亚硫酸钠通过脂肪细胞对共培养肝细胞脂质代谢的影响

发布时间：2018-04-12 14:09

本文选题：亚硫酸钠 + 脂肪细胞与肝脏细胞共培养　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:探讨亚硫酸钠是否通过脂肪细胞来影响肝脏细胞内甘油三酯(TG)与胆固醇含量,为研究亚硫酸钠的肝毒性作用机制提供参考。方法:将3T3-L1前脂肪细胞进行诱导分化,成为成熟脂肪细胞,并用油红O染色验证;用不同浓度(0.02、0.1、0.5、2.5 mmol/L)Na2SO3和1 mmol/L油酸(OA)染毒脂肪细胞12 h,收集脂肪细胞上清,再分别用上述收集的各组脂肪细胞上清染毒Hep G2细胞24 h,即共培养组。另外再用同上述相同浓度(0.02、0.1、0.5、2.5 mmol/L)Na2SO3和1 mmol/L油酸单独培养Hep G2细胞24 h,即单独培养组;采用游离脂肪酸(FFA)检测试剂盒测定脂肪细胞上清中FFA含量;采用ELISA法测定脂肪细胞上清中脂联素的含量;采用GPO-POD酶法测定脂肪细胞培养上清中的TG含量;采用有机抽提法提取肝细胞内TG、总胆固醇(TC)和游离胆固醇(FC),再用POD酶法测定其含量;采用western blot法检测Na2SO3对单独培养组和共培养组肝细胞内TG代谢相关蛋白腺苷酸活化蛋白激酶(AMPKα)、P-AMPKα、乙酰辅酶A羧化酶(ACC)、P-ACC、固醇调节元件结合蛋白-1c(SREBP-1c)、脂肪酸合成酶(FASN)、肉碱酯酰转移酶1(CPT-1)和胆固醇代谢相关蛋白3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGCR)、低密度脂蛋白受体(LDLR)、酰基辅酶A:胆固醇酰基转移酶(ACAT)的相对表达水平。结果:1、与阴性对照相比Na2SO3各处理组脂肪细胞培养上清中FFA和脂联素的含量都明显减少(P0.05),TG含量无明显变化(P0.05)。2、Na2SO3处理后,单独培养细胞内TG含量无明显变化(P0.05)。但是2.5 mmol/L亚硫酸钠可引起共培养Hep G2细胞内TG的增加(P0.05)。3、Western blot结果显示,Na2SO3处理后,单独培养肝细胞内SREBP-1c、AMPKα、P-ACC、CPT-1无明显变化(P0.05);2.5 mmol/L Na2SO3组和OA组FASN蛋白表达增高(P0.05);ACC除0.5 mmol/L Na2SO3变化不明显外,其余各组都升高(P0.05)。共培养组,P-ACC、SREBP-1c、FASN、CPT-1无明显变化(P0.05);未检测到P-AMPKα;AMPKα和ACC的蛋白表达均增高(P0.05)。4、Na2SO3处理后,单独培养肝细胞内TC和FC含量的变化不明显(P0.05);0.5 mmol/L Na2SO3、2.5 mmol/L Na2SO3和1 mmol/L OA可引起共培养组肝脏细胞内TC降低(P0.05);而2.5 mmol/L Na2SO3和1 mmol/L OA可引起共培养组肝脏细胞内FC含量增加(P0.05)。5、Western blot结果显示,Na2SO3处理后,单独培养组肝细胞内HMGCR表达无明显变化(P0.05);ACAT蛋白表达水平均增高(P0.05);2.5 mmol/L Na2SO3使LDLR表达升高(P0.05)。共培养组,HMGCR和LDLR蛋白表达无明显变化(P0.05);ACAT蛋白表达增高(P0.05)。结论:暴露在较低剂量的Na2SO3环境下时,不会引起肝脏TG和胆固醇的增加。但暴露于2.5 mmol/L较高浓度的Na2SO3环境下,机体不能代谢解毒Na2SO3,则可能通过脂肪细胞的作用使肝脏中TG和FC明显升高,TC的含量降低,对肝脏脂代谢产生影响。
[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.
【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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