IGF-1对脂肪变肝细胞p38MAPK表达和生化指标的影响

发布时间：2019-02-12 09:20

【摘要】：目的:研究IGF-1对油酸诱导的脂肪变肝细胞p38MAPK表达及其生化指标的影响,旨在探讨IGF-1在非酒精性脂肪性肝病中的作用,进而为NAFLD的临床防治提供新思路。方法:人肝癌细胞株HepG2,用含10%的胎牛血清的DMEM培养基培养。然后以不同浓度油酸诱导HepG2细胞发生脂肪变性,采用MTT法探索最佳造模浓度。进行所有实验之前,均将HepG2细胞置于无血清DMEM培养基中培养12 h。将HepG2细胞随机分为3组:正常对照组、油酸诱导组、油酸联合IGF-1组。采用油红O染色观察各组细胞内脂质沉积情况;Western Blot检测各组p38MAPK、p-p38MAPK蛋白的表达;采用生化试剂盒检测各组细胞内TG(甘油三酯)及上清液中ALT、AST的水平。结果:1、油酸对HepG2细胞株具有诱导作用,使其发生一定程度的脂肪变性,并可顺利构建肝细胞脂肪变模型。在油红O染色之后,通过光学显微镜进行观察,结果显示肝细胞脂质沉积并没有出现于正常对照组中;油酸诱导组在培养24h后可见细胞内少量脂质沉积,脂变率(10.37±1.79)%;随着培养时间的增加,脂肪变程度逐渐加重,48h后脂质沉积增多,脂变率(34.26±7.04)%;72h后则出现大量脂质沉积,脂变率(75.63±12.03)%。通过对上述3个培养时点的对比,差异具有统计学意义(P0.01)。油酸联合IGF-1组与油酸诱导组相比,培养24h后其脂变率无明显差异(P0.05),48h、72h后油酸联合IGF-1组的脂变率明显降低,分别为(28.76±7.12)%、(37.67±6.89)%,差异分别具有统计学意义(P0.05)。2、各组脂肪变肝细胞内TG水平的比较随着培养时间的增加,油酸诱导组、油酸联合IGF-1组细胞内TG含量均有所增高,油酸诱导组内各时点进行比较具有显著的统计学差异(P0.01)。油酸联合IGF-1组和油酸诱导组各时点的TG含量明显多于正常对照组,此差异比较明显,具有显著的统计学意义(P0.01)。油酸联合IGF-1组细胞在经过48h、72h培养之后,细胞内的TG含量明显低于油酸诱导组,此差异比较明显,具有显著的统计学意义(P0.05)。3、各组培养上清液中ALT、AST含量的比较随着培养时间的增加,油酸诱导组、油酸联合IGF-1组细胞上清液中的AST和ALT的含量明显增加,在油酸诱导组中各时点之间的差异比较明显,具有显著的统计学意义(P0.01)。油酸联合IGF-1组细胞在经过48h、72h培养之后,细胞上清液中ALT、AST含量明显低于油酸诱导组,此差异比较明显,具有显著的统计学意义(P0.01)。4、各组细胞p-p38MAPK和p38MAPK蛋白表达水平的比较各组细胞分别培养24小时、48小时和72小时之后,p38MAPK蛋白表达都比较明显,但是各组之间的差异不明显,不具有统计学意义(P0.05)。油酸联合IGF-1组以及油酸诱导组p-p38MAPK均有表达,正常对照组无表达。油酸诱导组72h时p-p38MAPK蛋白表达与24h、48h相比明显增高(P0.01);油酸联合IGF-1组内各时点进行比较,差异无统计学意义(P0.05),然而与油酸诱导组培养72h后p-p38MAPK的蛋白表达(2.93±0.13)相比较,油酸联合IGF-1组表达水平却显著降低(1.12±0.07),差异具有统计学意义(P0.01)。结论:IGF-1可以改善肝细胞脂肪变的程度,明显降低肪变肝细胞p-p38MAPK蛋白的表达水平,改善脂肪变肝细胞的生化指标,提示IGF-1可能通过p38MAPK信号通路参与肝细胞脂肪变的调节,在非酒精性脂肪性肝病的发生发展过程中发挥着重要作用。
[Abstract]:Objective: To study the effect of IGF-1 on the expression of p38MAPK and its biochemical index in the induced fatty liver cells, and to explore the role of IGF-1 in non-alcoholic fatty liver disease and to provide a new way for the clinical prevention and treatment of NAFLD. Methods: Human liver cancer cell line HepG2 was cultured with DMEM medium containing 10% fetal bovine serum. Then, the fat degeneration of HepG2 cells was induced with oleic acid at different concentrations, and the best model concentration was explored by MTT method. HepG2 cells were cultured in serum-free DMEM medium for 12 h prior to all experiments. HepG2 cells were randomly divided into three groups: normal control group, oleic acid induction group, and oleic acid group IGF-1 group. The expression of p38MAPK and p-p38MAPK in each group was observed by oil-red O-staining. The levels of ALT and AST in the cells of each group were detected by a biochemical kit. Results: 1. The oleic acid has an induction effect on the HepG2 cell line, which makes it a certain degree of fat degeneration, and can successfully construct the hepatocyte fat-variable model. After the oil red O was stained, the results showed that the lipid deposition of the hepatocyte was not present in the normal control group; the oleic acid-induced group showed a small amount of lipid deposition in the cells after the culture of 24h, the lipid rate (10.37% 1.79)%, and with the increase of the culture time, The degree of fat change was gradually increased, the lipid deposition was increased after 48h, and the lipid rate (34. 