甲醛对HepG2细胞脂质代谢相关通路影响的研究

发布时间：2018-03-18 23:22

本文选题：甲醛　切入点：HepG2细胞　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:以HepG2细胞作为模型,研究甲醛对肝细胞脂质代谢的影响,包括SREBP-1c-FAS通路(甘油三酯代谢相关)及Insig-1-SCAP-SREBPs通路(胆固醇代谢相关)的影响,从而为甲醛所引起的肝细胞脂质代谢紊乱提供依据。方法:分别以0.004、0.02、0.1、0.5、2.5、12.5 mmol/L的甲醛(formaldehyde,FA)、完全培养基组(阴性对照)、1 mmol/L油酸(oleic acid,OA)及2.5μg/m L布雷德菌素A(Brefeldin A,BFA)处理人肝癌HepG2细胞24 h和48 h,采用MTT法检测细胞活性。分别以0.004、0.02、0.1 mmol/L的FA处理人肝癌HepG2细胞24 h和48 h,用POD酶法来测定细胞内和培养上清中甘油三酯(TG)、游离胆固醇(FC)的含量;采用Western blot法检测甘油三酯代谢相关腺苷酸活化蛋白激酶a(AMPKα)、磷酸化腺苷酸活化蛋白激酶a(p-AMPKα)、固醇调节元件结合蛋白1c(SREBP-1c)、乙酰辅酶A羧化酶(ACC1)、磷酸化乙酰辅酶A羧化酶(p-ACC1)、脂肪酸合酶(FASN)、二酰甘油酰基转移酶1/2(DGAT1/2)、甘油三酯水解酶(CES3)、脂肪甘油三酯脂酶(ATGL)以及胆固醇代谢相关固醇调节元件结合蛋白2(SREBP-2)、羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、胆固醇酰基转移酶(ACAT)、胰岛素诱导基因1(Insig-1)、裂解激活蛋白(SCAP)、低密度脂蛋白受体(LDLR)的蛋白表达水平;采用ELISA法分别检测细胞内极低密度脂蛋白(VLDL)、载脂蛋白B100(apo B100)、位点1蛋白酶(S1P)及位点2蛋白酶(S2P)的表达水平。结果:1、甲醛染毒24 h和48 h后,与对照组相比,0.5~12.5 mmol/L FA染毒均可明显降低HepG2细胞活性(P㩳0.05)。2、甲醛染毒24 h后,上清中TG水平增加;SREBP-1c、ACC1和FASN蛋白表达在24 h处理后显著增加。DGAT1蛋白表达在0.1 mmol/L FA处理24 h后显著增加,但在其他组表达减少;24 h处理后,CES3蛋白表达显著升高。3、甲醛染毒48 h后,细胞内甘油三酯含量明显降低;p-AMPKα蛋白在0.1 mmol/L FA组表达显著增加;SREBP-1c、ACC1和FASN蛋白表达均没有明显的变化;DGAT1蛋白表达降低;ATGL蛋白表达0.004mmol/L FA组表达明显减少。4、细胞在染毒处理24 h后,细胞内的游离胆固醇含量升高;HMGCR蛋白在各个处理组表达均明显增加;ACAT蛋白表达在各个处理组均明显降低;SREBP-2蛋白表达在0.1 mmol/L FA及OA组明显降低;Insig-1蛋白在染毒在0.004 mmol/L FA组表达降低,而在其余组表达均明显增加;LDLR蛋白表达在BFA组除外的处理组均明显降低。5、细胞在染毒处理48 h后,细胞内游离胆固醇含量明显增加;上清中的游离胆固醇含量在0.004、0.02 mmol/L FA组明显降低;HMGCR蛋白在各个处理组表达均明显增加;ACAT蛋白表达在0.02、0.1 mmol/L FA组有明显的降低;Insig-1蛋白表达水平在0.02、0.1 mmol/L FA组明显降低;LDLR蛋白表达水平在各个处理组均明显降低。结论:1、高浓度甲醛可明显抑制肝细胞的活性。2、甲醛接触可能通过SREBP-1c-FAS通路影响肝细胞甘油三酯的合成及分泌外排。3、甲醛接触可能通过Insig-1-SCAP-SREBPs通路影响肝细胞胆固醇的代谢。
[Abstract]:Aim: to study the effects of formaldehyde on lipid metabolism in HepG2 cells, including the effects of SREBP-1c-FAS pathway (triglyceride metabolism related) and Insig-1-SCAP-SREBPs pathway (cholesterol metabolism related). Methods: human hepatoma HepG2 cells were treated with 0.004 渭 g / ml of 0. 004 ~ 0. 02 ~ 0. 01 ~ 0. 5 ~ 2. 5 mmol/L of formaldehyde formaldehydea (12.5 mmol/L), complete medium group (1 mmol/L oleic acididine) and 2.5 渭 g / m L bradymycin ABFAA respectively. At 24 h and 48 h, the cell activity was detected by MTT assay, and the content of triglyceride triglyceride (TGG) and free cholesterol (FCc) in the cells and supernatants were determined by POD enzymatic method after treated with 0. 004- 0. 