本文關鍵詞： 缺血預處理 再灌注損傷 肝臟 Leptin 放射免疫分析法 逆轉錄-聚合酶鏈反應 rh-leptin 克隆 表達 融合蛋白 大腸桿菌 復性 蛋白純化 生物活性　出處：《中國人民解放軍軍醫(yī)進修學院》2007年碩士論文　論文類型：學位論文

【摘要】： 目的：建立大鼠肝臟缺血-再灌注(Ischemia-Reperfusion，I／R)損傷模型，探討大鼠肝臟I／R損傷時血清Leptin蛋白及脂肪組織Leptin mRNA水平的變化規(guī)律，探討影響血清Leptin水平變化的因素，以及此變化規(guī)律對機體的影響。 方法：將45只體重220g左右的雄性SD大鼠隨機分為5組，，第一組為假手術組(Sham組)，第二組為肝臟缺血60min、再灌注60min(160R60)組，第三組為I60R150組，第四組為I60R240組，第五組為I60R360組。麻醉后，在門靜脈分出發(fā)向肝臟尾葉的分支上，以動脈夾夾閉肝動脈、門靜脈及膽管，建立大鼠肝臟70％I／R損傷模型。動脈夾置入后，關閉腹腔。缺血60min后，取出動脈夾進行再灌注。采用高靈敏鼠Leptin放射免疫分析法測定血清Leptin濃度變化，并采用逆轉錄-聚合酶鏈反應(RT-PCR)方法檢測脂肪組織Leptin mRNA表達水平的變化。 結果：(1)與Sham組(12.01±2.73 ng／ml)相比，I60R60組(17.77±5.61 ng／ml)和I60R150組(17.26±6.13 ng／ml)血清Leptin濃度即出現(xiàn)增高趨勢，I60R360組(19.54±6.04 ng／ml)血清Leptin濃度顯著增高(q=4.39，P＜0.05)。(2)與Sham組相比，I60R60組、I60R150組和I60R240組Leptin mRNA表達水平無明顯改變，I60R360組Leptin mRNA表達水平降低(輝度比值q=4.56，P＜0.05)。 結論：大鼠70％肝臟缺血-再灌注損傷模型建立成功。Leptin作為一種炎性因子，參與了大鼠肝臟I／R損傷的病理生理改變過程。大鼠血清Leptin濃度在肝臟I／R損傷早期和中期呈升高趨勢，但脂肪組織Leptin mRNA表達水平在損傷后期呈下降趨勢。 目的：為了獲得重組人Leptin(rh-leptin)蛋白高表達的菌株及大量具有生物活性的rh-leptin蛋白，構建Leptin重組表達載體，篩選陽性質粒，轉化感受態(tài)細菌進行表達，對rh-leptin蛋白進行復性及純化，并鑒定其免疫學及生物學活性。 方法：提取脂肪組織RNA，通過RT-PCR方法獲得用PCR方法擴增人Leptin的編碼區(qū)，使產物與pGEM-T easy載體相連，轉染DH5α。測序后，選出陽性克隆質粒擴增。將酶切后的leptin片段與pET-28a(+)載體連接，獲得leptin的表達質粒。將重組表達質粒轉入BL21(DE3)，測序結果顯示Leptin編碼區(qū)沒有突變產生，用IPTG誘導leptin重組蛋白表達。表達產物進行SDS-PAGE電泳及Western印跡雜交進行免疫學分析。并通過Km小鼠減肥實驗及胃腸推進率測定實驗檢驗其生物學活性。 結果：在包涵體蛋白22 kDa處有明顯的新增蛋白帶，其含量超過總蛋白的50％：經Western-blot證實為重組的人leptin蛋白。大量表達后，經蛋白復性和純化，通過Km小鼠減肥實驗及胃腸推進率測定實驗證實重組表達的人Leptin蛋白具有生物學活性。 結論：成功構建了rh-leptin的高表達菌株，建立了rh-leptin的純化與復性方法，經驗證表達產物具有免疫學及生物學活性，可供進一步基礎及臨床研究應用。
[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 鹵2.73 ng / ml). The serum Leptin levels in I60R60 group (17.77 鹵5.61 ng / ml) and I60R150 group (17.26 鹵6.13 ng / ml) were increased. Serum Leptin concentration in I60R360 group (19.54 鹵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 偽 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.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363

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