Leptin在肝脏缺血—再灌注损伤中的变化规律及其重组、表达

发布时间：2018-02-04 03:57

本文关键词： 缺血预处理再灌注损伤肝脏 Leptin 放射免疫分析法逆转录-聚合酶链反应 rh-leptin 克隆表达融合蛋白大肠杆菌复性蛋白纯化生物活性　出处：《中国人民解放军军医进修学院》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：建立大鼠肝脏缺血-再灌注(Ischemia-Reperfusion，I／R)损伤模型，探讨大鼠肝脏I／R损伤时血清Leptin蛋白及脂肪组织Leptin mRNA水平的变化规律，探讨影响血清Leptin水平变化的因素，以及此变化规律对机体的影响。方法：将45只体重220g左右的雄性SD大鼠随机分为5组，，第一组为假手术组(Sham组)，第二组为肝脏缺血60min、再灌注60min(160R60)组，第三组为I60R150组，第四组为I60R240组，第五组为I60R360组。麻醉后，在门静脉分出发向肝脏尾叶的分支上，以动脉夹夹闭肝动脉、门静脉及胆管，建立大鼠肝脏70％I／R损伤模型。动脉夹置入后，关闭腹腔。缺血60min后，取出动脉夹进行再灌注。采用高灵敏鼠Leptin放射免疫分析法测定血清Leptin浓度变化，并采用逆转录-聚合酶链反应(RT-PCR)方法检测脂肪组织Leptin mRNA表达水平的变化。结果：(1)与Sham组(12.01±2.73 ng／ml)相比，I60R60组(17.77±5.61 ng／ml)和I60R150组(17.26±6.13 ng／ml)血清Leptin浓度即出现增高趋势，I60R360组(19.54±6.04 ng／ml)血清Leptin浓度显著增高(q=4.39，P＜0.05)。(2)与Sham组相比，I60R60组、I60R150组和I60R240组Leptin mRNA表达水平无明显改变，I60R360组Leptin mRNA表达水平降低(辉度比值q=4.56，P＜0.05)。结论：大鼠70％肝脏缺血-再灌注损伤模型建立成功。Leptin作为一种炎性因子，参与了大鼠肝脏I／R损伤的病理生理改变过程。大鼠血清Leptin浓度在肝脏I／R损伤早期和中期呈升高趋势，但脂肪组织Leptin mRNA表达水平在损伤后期呈下降趋势。目的：为了获得重组人Leptin(rh-leptin)蛋白高表达的菌株及大量具有生物活性的rh-leptin蛋白，构建Leptin重组表达载体，筛选阳性质粒，转化感受态细菌进行表达，对rh-leptin蛋白进行复性及纯化，并鉴定其免疫学及生物学活性。方法：提取脂肪组织RNA，通过RT-PCR方法获得用PCR方法扩增人Leptin的编码区，使产物与pGEM-T easy载体相连，转染DH5α。测序后，选出阳性克隆质粒扩增。将酶切后的leptin片段与pET-28a(+)载体连接，获得leptin的表达质粒。将重组表达质粒转入BL21(DE3)，测序结果显示Leptin编码区没有突变产生，用IPTG诱导leptin重组蛋白表达。表达产物进行SDS-PAGE电泳及Western印迹杂交进行免疫学分析。并通过Km小鼠减肥实验及胃肠推进率测定实验检验其生物学活性。结果：在包涵体蛋白22 kDa处有明显的新增蛋白带，其含量超过总蛋白的50％：经Western-blot证实为重组的人leptin蛋白。大量表达后，经蛋白复性和纯化，通过Km小鼠减肥实验及胃肠推进率测定实验证实重组表达的人Leptin蛋白具有生物学活性。结论：成功构建了rh-leptin的高表达菌株，建立了rh-leptin的纯化与复性方法，经验证表达产物具有免疫学及生物学活性，可供进一步基础及临床研究应用。
[Abstract]:Objective: to establish an Ischemia-reperfusion I / R injury model of rat liver. To investigate the changes of serum Leptin protein and Leptin mRNA level in adipose tissue during liver I / R injury in rats, and to explore the factors influencing the changes of serum Leptin level. And the influence of this change law on the body. Methods: 45 male Sprague-Dawley rats weighing about 220 g were randomly divided into 5 groups. The first group was sham-operated group and the second group was liver ischemia for 60 minutes. The third group was I60R150 group, 4th group was I60R240 group and 5th group was I60R360 group. The hepatic artery, portal vein and bile duct were clamped in the branch of portal vein to the caudal lobe of liver, and the rat liver injury model of 70I / R was established. The abdominal cavity was closed. After ischemia for 60 minutes, the artery clamp was taken out for reperfusion. The serum Leptin concentration was measured by Leptin radioimmunoassay (RIA). The expression of Leptin mRNA in adipose tissue was detected by reverse transcriptase polymerase chain reaction (RT PCR). Results (1) compared with Sham group (12.01 卤2.73 ng / ml). The serum Leptin levels in I60R60 group (17.77 卤5.61 ng / ml) and I60R150 group (17.26 卤6.13 ng / ml) were increased. Serum Leptin concentration in I60R360 group (19.54 卤6.04 ng / ml) was significantly higher than that in Sham group (P < 0.05). There was no significant change in Leptin mRNA expression in I60R60 group and I60R240 group. The expression of Leptin mRNA in I60R360 group was decreased (P < 0.05). Conclusion: rat model of 70% hepatic ischemia-reperfusion injury was established successfully. Leptin as an inflammatory factor. The level of serum Leptin increased in the early and middle stages of liver I / R injury. However, the expression of Leptin mRNA in adipose tissue decreased at the late stage of injury. Objective: to obtain a high expression strain of recombinant human Leptin rh-leptin protein and a large number of bioactive rh-leptin proteins. The recombinant expression vector of Leptin was constructed, the positive plasmid was screened, the rh-leptin protein was renatured and purified, and its immunological and biological activity was identified. Methods: RT-PCR was used to amplify the coding region of human Leptin by RT-PCR, and the product was linked to pGEM-T easy vector. DH5 伪 was transfected. After sequencing, the positive clone plasmid was amplified. The leptin fragment was ligated with pET-28a () vector. The expression plasmid of leptin was obtained. The recombinant expression plasmid was transformed into BL21DE-3. The sequencing results showed that there was no mutation in the coding region of Leptin. The expression of leptin recombinant protein was induced by IPTG. The expression product was analyzed by SDS-PAGE electrophoresis and Western blotting. Its biological activity was tested by propulsive rate test. Results: there were significant new protein bands at the site of inclusion body protein 22 kDa. Its content is more than 50% of the total protein: confirmed by Western-blot as a recombinant human leptin protein. After a large number of expression, protein renaturation and purification. The biological activity of recombinant human Leptin protein was confirmed by weight loss test and gastrointestinal propulsive rate test in km mice. Conclusion: the high expression strain of rh-leptin was successfully constructed, and the purification and renaturation method of rh-leptin was established. The expressed product was proved to have immunological and biological activity. It can be used for further basic and clinical research.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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