02 mmol/L FA for 24 h and 48 h, respectively. Western blot method was used to detect triglyceride metabolization-associated adenylate activated protein kinase (AMPK 伪), phosphorylated adenylate activated protein kinase (AP-AMPK 伪), steroid regulatory element binding protein (1cctr) SREBP-1cn, acetyl coA carboxylase (ACC1), phosphorylated acetylcoA carboxylase (p-ACC1). Fatty acid synthase (FASN), diacylglycerol acyltransferase (1 / 2) DGAT1 / 2, triglyceride hydrolase (CES3), fatty triglyceridase (ATGLL) and cholesterol metabolism-related steroid regulatory element binding protein 2rSREBP-2, hydroxymethyl glutaryl glutaryl coenzyme A reductase HMGCRE, gall fixation. The protein expression level of acyltransferase A, insulin inducible gene 1 (Insig 1), lytic activator protein (SCAP), low density lipoprotein receptor (LDLR); The expression levels of very low density lipoprotein (VLDL), apolipoprotein B100 apo B100, site 1 protease S1Pand site 2 protease S2P were detected by ELISA assay. Compared with the control group, 0.5 mmol/L FA exposure significantly decreased the activity of HepG2 cells. After exposure to formaldehyde for 24 h, the levels of TG in supernatant increased significantly, and the expression of ACC1 and FASN protein in supernatant increased significantly after 24 h treatment. The expression of DGAT1 protein increased significantly after treatment with 0.1 mmol/L FA for 24 h. However, the expression of CES3 protein in other groups increased significantly after 24 h treatment, and 48 h after formaldehyde exposure, the expression of CES3 protein increased significantly in other groups. The expression of p-AMPK 伪 protein in the 0.1 mmol/L FA group was significantly increased. There was no significant change in the expression of ACC1 and FASN protein. The expression of ACC1 and FASN protein in the #number0# mmol/L FA group was significantly decreased. The expression of AMPK 伪 protein decreased significantly in the 0.004 mmol / L FA group, and the cells were treated 24 hours after exposure. The expression of HMGCR protein increased significantly in each treatment group. In each treatment group, the expression of SSREBP-2 protein decreased significantly in 0. 1 mmol/L FA group and OA group significantly decreased the expression of Insig-1 protein in 0. 004 mmol/L FA group. In the other groups, the expression of LDLR protein decreased significantly in all the other groups except in the BFA group, and the intracellular free cholesterol content increased significantly after 48 h of exposure to BFA. The content of free cholesterol in supernatant in 0.0040.02 mmol/L FA group significantly decreased the expression of HMGCR protein in all treatment groups, and increased the expression of catalase protein in 0.02n0.1 mmol/L FA group significantly decreased the expression level of Insig-1 protein in 0.02o0. 1 mmol/L FA group. Conclusion: high concentration of formaldehyde can significantly inhibit the activity of hepatocytes. Formaldehyde exposure may affect the synthesis and secretion of triglyceride in hepatocytes by SREBP-1c-FAS pathway. The cholesterol metabolism of hepatocytes may be affected by Insig-1-SCAP-SREBPs pathway.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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