本文選題：甲醛　切入點(diǎn)：HepG2細(xì)胞　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:以HepG2細(xì)胞作為模型,研究甲醛對(duì)肝細(xì)胞脂質(zhì)代謝的影響,包括SREBP-1c-FAS通路(甘油三酯代謝相關(guān))及Insig-1-SCAP-SREBPs通路(膽固醇代謝相關(guān))的影響,從而為甲醛所引起的肝細(xì)胞脂質(zhì)代謝紊亂提供依據(jù)。方法:分別以0.004、0.02、0.1、0.5、2.5、12.5 mmol/L的甲醛(formaldehyde,FA)、完全培養(yǎng)基組(陰性對(duì)照)、1 mmol/L油酸(oleic acid,OA)及2.5μg/m L布雷德菌素A(Brefeldin A,BFA)處理人肝癌HepG2細(xì)胞24 h和48 h,采用MTT法檢測(cè)細(xì)胞活性。分別以0.004、0.02、0.1 mmol/L的FA處理人肝癌HepG2細(xì)胞24 h和48 h,用POD酶法來測(cè)定細(xì)胞內(nèi)和培養(yǎng)上清中甘油三酯(TG)、游離膽固醇(FC)的含量;采用Western blot法檢測(cè)甘油三酯代謝相關(guān)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)、固醇調(diào)節(jié)元件結(jié)合蛋白1c(SREBP-1c)、乙酰輔酶A羧化酶(ACC1)、磷酸化乙酰輔酶A羧化酶(p-ACC1)、脂肪酸合酶(FASN)、二酰甘油�；D(zhuǎn)移酶1/2(DGAT1/2)、甘油三酯水解酶(CES3)、脂肪甘油三酯脂酶(ATGL)以及膽固醇代謝相關(guān)固醇調(diào)節(jié)元件結(jié)合蛋白2(SREBP-2)、羥甲基戊二酸單酰輔酶A還原酶(HMGCR)、膽固醇�；D(zhuǎn)移酶(ACAT)、胰島素誘導(dǎo)基因1(Insig-1)、裂解激活蛋白(SCAP)、低密度脂蛋白受體(LDLR)的蛋白表達(dá)水平;采用ELISA法分別檢測(cè)細(xì)胞內(nèi)極低密度脂蛋白(VLDL)、載脂蛋白B100(apo B100)、位點(diǎn)1蛋白酶(S1P)及位點(diǎn)2蛋白酶(S2P)的表達(dá)水平。結(jié)果:1、甲醛染毒24 h和48 h后,與對(duì)照組相比,0.5~12.5 mmol/L FA染毒均可明顯降低HepG2細(xì)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蛋白表達(dá)在24 h處理后顯著增加。DGAT1蛋白表達(dá)在0.1 mmol/L FA處理24 h后顯著增加,但在其他組表達(dá)減少;24 h處理后,CES3蛋白表達(dá)顯著升高。3、甲醛染毒48 h后,細(xì)胞內(nèi)甘油三酯含量明顯降低;p-AMPKα蛋白在0.1 mmol/L FA組表達(dá)顯著增加;SREBP-1c、ACC1和FASN蛋白表達(dá)均沒有明顯的變化;DGAT1蛋白表達(dá)降低;ATGL蛋白表達(dá)0.004mmol/L FA組表達(dá)明顯減少。4、細(xì)胞在染毒處理24 h后,細(xì)胞內(nèi)的游離膽固醇含量升高;HMGCR蛋白在各個(gè)處理組表達(dá)均明顯增加;ACAT蛋白表達(dá)在各個(gè)處理組均明顯降低;SREBP-2蛋白表達(dá)在0.1 mmol/L FA及OA組明顯降低;Insig-1蛋白在染毒在0.004 mmol/L FA組表達(dá)降低,而在其余組表達(dá)均明顯增加;LDLR蛋白表達(dá)在BFA組除外的處理組均明顯降低。5、細(xì)胞在染毒處理48 h后,細(xì)胞內(nèi)游離膽固醇含量明顯增加;上清中的游離膽固醇含量在0.004、0.02 mmol/L FA組明顯降低;HMGCR蛋白在各個(gè)處理組表達(dá)均明顯增加;ACAT蛋白表達(dá)在0.02、0.1 mmol/L FA組有明顯的降低;Insig-1蛋白表達(dá)水平在0.02、0.1 mmol/L FA組明顯降低;LDLR蛋白表達(dá)水平在各個(gè)處理組均明顯降低。結(jié)論:1、高濃度甲醛可明顯抑制肝細(xì)胞的活性。2、甲醛接觸可能通過SREBP-1c-FAS通路影響肝細(xì)胞甘油三酯的合成及分泌外排。3、甲醛接觸可能通過Insig-1-SCAP-SREBPs通路影響肝細(xì)胞膽固醇的代謝。
[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 偽), phosphorylated adenylate activated protein kinase (AP-AMPK 偽), 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 偽 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 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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