本文選題：亞硫酸鈉 + 脂肪細胞與肝臟細胞共培養(yǎng)�。� 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：目的:探討亞硫酸鈉是否通過脂肪細胞來影響肝臟細胞內甘油三酯(TG)與膽固醇含量,為研究亞硫酸鈉的肝毒性作用機制提供參考。方法:將3T3-L1前脂肪細胞進行誘導分化,成為成熟脂肪細胞,并用油紅O染色驗證;用不同濃度(0.02、0.1、0.5、2.5 mmol/L)Na2SO3和1 mmol/L油酸(OA)染毒脂肪細胞12 h,收集脂肪細胞上清,再分別用上述收集的各組脂肪細胞上清染毒Hep G2細胞24 h,即共培養(yǎng)組。另外再用同上述相同濃度(0.02、0.1、0.5、2.5 mmol/L)Na2SO3和1 mmol/L油酸單獨培養(yǎng)Hep G2細胞24 h,即單獨培養(yǎng)組;采用游離脂肪酸(FFA)檢測試劑盒測定脂肪細胞上清中FFA含量;采用ELISA法測定脂肪細胞上清中脂聯素的含量;采用GPO-POD酶法測定脂肪細胞培養(yǎng)上清中的TG含量;采用有機抽提法提取肝細胞內TG、總膽固醇(TC)和游離膽固醇(FC),再用POD酶法測定其含量;采用western blot法檢測Na2SO3對單獨培養(yǎng)組和共培養(yǎng)組肝細胞內TG代謝相關蛋白腺苷酸活化蛋白激酶(AMPKα)、P-AMPKα、乙酰輔酶A羧化酶(ACC)、P-ACC、固醇調節(jié)元件結合蛋白-1c(SREBP-1c)、脂肪酸合成酶(FASN)、肉堿酯酰轉移酶1(CPT-1)和膽固醇代謝相關蛋白3-羥基-3-甲基戊二酸單酰輔酶A還原酶(HMGCR)、低密度脂蛋白受體(LDLR)、�；o酶A:膽固醇酰基轉移酶(ACAT)的相對表達水平。結果:1、與陰性對照相比Na2SO3各處理組脂肪細胞培養(yǎng)上清中FFA和脂聯素的含量都明顯減少(P0.05),TG含量無明顯變化(P0.05)。2、Na2SO3處理后,單獨培養(yǎng)細胞內TG含量無明顯變化(P0.05)。但是2.5 mmol/L亞硫酸鈉可引起共培養(yǎng)Hep G2細胞內TG的增加(P0.05)。3、Western blot結果顯示,Na2SO3處理后,單獨培養(yǎng)肝細胞內SREBP-1c、AMPKα、P-ACC、CPT-1無明顯變化(P0.05);2.5 mmol/L Na2SO3組和OA組FASN蛋白表達增高(P0.05);ACC除0.5 mmol/L Na2SO3變化不明顯外,其余各組都升高(P0.05)。共培養(yǎng)組,P-ACC、SREBP-1c、FASN、CPT-1無明顯變化(P0.05);未檢測到P-AMPKα;AMPKα和ACC的蛋白表達均增高(P0.05)。4、Na2SO3處理后,單獨培養(yǎng)肝細胞內TC和FC含量的變化不明顯(P0.05);0.5 mmol/L Na2SO3、2.5 mmol/L Na2SO3和1 mmol/L OA可引起共培養(yǎng)組肝臟細胞內TC降低(P0.05);而2.5 mmol/L Na2SO3和1 mmol/L OA可引起共培養(yǎng)組肝臟細胞內FC含量增加(P0.05)。5、Western blot結果顯示,Na2SO3處理后,單獨培養(yǎng)組肝細胞內HMGCR表達無明顯變化(P0.05);ACAT蛋白表達水平均增高(P0.05);2.5 mmol/L Na2SO3使LDLR表達升高(P0.05)。共培養(yǎng)組,HMGCR和LDLR蛋白表達無明顯變化(P0.05);ACAT蛋白表達增高(P0.05)。結論:暴露在較低劑量的Na2SO3環(huán)境下時,不會引起肝臟TG和膽固醇的增加。但暴露于2.5 mmol/L較高濃度的Na2SO3環(huán)境下,機體不能代謝解毒Na2SO3,則可能通過脂肪細胞的作用使肝臟中TG和FC明顯升高,TC的含量降低,對肝臟脂代謝產生影響。
[Abstract]:Aim: to investigate whether sodium sulfite affects the content of triglyceride (TGG) and cholesterol in hepatic cells through adipocytes, and to provide reference for studying the mechanism of hepatic toxicity of sodium sulfite.Methods: the preadipocytes of 3T3-L1 were induced to differentiate into mature adipocytes. The supernatants of adipocytes were collected after 12 hours of exposure to different concentrations of 0. 02 and 0. 52.5 mmol/L)Na2SO3 and 1 mmol/L oleic acid respectively.Hep G2 cells were exposed to the supernatant of adipocytes collected above for 24 h, that is, co-culture group.In addition, the cells of Hep G2 were cultured alone for 24 h with the same concentration of 0. 02 and 0. 5 mmol/L)Na2SO3 and 1 mmol/L oleic acid for 24 h, and the content of FFA in the supernatant of adipocytes was detected by free fatty acid assay kit.The content of adiponectin in adipocyte supernatant and TG in adipocyte culture supernatant were determined by ELISA assay and GPO-POD enzymatic method respectively.TG-, TC- and FC- (free cholesterol) in hepatocytes were extracted by organic extraction method, and their contents were determined by POD enzymatic method.Results compared with the negative control group, the content of FFA and adiponectin in the supernatant of adipocyte culture in Na2SO3 treatment group was significantly decreased. There was no significant change in TG content in the adipocyte culture supernatant treated with P0.05N. 2Na2SO3, but there was no significant change in TG content in the cultured cells.All the other groups increased P0.05A.Blot results showed that after Na2SO3 treatment,There was no significant change in the expression of HMGCR in hepatocytes in the isolated culture group. The expression level of P0.05ACAT protein was increased. The expression of LDLR was increased by P0.05ACAT 2.5 mmol/L Na2SO3.There was no significant change in the expression of HMGCR and LDLR in co-culture group.Conclusion: when exposed to lower dose of Na2SO3, TG and cholesterol in liver were not increased.However, when exposed to 2.5 mmol/L high concentration of Na2SO3, the body could not metabolize detoxified Na _ 2SO _ 3, which might increase the content of TG and FC in liver and decrease the content of TC through the action of adipocytes, and affect the lipid metabolism of liver.
【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

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