組相比,突變組炎癥因子IL-2、IL-6 m RNA的表達(dá)明顯增高(野生組IL-2:1.00±0.05,野生組IL-6:1.00±0.09,t值分別為4.829,3.089,P值均0.05)。突變組炎癥因子TNF-βm RNA的表達(dá)與對照組相比不存在統(tǒng)計(jì)學(xué)差異(1.04±0.05 VS 1.03±0.03,t值為0.297,P0.05)。突變組炎癥因子IL-8 m RNA的表達(dá)與野生組相比不存在統(tǒng)計(jì)學(xué)差異(1.17±0.02 VS 1.13±0.04,t值為1.550,P0.05)。(3)油紅O染色結(jié)果顯示突變組甘油三酯含量較野生組與對照組明顯增加。突變組的甘油三酯含量較野生組和對照組明顯升高(0.54±0.02 VS 0.20±0.02 VS 0.24±0.02,t值分別為20.859,18.405,P值均0.01)。與野生型和對照組相比,突變組SREBP-lc、FASN m RNA表達(dá)量明顯增加(P值均0.01),突變組SREBP-lc、FASN蛋白表達(dá)較野生組與對照組明顯增加(P值均0.01)。突變組SREBP-lc與甘油三酯水平均呈正相關(guān)(r=0.913,P0.01)。結(jié)論:(1)TM6SF2基因E167K多態(tài)性通過上調(diào)Cyclin D1、p53、Rb的表達(dá)和下調(diào)P27的表達(dá)引起HEPA1-6細(xì)胞周期紊亂。(2)TM6SF2基因E167K多態(tài)性可促進(jìn)炎癥因子IL-2、IL-6的表達(dá),可促進(jìn)肝損傷的進(jìn)展。(3)TM6SF2基因E167K多態(tài)性可促進(jìn)脂肪合成增加,甘油三酯的合成增加可能通過脂質(zhì)代謝密切相關(guān)基因SREBP-lc、FASN起作用,此研究為探討非酒精性脂肪性肝病的發(fā)病機(jī)制提供了新線索。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575

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