HGF对NASH小鼠肝组织内质网应激的影响及意义
发布时间：2018-11-14 15:52
【摘要】：目的:建立NASH小鼠模型,通过观察NASH小鼠模型证实NASH疾病发展过程中存在内质网应激(endoplasmic reticulum stress,ER stress);通过注射重组人肝细胞生长因子(recombinant human hepatocyte growth factor,rhHGF),探讨HGF对NASH肝组织ER stress的影响,为HGF在NASH患者中可能的临床应用提供理论依据。方法:选用6W雄性SPF级BALB/c小鼠18只,经纯化标准饲料AIN93M(10%kcal Fat)适应性喂养1周,第2周开始造模,定义为正常对照组(6只)和高脂组(12只)。对照组仍旧使用AIN93M(10%kcal Fat)进行喂养,高脂组纯化饮食选用TP23400(60%kcal Fat)喂养16周,在第17周末将高脂组分为NASH模型组(6只)和HGF组(6只),对HGF组小鼠进行尾静脉注射rhHGF,正常对照组和NASH模型组分别经小鼠尾静脉注射生理盐水,注射剂量均为0.5μg/(kg·d),连续注射14天,在隔夜禁食12小时后,测量小鼠体重;内眦静脉取血,离心取血清,测定丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate transaminase,AST)、甘油三酯(triglyceride,TG)的水平;ELISA法及放射免疫法测炎性因子白介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平;处死小鼠,观察肝脏形态并测量肝湿重,计算肝指数;取部分中叶肝组织进行苏木精-伊红染色(hematoxylin and eosin staining,HE)染色,显微镜下观察其病理变化并选用NAFLD活动度积分(NAFLD activity score,NAS)评分;使用凯基全蛋白提取试剂盒进行肝脏组织全蛋白的提取,ELISA法测ER stress相关蛋白即葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)和C/EBP同源蛋白(C/EBP homologous protein,CHOP)的水平。结果:1.成功制造NASH小鼠模型;2.NASH模型组体重及肝指数与正常对照组相比升高明显(P￡0.05);3.NASH模型组小鼠的ALT、AST、IL-6、IL-1β及TNF-α水平均明显高于正常对照组(P￡0.05),HGF组比NASH模型组明显降低(P￡0.05);4.NASH模型组的TG水平较正常对照组明显升高(P￡0.05),HGF组高于NASH模型组,差异有统计学意义(P￡0.05);5.组织学变化:正常对照组小鼠肝组织的HE染色切片可见清晰的肝脏小叶结构,肝细胞与肝血窦并行呈放射样整齐排列,未见脂滴和炎细胞,NASH模型组的小叶结构紊乱,肝细胞排列无序,可见大量脂滴和炎细胞浸润;HGF组较NASH模型组的病理改变减轻;6.ELISA法测得NASH模型组GRP78水平高于正常对照组(P￡0.05),NASH模型组CHOP水平高于正常对照组,但差异无统计学意义;HGF组两种蛋白水平较NASH模型组均有所降低(P￡0.05)。结论:通过建立NASH小鼠模型,观察到在NASH疾病的发生发展过程中,ER stress参与肝脏的损伤机制;HGF可通过抑制NASH疾病中的ER stress反应,起到保护肝脏的作用。
[Abstract]:Objective: to establish NASH mouse model and to confirm that endoplasmic reticulum stress (endoplasmic reticulum stress,ER stress);) exists in the development of NASH by observing NASH mouse model. By injecting recombinant human hepatocyte growth factor (recombinant human hepatocyte growth factor,rhHGF) to investigate the effect of HGF on ER stress in NASH liver tissue, this study provides a theoretical basis for the possible clinical application of HGF in NASH patients. Methods: eighteen 6W male BALB/c mice of SPF grade were fed with purified standard feed AIN93M (10%kcal Fat) for one week, and then the model was established at the second week. The mice were divided into normal control group (6 mice) and hyperlipidemia group (12 mice). The control group was still fed with AIN93M (10%kcal Fat), and the high-fat group was fed with TP23400 (60%kcal Fat) for 16 weeks. At the end of the 17th week, the high-fat group was divided into NASH model group (6 rats) and HGF group (6 rats). The mice in HGF group were injected with normal control group and NASH model group by tail vein injection of normal saline, respectively. The injection dose was 0.5 渭 g / (kg d), for 14 days. After 12 hours of overnight fasting, the mice in the HGF group were injected with normal saline respectively. The weight of mice was measured. The levels of alanine aminotransferase (alanine aminotransferase,ALT), aspartate aminotransferase (aspartate transaminase,AST) and triglyceride (triglyceride,TG) were measured. The levels of interleukin-1 尾 (IL-1 尾), interleukin-6 (interleukin-6,IL-6) and tumor necrosis factor- 伪 (TNF- 伪) were measured by ELISA and radioimmunoassay. Mice were killed, liver morphology was observed, liver wet weight was measured and liver index was calculated. Some middle lobe liver tissues were stained with hematoxylin-eosin staining (hematoxylin and eosin staining,HE). The pathological changes were observed under microscope and NAFLD activity score (NAFLD activity score,NAS) was selected. The total protein of liver tissue was extracted by Keti whole protein extraction kit, and the levels of ER stress related proteins (glucose regulatory protein 78 (glucoseregulated protein 78 GRP 78) and C/EBP homologous protein (C/EBP homologous protein,CHOP) were measured by ELISA method. The result is 1: 1. NASH mouse model was established successfully, the body weight and liver index of 2.NASH model group were significantly higher than that of normal control group (P0.05). The levels of ALT,AST,IL-6,IL-1 尾 and TNF- 伪 in the 3.NASH model group were significantly higher than those in the normal control group (P0.05). The levels of ALT,AST,IL-6,IL-1 尾 and TNF- 伪 in the), HGF group were significantly lower than those in the NASH model group (P0.05). The TG level of 4.NASH model group was significantly higher than that of normal control group (P0.05), HGF group was higher than NASH model group, the difference was statistically significant (P0.05). Histological changes: the HE staining sections of normal control mice showed clear hepatic lobular structure, hepatic cells and hepatic sinusoid cells were arranged in radioactivity order, no lipid droplets and inflammatory cells were found, and the lobular structure of NASH model group was disordered. Liver cells were disordered, and a large number of lipid droplets and inflammatory cells were observed. The GRP78 level of NASH model group was higher than that of normal control group (P0.05) the CHOP level of), NASH model group was higher than that of normal control group, but the difference was not statistically significant. The level of two proteins in HGF group was lower than that in NASH model group (P0.05). Conclusion: through the establishment of NASH mouse model, it is observed that, ER stress participates in the mechanism of liver injury during the occurrence and development of NASH disease, and HGF can protect the liver by inhibiting the ER stress response in NASH disease.
