GLP-1受体在大鼠肝星状细胞的表达及利拉鲁肽对高糖条件下大鼠肝星状细胞增殖的作用

发布时间：2019-05-15 16:29

【摘要】：目的:目前非酒精性脂肪性肝病(non-alcoholic fatty liver disease NAFLD)的发生日益增加。NAFLD包括从肝脏脂肪变性到脂肪性肝炎,进而发展为肝脏纤维化,最终至肝硬化的一系列病变。但是目前尚没有药物可以治疗NAFLD。胰高血糖素样肽-1(Glucagon-like peptide-1 GLP-1)是由肠道内分泌细胞L细胞分泌的一种重要的肠促胰岛素,GLP-1类似物利拉鲁肽作为降糖药物已广泛的应用于临床,其胰腺外作用是目前糖尿病领域关注的热点。GLP-1受体介导的抗脏器纤维化作用已得到初步证实,其机制非常复杂,在不同的脏器可能存在不同的作用通路,并且一些参与脏器纤维化的关键细胞如肝星状细胞(hepatic stellate cells HSC)等最有可能参与其中。那么作为肝纤维化的关键细胞HSC上是否存在GLP-1受体的表达呢?本实验旨在探讨GLP-1受体在大鼠HSC是否表达及GLP-1类似物利拉鲁肽对高糖条件下大鼠HSC的作用,从而探讨其对于NAFLD及肝脏纤维化的作用。方法:1大鼠HSC的培养:大鼠HSC复苏后接种于无菌塑料培养瓶中,37℃、体积分数为5%的CO2饱和湿度培养箱中常规培养,培养液为含体积分数为10%胎牛血清的1640,每天换液1次,48小时后提取样本于显微镜下进行形态学鉴定,并进行后续实验分析。2 Western法检测GLP-1受体蛋白在大鼠HSC的表达。3 Real-time PCR法检测大鼠HSCGLP-1受体m RNA的表达。4分组干预:将培养48小时的大鼠HSC分7组分别为正常糖对照组(5.5mmol/L)、高渗对照组(5.5mmol/L低糖+19.5mmol/L甘露醇)、高糖对照组(25.0mmol/L)、高糖+低浓度利拉鲁肽组(50nmol/L)、高糖+中浓度利拉鲁肽组(100nmol/L)、高糖+高浓度利拉鲁肽组(200nmol/L)、正常糖+高浓度利拉鲁肽组(200nmol/L)。5 MTS法检测上述7组细胞的增殖情况。6数据分析:所得数据采用均数±标准差(sx?)表示,统计分析运用SPSS 13.0软件。采用方差分析对各个时间点的7组的光密度(optical density OD)值进行比较,采用Bonferroni法进行两两比较,探讨同一时间点具体不同的组的差别。对不同时间点的高糖+高浓度利拉鲁肽采取配对t检验,探讨时间对该组OD值得影响。P0.05为统计学差异有意义。结果:1大鼠HSC形态学鉴定倒置显微镜下可以观察到活化的HSC为扁平状,胞体大,胞体周围有突起。2 Western blot及Real-time PCR检测GLP-1受体均在大鼠HSC上有表达。5个样本均测得GLP-1受体蛋白及m RNA表达。3 HSC的增殖:高糖组与正常糖组差异有统计学意义(p0.05)。高糖+高浓度利拉鲁肽干预组作用48小时、72小时时较高糖对照组增殖降低(p0.05),但高于正常糖对照组(p0.05),高浓度利拉鲁肽组(200nmol/L)增殖低于低(50nmol/L)、中(100nmol/L)浓度利拉鲁肽组(p0.05),但低、中浓度的利拉鲁肽组增殖差异无统计学意义(p0.05)。高渗对照组及正常糖+高浓度利拉鲁肽组同正常糖对照组比较增殖差异无统计学意义(p0.05)。结论:1大鼠HSC上有GLP-1受体的表达。2高糖可促进大鼠HSC增殖,而利拉鲁肽可抑制高糖作用下其增殖,并与其浓度和作用时间紧密相关。
[Abstract]:Objective: To study the increasing of non-alcoholic fatty liver disease (NAFLD). NAFLD includes a range of pathological changes from hepatic steatosis to steatohepatitis, further developing to liver fibrosis, and ultimately to cirrhosis. However, no drug can be used to treat NAFLD at present. Glucagon-like peptide-1GLP-1 is an important intestinal proinsulin, which is secreted by the L-cells of the intestinal endocrine cell. The mechanism of anti-organ fibrosis mediated by GLP-1 receptor has been proved. The mechanism is very complicated. There may be different pathways in different organs, and some of the key cells involved in organ fibrosis, such as hepatic stellate cells (HSC), may be involved. Is there a GLP-1 receptor expression on the key cell HSC as a liver fibrosis? The purpose of this study was to investigate the expression of GLP-1 receptor in rat HSC and the effect of GLP-1 analog liraglutide on HSC in rats with high glucose, so as to study the effect of GLP-1 receptor on NAFLD and hepatic fibrosis. Method:1 rat HSCs were cultured: the rat HSC was recovered and then inoculated into a sterile plastic culture flask, and the culture medium was 1640 with a volume fraction of 10% fetal bovine serum in a CO2 saturated humidity incubator with a volume fraction of 5% at 37 & deg; C. The expression of the rat HSCGLP-1 receptor m RNA was detected by the real-time PCR method. The rats were divided into normal sugar control group (5.5 mmol/ L), hypertonic control group (5.5 mmol/ L low-sugar + 19.5 mmol/ L mannitol), high-sugar control group (25.0 mmol/ L), high-sugar + low-concentration liraglutide group (50 nmol/ L) and high-sugar + medium-concentration liraglutide group (100 nmol/ L), respectively. The proliferation of the above-mentioned 7-group cells was detected by high-glucose + high-concentration liraglutide (200 nmol/ L), normal sugar + high-concentration liraglutide (200 nmol/ L).5 MTS assay. ) SPSS 13.0 software was used for statistical analysis. The optical density OD values of 7 groups of time points were compared by analysis of variance, and the difference of different groups at the same time was discussed by using the Bonferroni method. The paired t-test was carried out on the high-glucose + high-concentration liraglutide at different time points, and the effect of time on the set OD was discussed. The difference of P0.05 was significant. Results: The expression of GLP-1 receptor and the expression of m-RNA were measured by Western blot and Real-time PCR. The difference between the high-sugar group and the normal sugar group was statistically significant (p0.05). The proliferation of high-glucose + high-concentration liraglutide was decreased (p0.05) at 48 h and 72 h, but higher than that of normal sugar control group (p0.05), and the proliferation of high-concentration liraglutide group (200 nmol/ L) was lower than that of low (50 nmol/ L), medium (100 nmol/ L) concentration of liraglutide (p0.05), but low, There was no significant difference in the proliferation of the liraglutide group at medium concentration (p0.05). There was no significant difference in the proliferation of the high-permeability control group and the normal sugar + high-concentration liraglutide group compared with the normal sugar control group (p0.05). Conclusion:1. The expression of GLP-1 receptor in rat HSC can promote the proliferation of HSC in rat, and the liraglutide can inhibit the proliferation of HSC, and it is closely related to its concentration and time of action.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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