CMKLR1在NASH大鼠肝组织过表达的意义

发布时间：2018-04-19 14:30

本文选题：非酒精性脂肪性肝炎 + CMKLR1　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:构建NASH大鼠模型,并以慢病毒为媒介实施在体干预,介导CMKLR1在大鼠肝组织中过表达,以期通过基础研究为临床治疗NAFLD提供方向。方法:以慢病毒为载体,鉴定其成功整合CMKLR1基因后以备在体干预。以42只雄性SD大鼠为研究对象,在适宜的光照、温度、湿度环境中适应性喂养1w后随机分为4组,模型组、对照组、转染组(SV-CMKLR1)各12只,空转染(CSV)6只,除对照组给予普通饮食外,其余各组均给予高脂饮食。在实验第1d,转染组于尾静脉注射携带CMKLR1基因的慢病毒2×109pfu 100微升;CSV组于尾静脉注射不携带CMKLR1基因的慢病毒2×109pfu 100微升;PBS作为尾静脉外源性注射物的对照体,各自给予模型组、对照组100微升。在实验的第7、14、21天分别从空染组取2只大鼠处死,显微镜下观察其转染细胞效率。跟踪大鼠一般情况,每周进行称重;分别在8W末、12W末随机从各组取6只大鼠,禁食12h后腹腔麻醉(麻醉药物选用水合氯醛),打开腹腔自腹主动脉取血,离心后留取上清液保存,观察肝脏大体形态变化,留取部分肝组织液氮冷冻后置-80℃冰箱冻存备用,同时取部分肝组织行HE染色,显微镜下观察并行NAFLD活动度评分;Real-time PCR检测各组大鼠肝组织中脂联素、CMKLR1 m RNA水平;Western Blot检测各组大鼠肝组织中CMKLR1、脂联素的蛋白水平。结果:(1)同期大鼠体重及肝指数相比较:模型组对照组、模型组转染组,差异均有统计学意义(P￡0.05)。(2)肝组织HE染色结果:对照组大鼠肝组织肝细胞排列整齐、沿条索呈放射状整齐分布、未见炎症细胞浸润或空泡样变;8w末时,可见模型组肝组织肝细胞体积呈现不同程度的增大、肝条索排列紊乱、可见气球样变,表现为单纯性脂肪变;12w末时,观察视野中几乎所有的肝细胞肿大,胞浆疏松,大量球形脂滴出现及大量炎细胞浸润,可见点状坏死及桥接坏死,进展为NASH;8w末时,转染组肝细胞镜下也表现为单纯性脂肪变、12w末时亦进展为NASH,但脂肪变性程度均较模型组轻。(3)同期血清中凯莫瑞水平作比较:模型组对照组,但差异无统计学意义,转染组模型组,差异有统计学意义(P￡0.05)。(4)同期血清中脂联素水平作比较:模型组对照组;转染组模型组,差异均有统计学意义(P￡0.05)。(5)PCR检测肝组织中CMKLR1mRNA及脂联素mRNA表达情况,同期比较结果显示:大鼠肝组织中CMKLR1水平:模型组对照组,无显著统计学差异;转染组模型组,存在显著统计学差异(P￡0.05);大鼠肝组织中脂联素表达情况:对照组模型组,转染组模型组,差异均有统计学意义(P￡0.05)。(6)Western Blot检测大鼠肝组织中CMKLR1及脂联素蛋白含量,同期比较结果显示:大鼠肝组织中CMKLR1蛋白含量模型组对照组,无显著统计学差异;转染组模型组,存在显著统计学差异(P￡0.05);大鼠肝组织中脂联素蛋白表达情况:对照组模型组,转染组模型组,差异均有统计学意义(P￡0.05)。结论:外源性注射携带CMKLR1基因的慢病毒,使CMKLR1在大鼠肝组织内过表达,可显著改善NASH时肝脏的病理学改变。
[Abstract]:Objective: to construct NASH rat model, and to intervene in vivo with lentivirus as medium, mediate the overexpression of CMKLR1 in rat liver tissue, in order to provide direction for clinical treatment of NAFLD through basic research. Method: using lentivirus as the carrier to identify the successful integration of CMKLR1 gene for intervention in vivo. 42 male SD rats were used as the research object. Suitable light, temperature, humidity environment after adaptive feeding of 1W into 4 groups randomly, model group, control group, transfection group (SV-CMKLR1) 12, empty transfection (CSV) 6, except the control group to the ordinary diet, the other groups were given high fat diet. In the experiment 1D, the tail vein injection of CMKLR1 gene lentivirus 2 x 109pfu 100 micro The CSV group was injected with the lentivirus 2 x 109pfu 100 microliters without CMKLR1 gene in the tail vein, and PBS was given as the control body of the exogenous injections of the tail vein. Each group was given the model group and the control group was 100 microliters. 2 rats were killed from the air Dyeing Group on the day 7,14,21 of the experiment, and the transfection efficiency of the transfected cells was observed under the microscope. The general situation of the rats was followed. At the end of 8W and at the end of 12W, 6 rats were taken randomly from each group. After fasting 12h, the abdominal anaesthesia (anaesthetized drug chloral chloral) was used to open the abdominal aorta and take the blood from the abdominal aorta. After centrifugation, the supernatant was retained, and the gross morphological changes of the liver were observed, and some liver tissues were frozen and stored