本文選題：甲醛　切入點：HepG2細胞　出處：《山西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:以HepG2細胞作為模型,研究甲醛對肝細胞脂質(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細胞24 h和48 h,采用MTT法檢測細胞活性。分別以0.004、0.02、0.1 mmol/L的FA處理人肝癌HepG2細胞24 h和48 h,用POD酶法來測定細胞內(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)、二酰甘油酰基轉(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)的蛋白表達水平;采用ELISA法分別檢測細胞內(nèi)極低密度脂蛋白(VLDL)、載脂蛋白B100(apo B100)、位點1蛋白酶(S1P)及位點2蛋白酶(S2P)的表達水平。結(jié)果:1、甲醛染毒24 h和48 h后,與對照組相比,0.5~12.5 mmol/L FA染毒均可明顯降低HepG2細胞活性(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蛋白表達在24 h處理后顯著增加。DGAT1蛋白表達在0.1 mmol/L FA處理24 h后顯著增加,但在其他組表達減少;24 h處理后,CES3蛋白表達顯著升高。3、甲醛染毒48 h后,細胞內(nèi)甘油三酯含量明顯降低;p-AMPKα蛋白在0.1 mmol/L FA組表達顯著增加;SREBP-1c、ACC1和FASN蛋白表達均沒有明顯的變化;DGAT1蛋白表達降低;ATGL蛋白表達0.004mmol/L FA組表達明顯減少。4、細胞在染毒處理24 h后,細胞內(nèi)的游離膽固醇含量升高;HMGCR蛋白在各個處理組表達均明顯增加;ACAT蛋白表達在各個處理組均明顯降低;SREBP-2蛋白表達在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組表達降低,而在其余組表達均明顯增加;LDLR蛋白表達在BFA組除外的處理組均明顯降低。5、細胞在染毒處理48 h后,細胞內(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蛋白在各個處理組表達均明顯增加;ACAT蛋白表達在0.02、0.1 mmol/L FA組有明顯的降低;Insig-1蛋白表達水平在0.02、0.1 mmol/L FA組明顯降低;LDLR蛋白表達水平在各個處理組均明顯降低。結(jié)論:1、高濃度甲醛可明顯抑制肝細胞的活性。2、甲醛接觸可能通過SREBP-1c-FAS通路影響肝細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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

【參考文獻】

相關(guān)期刊論文 前9條

1 閆丹丹;白劍英;梁瑞峰;王幼萍;雷佩玉;;甲醛對HepG2細胞脂肪代謝的影響[J];癌變.畸變.突變;2013年06期

2 李亞琳;;甲醛對小鼠肝臟毒性作用的研究[J];動物醫(yī)學進展;2013年04期

3 徐浩;崔立然;張玲;;甲醛對機體整體內(nèi)源性代謝產(chǎn)物的影響[J];解剖學報;2012年03期

4 薛來俊;曉開提·依不拉音;張彥紅;;甲醛染毒小鼠肝臟、腎臟、脾臟的病理變化[J];中國職業(yè)醫(yī)學;2007年03期

5 姚曉敏;宋保亮;王燦華;張文靜;林志新;李伯良;;人酰基輔酶A:膽固醇�；D(zhuǎn)移酶(ACAT)[J];上海交通大學學報(農(nóng)業(yè)科學版);2006年01期

6 于立群;甲醛的健康效應(yīng)[J];國外醫(yī)學(衛(wèi)生學分冊);2004年02期

7 楊振洲,蔡同建;室內(nèi)甲醛的危害及其預(yù)防[J];中國公共衛(wèi)生;2003年06期

8 庚晉 ,周潔;甲醛污染的危害、來源及預(yù)防[J];吉林建材;2002年04期

9 蔣學之,張瑞穩(wěn),吳逸人,苗德林,張曉莉,管小琴;甲醛對鼠的肝臟毒性研究[J];上海醫(yī)科大學學報;1990年02期

，

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