摘要
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。
BACKGROUND： With the development and application of molecular biology and molecular genetic techniques, people research the production of human insulin by means of genetic engineering, and consider from the molecular level to reconstruct the cloning cell line which excretes insulin, so as to replace insulin injection and islet transplantation to treat diabetes mellitus.
OBJECTIVE： To construct and screen human insulin gene eukaryon expression carrier of high performance.
DESIGN： A randomized control experiment.
SETTING： Experimental Animal Department of China Medical University.
MATERIALS： Forty healthy adult Kunming mice of clean degree, weighing 20-30 g, half males and half females, were provided by the experimental animal center of Munitions University of Chinese PLA, and they. were free to the access of the artificial granule food and water, and all the mice were lighted for 14 hours every day. Escherichia coli DH5α and BHK cell line were preserved by the experimental animal center.
METHODS： Recombinant plasmid pEF1α-ImINS was constructed with routine method. The baby hamster kidney （BHK） cells were transfected with recombinant plasmid pEF1α-ImINS and other 3 recombinant plasmids of insulin, then screened with G418, and the positive clone ceils were passaged to the 20^th generation, the expressions of insulin and/or proinsulin in BHK cells were detected with radioimmunoassay and immunohistochemical technique. The PBS suspension （5×10^7） and the supernatant （0.5 mL） of the 20^th generation BHK positive cells trasnfected with pEF1α-ImINS were injected intraperitoneally to each mice, the blood glucose was detected before and after injection to analyze the biological effect of the transgeneic product in decreasing blood glucose.
MAIN OUTCOME MEASURES： The integration and expression of insulin gene in BHK cells and the changes of blood glucose before and after injection were mainly observed.
RESULTS： The highest insulin expression was 7.984 mIU/L, the gray value of in
出处
《中国临床康复》
CSCD
北大核心
2006年第40期178-180,共3页
Chinese Journal of Clinical Rehabilitation
基金
吉林省科委基金项目(98579)~~
