發(fā)布時(shí)間：2018-04-19 14:30

  本文選題：非酒精性脂肪性肝炎 + CMKLR1�。� 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:構(gòu)建NASH大鼠模型,并以慢病毒為媒介實(shí)施在體干預(yù),介導(dǎo)CMKLR1在大鼠肝組織中過表達(dá),以期通過基礎(chǔ)研究為臨床治療NAFLD提供方向。方法:以慢病毒為載體,鑒定其成功整合CMKLR1基因后以備在體干預(yù)。以42只雄性SD大鼠為研究對象,在適宜的光照、溫度、濕度環(huán)境中適應(yīng)性喂養(yǎng)1w后隨機(jī)分為4組,模型組、對照組、轉(zhuǎn)染組(SV-CMKLR1)各12只,空轉(zhuǎn)染(CSV)6只,除對照組給予普通飲食外,其余各組均給予高脂飲食。在實(shí)驗(yàn)第1d,轉(zhuǎn)染組于尾靜脈注射攜帶CMKLR1基因的慢病毒2×109pfu 100微升;CSV組于尾靜脈注射不攜帶CMKLR1基因的慢病毒2×109pfu 100微升;PBS作為尾靜脈外源性注射物的對照體,各自給予模型組、對照組100微升。在實(shí)驗(yàn)的第7、14、21天分別從空染組取2只大鼠處死,顯微鏡下觀察其轉(zhuǎn)染細(xì)胞效率。跟蹤大鼠一般情況,每周進(jìn)行稱重;分別在8W末、12W末隨機(jī)從各組取6只大鼠,禁食12h后腹腔麻醉(麻醉藥物選用水合氯醛),打開腹腔自腹主動(dòng)脈取血,離心后留取上清液保存,觀察肝臟大體形態(tài)變化,留取部分肝組織液氮冷凍后置-80℃冰箱凍存?zhèn)溆?同時(shí)取部分肝組織行HE染色,顯微鏡下觀察并行NAFLD活動(dòng)度評分;Real-time PCR檢測各組大鼠肝組織中脂聯(lián)素、CMKLR1 m RNA水平;Western Blot檢測各組大鼠肝組織中CMKLR1、脂聯(lián)素的蛋白水平。結(jié)果:(1)同期大鼠體重及肝指數(shù)相比較:模型組對照組、模型組轉(zhuǎn)染組,差異均有統(tǒng)計(jì)學(xué)意義(P￡0.05)。(2)肝組織HE染色結(jié)果:對照組大鼠肝組織肝細(xì)胞排列整齊、沿條索呈放射狀整齊分布、未見炎癥細(xì)胞浸潤或空泡樣變;8w末時(shí),可見模型組肝組織肝細(xì)胞體積呈現(xiàn)不同程度的增大、肝條索排列紊亂、可見氣球樣變,表現(xiàn)為單純性脂肪變;12w末時(shí),觀察視野中幾乎所有的肝細(xì)胞腫大,胞漿疏松,大量球形脂滴出現(xiàn)及大量炎細(xì)胞浸潤,可見點(diǎn)狀壞死及橋接壞死,進(jìn)展為NASH;8w末時(shí),轉(zhuǎn)染組肝細(xì)胞鏡下也表現(xiàn)為單純性脂肪變、12w末時(shí)亦進(jìn)展為NASH,但脂肪變性程度均較模型組輕。(3)同期血清中凱莫瑞水平作比較:模型組對照組,但差異無統(tǒng)計(jì)學(xué)意義,轉(zhuǎn)染組模型組,差異有統(tǒng)計(jì)學(xué)意義(P￡0.05)。(4)同期血清中脂聯(lián)素水平作比較:模型組對照組;轉(zhuǎn)染組模型組,差異均有統(tǒng)計(jì)學(xué)意義(P￡0.05)。(5)PCR檢測肝組織中CMKLR1mRNA及脂聯(lián)素mRNA表達(dá)情況,同期比較結(jié)果顯示:大鼠肝組織中CMKLR1水平:模型組對照組,無顯著統(tǒng)計(jì)學(xué)差異;轉(zhuǎn)染組模型組,存在顯著統(tǒng)計(jì)學(xué)差異(P￡0.05);大鼠肝組織中脂聯(lián)素表達(dá)情況:對照組模型組,轉(zhuǎn)染組模型組,差異均有統(tǒng)計(jì)學(xué)意義(P￡0.05)。(6)Western Blot檢測大鼠肝組織中CMKLR1及脂聯(lián)素蛋白含量,同期比較結(jié)果顯示:大鼠肝組織中CMKLR1蛋白含量模型組對照組,無顯著統(tǒng)計(jì)學(xué)差異;轉(zhuǎn)染組模型組,存在顯著統(tǒng)計(jì)學(xué)差異(P￡0.05);大鼠肝組織中脂聯(lián)素蛋白表達(dá)情況:對照組模型組,轉(zhuǎn)染組模型組,差異均有統(tǒng)計(jì)學(xué)意義(P￡0.05)。結(jié)論:外源性注射攜帶CMKLR1基因的慢病毒,使CMKLR1在大鼠肝組織內(nèi)過表達(dá),可顯著改善NASH時(shí)肝臟的病理學(xué)改變。
[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.

【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

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 譚麗萍;石安華;馬潔;姚政;殷華;陳文慧;;高脂致非酒精性脂肪肝動(dòng)物模型肝臟形態(tài)學(xué)動(dòng)態(tài)研究[J];中國民族民間醫(yī)藥;2015年13期

2 董志霞;陸倫根;;2015 JSGE NAFLD/NASH循證臨床實(shí)踐指南解讀[J];浙江醫(yī)學(xué);2015年12期

3 Mustafa Koplay;Mesut Sivri;Hasan Erdogan;Alaaddin Nayman;;Importance of imaging and recent developments in diagnosis of nonalcoholic fatty liver disease[J];World Journal of Hepatology;2015年05期

4 Nancy Khov;Amol Sharma;Thomas R Riley;;Bedside ultrasound in the diagnosis of nonalcoholic fatty liver disease[J];World Journal of Gastroenterology;2014年22期

5 高麗;Robert Gyurko;Thomas Van Dyke;;人類趨化因子受體1(CMKLR1/hChemR23)轉(zhuǎn)基因小鼠對實(shí)驗(yàn)性腹膜炎和牙周炎的炎癥抑制效應(yīng)[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2012年03期

，

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