26% 7. 04)%; after 72h, a large amount of lipid deposition was observed, and the lipid rate (70.63% 12.03)%. The difference of the three culture time points was statistically significant (P0.01). Compared with the group of oleic acid in the combination of oleic acid and IGF-1, there was no significant difference in the lipid rate of the combined group of oleic acid and the group of oleic acid (P0.05), and the lipid rate of the combined group of oleic acid and IGF-1 in the group was significantly lower than that of the group of oleic acid (P 0.05), 48 h, and 72h, respectively (28. 76% 7. 12)%, (37. 67% 6.89)%, and the difference was statistically significant (P0.05). The TG levels in the group of oleic acid and the group of oleic acid and IGF-1 increased with the increase of the culture time, and the TG level in the oleic acid group and the oleic acid group increased with the increase of the culture time, and there was a significant difference in the time points in the oleic acid-induced group (P0.01). The TG content of oleic acid combined with IGF-1 and oleic acid group was significantly higher than that in the normal control group, and the difference was significant (P0.01). The TG content of oleic acid combined with IGF-1 group was significantly lower than that of oleic acid induction group after 48 h and 72h culture, and the difference was significant (P0.05). The contents of AST and ALT in the supernatant of the combination of oleic acid and IGF-1 were significantly increased, and the difference between the time points in the oleic acid group was significant (P0.01). The content of ALT and AST in the supernatant of oleic acid combined with IGF-1 was significantly lower than that of the induction group of oleic acid, and the difference was significant (P0.01). The expression level of p-p38MAPK and p38 MAPK in each group was 24 h, 48 h and 72 h, respectively. The expression of p-p38 MAPK in the combination of oleic acid and IGF-1 and oleic acid group was not expressed in the normal control group. The expression of p-p38MAPK in oleic acid-induced group was significantly higher than that of 24h and 48h (P0.01), and the expression of p-p38MAPK in the group of oleic acid and IGF-1 was not statistically significant (P0.05). However, the expression of p-p38MAPK was compared with the expression of p-p38MAPK (2.93. The expression level of the combined oleic acid and IGF-1 group was significantly lower (1. 12-0. 07), and the difference was statistically significant (P0.01). Conclusion: IGF-1 can improve the level of hepatic steatosis, decrease the expression level of p-p38MAPK in the hepatocyte, and improve the biochemical index of the fat-variable hepatocytes. It is suggested that IGF-1 may be involved in the regulation of the changes of the liver cell fat through the p38 MAPK signal pathway. It plays an important role in the development of nonalcoholic fatty liver disease.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.5

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