【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】：R575
本文编号：2331610
[Abstract]:Objective: to establish NASH mouse model and to confirm that endoplasmic reticulum stress (endoplasmic reticulum stress,ER stress);) exists in the development of NASH by observing NASH mouse model. By injecting recombinant human hepatocyte growth factor (recombinant human hepatocyte growth factor,rhHGF) to investigate the effect of HGF on ER stress in NASH liver tissue, this study provides a theoretical basis for the possible clinical application of HGF in NASH patients. Methods: eighteen 6W male BALB/c mice of SPF grade were fed with purified standard feed AIN93M (10%kcal Fat) for one week, and then the model was established at the second week. The mice were divided into normal control group (6 mice) and hyperlipidemia group (12 mice). The control group was still fed with AIN93M (10%kcal Fat), and the high-fat group was fed with TP23400 (60%kcal Fat) for 16 weeks. At the end of the 17th week, the high-fat group was divided into NASH model group (6 rats) and HGF group (6 rats). The mice in HGF group were injected with normal control group and NASH model group by tail vein injection of normal saline, respectively. The injection dose was 0.5 渭 g / (kg d), for 14 days. After 12 hours of overnight fasting, the mice in the HGF group were injected with normal saline respectively. The weight of mice was measured. The levels of alanine aminotransferase (alanine aminotransferase,ALT), aspartate aminotransferase (aspartate transaminase,AST) and triglyceride (triglyceride,TG) were measured. The levels of interleukin-1 尾 (IL-1 尾), interleukin-6 (interleukin-6,IL-6) and tumor necrosis factor- 伪 (TNF- 伪) were measured by ELISA and radioimmunoassay. Mice were killed, liver morphology was observed, liver wet weight was measured and liver index was calculated. Some middle lobe liver tissues were stained with hematoxylin-eosin staining (hematoxylin and eosin staining,HE). The pathological changes were observed under microscope and NAFLD activity score (NAFLD activity score,NAS) was selected. The total protein of liver tissue was extracted by Keti whole protein extraction kit, and the levels of ER stress related proteins (glucose regulatory protein 78 (glucoseregulated protein 78 GRP 78) and C/EBP homologous protein (C/EBP homologous protein,CHOP) were measured by ELISA method. The result is 1: 1. NASH mouse model was established successfully, the body weight and liver index of 2.NASH model group were significantly higher than that of normal control group (P0.05). The levels of ALT,AST,IL-6,IL-1 尾 and TNF- 伪 in the 3.NASH model group were significantly higher than those in the normal control group (P0.05). The levels of ALT,AST,IL-6,IL-1 尾 and TNF- 伪 in the), HGF group were significantly lower than those in the NASH model group (P0.05). The TG level of 4.NASH model group was significantly higher than that of normal control group (P0.05), HGF group was higher than NASH model group, the difference was statistically significant (P0.05). Histological changes: the HE staining sections of normal control mice showed clear hepatic lobular structure, hepatic cells and hepatic sinusoid cells were arranged in radioactivity order, no lipid droplets and inflammatory cells were found, and the lobular structure of NASH model group was disordered. Liver cells were disordered, and a large number of lipid droplets and inflammatory cells were observed. The GRP78 level of NASH model group was higher than that of normal control group (P0.05) the CHOP level of), NASH model group was higher than that of normal control group, but the difference was not statistically significant. The level of two proteins in HGF group was lower than that in NASH model group (P0.05). Conclusion: through the establishment of NASH mouse model, it is observed that, ER stress participates in the mechanism of liver injury during the occurrence and development of NASH disease, and HGF can protect the liver by inhibiting the ER stress response in NASH disease.
【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】：R575
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