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背景:随着分子生物学和分子遗传学技术的发展和应用,人们利用基因工程方法进行人胰岛素的生产研究,并从分子水平再造分泌胰岛素的克隆细胞系来替代胰岛素注射和胰岛移植治疗糖尿病。目的:构建并筛选人胰岛素基因真核高效表达载体。设计:随机对照实验研究。单位:中国医科大学实验动物部。材料:实验选取健康成年清洁级昆明小鼠40只,雌雄各半,自由饮水,自由采食人工颗粒料,每日光照14h,实验动物由长春解放军军需大学实验动物中心提供。大肠肝菌DH5α和幼仓鼠细胞系由实验动物中心保存。方法:常规法构建重组质粒pEF1α-ImINS。将重组质粒pEF1α-ImINS和胰岛素其他3个重组质粒分别转染幼仓鼠肾细胞,经G418筛选,阳性克隆传至20代,分别用放射免疫和免疫组织化学技术方法分析胰岛素和/或胰岛素原在幼仓鼠肾细胞中表达的情况。并用重组质粒pEF1α-ImINS转染幼仓鼠肾细胞的阳性克隆20代的PBS细胞悬浮液(5×107细胞/只)及其培养上清(0.5mL/只)对昆明小鼠行腹腔注射,通过对注射前后小鼠血糖值测定,分析转基因产物降血糖的生物学效应。主要观察指标:胰岛素基因在幼仓鼠肾细胞中整合和表达的结果,以及注射前后血糖的变化。结果:胰岛素的表达量最高为7.984mIU/L,胰岛素表达水平的灰度值在幼仓鼠肾细胞质中为177.50±15.10,细胞核中为150.30±21.43;昆明小鼠注射pEF1α-ImINS转染幼仓鼠肾克隆细胞株的悬浮液后6h血糖值明显下降,与注射前24h比较,差异有极显著性意义(P<0.01)。结论:幼仓鼠肾细胞中重组质粒pEF1α-ImINS是胰岛素表达量最高的质粒;pEF1α-ImINS转染的克隆细胞株在小鼠体内有胰岛素表达,并且对正常小鼠有降血糖的作用。
Abstract BACKGROUND: With the development and application of molecular biology and molecular genetics technology, human genetic engineering has been used to study the production of human insulin and to rebuild insulin-producing clonal cell lines at the molecular level to replace insulin injections and islet transplantation for the treatment of diabetes. Objective: To construct and screen eukaryotic expression vector of human insulin gene. Design: Randomized controlled trial. Unit: Experimental Animal Department of China Medical University. MATERIALS: Forty Kunming mice of healthy adult clean grade were selected from the experiment. They were divided into male and female respectively. They were free to drink water and were allowed to eat artificial pellets for 14 hours. The experimental animals were provided by Experimental Animal Center of Changchun Military Liberation Army in Changchun. E. coli DH5α and baby hamster cell lines were kept in laboratory animals. Methods: The recombinant plasmid pEF1α-ImINS was constructed by routine method. The recombinant plasmid pEF1α-ImINS and insulin three other recombinant plasmids were transfected into baby hamster kidney cells, screening by G418, the positive clones were transmitted to 20 generations, respectively, by radioimmunoassay and immunohistochemical analysis of insulin and / or proinsulin Expression in baby hamster kidney cells. Kunming mice were intraperitoneally injected with PBS suspension (5 × 10 7 cells / mouse) and their culture supernatant (0.5 mL / mouse) of the positive clones of the hamster kidney cells transfected with the recombinant plasmid pEF1α-ImINS for 20 generations The blood glucose level of mice before and after injection was measured to analyze the biological effect of the hypoglycemic products of the transgenic products. MAIN OUTCOME MEASURES: The results of the integration and expression of insulin genes in baby hamster kidney cells, and changes in blood glucose before and after injection. Results: The highest expression of insulin was 7.984 mIU / L, the gray value of insulin expression was 177.50 ± 15.10 in the cytoplasm of baby hamster and 150.30 ± 21.43 in the nucleus; Kunming mice were injected with pEF1α-ImINS transfected baby hamster kidney The blood glucose level decreased 6h after the suspension of cloned cell line, and the difference was significant (P <0.01) compared with that before 24h. CONCLUSION: The recombinant plasmid pEF1α-ImINS is the highest expression plasmid in baby hamster kidney cells. The pEF1α-ImINS transfected clone has insulin expression in mice and hypoglycemic effect on normal mice.