HGF對NASH小鼠肝組織內(nèi)質(zhì)網(wǎng)應(yīng)激的影響及意義
發(fā)布時間：2018-11-14 15:52
【摘要】：目的:建立NASH小鼠模型,通過觀察NASH小鼠模型證實(shí)NASH疾病發(fā)展過程中存在內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ER stress);通過注射重組人肝細(xì)胞生長因子(recombinant human hepatocyte growth factor,rhHGF),探討HGF對NASH肝組織ER stress的影響,為HGF在NASH患者中可能的臨床應(yīng)用提供理論依據(jù)。方法:選用6W雄性SPF級BALB/c小鼠18只,經(jīng)純化標(biāo)準(zhǔn)飼料AIN93M(10%kcal Fat)適應(yīng)性喂養(yǎng)1周,第2周開始造模,定義為正常對照組(6只)和高脂組(12只)。對照組仍舊使用AIN93M(10%kcal Fat)進(jìn)行喂養(yǎng),高脂組純化飲食選用TP23400(60%kcal Fat)喂養(yǎng)16周,在第17周末將高脂組分為NASH模型組(6只)和HGF組(6只),對HGF組小鼠進(jìn)行尾靜脈注射rhHGF,正常對照組和NASH模型組分別經(jīng)小鼠尾靜脈注射生理鹽水,注射劑量均為0.5μg/(kg·d),連續(xù)注射14天,在隔夜禁食12小時后,測量小鼠體重;內(nèi)眥靜脈取血,離心取血清,測定丙氨酸氨基轉(zhuǎn)移酶(alanine aminotransferase,ALT)、天冬氨酸氨基轉(zhuǎn)移酶(aspartate transaminase,AST)、甘油三酯(triglyceride,TG)的水平;ELISA法及放射免疫法測炎性因子白介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)和腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)的水平;處死小鼠,觀察肝臟形態(tài)并測量肝濕重,計算肝指數(shù);取部分中葉肝組織進(jìn)行蘇木精-伊紅染色(hematoxylin and eosin staining,HE)染色,顯微鏡下觀察其病理變化并選用NAFLD活動度積分(NAFLD activity score,NAS)評分;使用凱基全蛋白提取試劑盒進(jìn)行肝臟組織全蛋白的提取,ELISA法測ER stress相關(guān)蛋白即葡萄糖調(diào)節(jié)蛋白78(glucoseregulated protein 78,GRP78)和C/EBP同源蛋白(C/EBP homologous protein,CHOP)的水平。結(jié)果:1.成功制造NASH小鼠模型;2.NASH模型組體重及肝指數(shù)與正常對照組相比升高明顯(P￡0.05);3.NASH模型組小鼠的ALT、AST、IL-6、IL-1β及TNF-α水平均明顯高于正常對照組(P￡0.05),HGF組比NASH模型組明顯降低(P￡0.05);4.NASH模型組的TG水平較正常對照組明顯升高(P￡0.05),HGF組高于NASH模型組,差異有統(tǒng)計學(xué)意義(P￡0.05);5.組織學(xué)變化:正常對照組小鼠肝組織的HE染色切片可見清晰的肝臟小葉結(jié)構(gòu),肝細(xì)胞與肝血竇并行呈放射樣整齊排列,未見脂滴和炎細(xì)胞,NASH模型組的小葉結(jié)構(gòu)紊亂,肝細(xì)胞排列無序,可見大量脂滴和炎細(xì)胞浸潤;HGF組較NASH模型組的病理改變減輕;6.ELISA法測得NASH模型組GRP78水平高于正常對照組(P￡0.05),NASH模型組CHOP水平高于正常對照組,但差異無統(tǒng)計學(xué)意義;HGF組兩種蛋白水平較NASH模型組均有所降低(P￡0.05)。結(jié)論:通過建立NASH小鼠模型,觀察到在NASH疾病的發(fā)生發(fā)展過程中,ER stress參與肝臟的損傷機(jī)制;HGF可通過抑制NASH疾病中的ER stress反應(yīng),起到保護(hù)肝臟的作用。
[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- 偽 (TNF- 偽) 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- 偽 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- 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575
本文編號：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- 偽 (TNF- 偽) 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- 偽 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- 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575
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