at -80 centigrade after cryopreservation, and at the same time taking the part of the liver. The liver tissue was stained with HE, and the parallel NAFLD activity score was observed under microscope; Real-time PCR was used to detect the adiponectin, CMKLR1 m RNA level in the liver tissues of the rats and the protein level of CMKLR1 and adiponectin in the liver tissues of each group by Western Blot. Results: (1) the body weight and liver index of rats in the same period were compared: model group control group and model group transfection Group, the difference was statistically significant (P 0.05). (2) liver tissue HE staining results: in the control group, the liver cells in the control group were arranged neatly, the cord of the liver was neatly distributed, and no inflammatory cell infiltration or vacuolar change was found. At the end of 8W, the liver cell volume of the liver tissue in the model group showed a different degree of increase, the disorder of hepatic cord arrangement, and the balloon could be seen. At the end of 12W, almost all liver cells were enlarged, cytoplasm was loose, a large number of spherical lipid droplets were found and a large number of inflammatory cells were infiltrated, and the necrosis and bridging necrosis were seen in NASH. At the end of 8W, the liver cells in the transfected group also showed simple fatty change and NASH was progressed at the end of 12W, but fat was also progressed, but fat at the end of 12W. The degree of fatty degeneration was less than that of the model group. (3) the level of sera in the serum was compared in the same period, but there was no significant difference in the model group, but the difference was statistically significant (P 0.05). (4) the serum adiponectin levels were compared in the same period: the model group was compared with the model group, and the difference was statistically significant (5 P 0.05) (5). The expression of CMKLR1mRNA and adiponectin mRNA in liver tissue was detected by PCR. The comparison results showed that there was no significant difference in the level of CMKLR1 in the rat liver tissue: there was no significant difference between the model group and the model group (P 0.05), and the expression of adiponectin in the rat liver tissue: the control group, the model group of transfection group, and the difference in the transfection group The difference was statistically significant (P 0.05). (6) the content of CMKLR1 and adiponectin protein in liver tissues of rats was detected by Western Blot. The results showed that there was no significant difference between the CMKLR1 protein content model group in the rat liver tissue and the model group of the transfected group (P 0.05), and the adiponectin eggs in the rat liver tissues were significantly different. White expression: the control group model group, transfection group model group, the difference was statistically significant (P 0.05). Conclusion: exogenous injection of CMKLR1 gene lentivirus, CMKLR1 in rat liver tissue overexpression, can significantly improve the pathological changes in the liver of NASH.

【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

【参考文献】