·糖尿病分子生物学与遗传学·

　　陈存仁 刘艳霞 胡萍 欧阳军 周菲栗 夏莲

　　作者单位：450052 郑州大学第一附属医院内分泌科(第一作者现在海南省人民医院内分泌科工作)

　　通讯作者：栗夏莲，

　　E-mail: xialianli@hotmail.com

　　基金项目： 河南省医学高新技术发展和扶持项目(2006003);河南省医学科技攻关项目(200803034)

　　【摘要】 目的 构建可随ATP浓度变化而在内质网与葡萄糖反应蛋白78(GRP78)可逆性结合与解聚的人胰岛素原基因表达载体,并研究其在HepG2细胞中的表达及葡萄糖对其分泌的影响。 方法 人工合成含胰岛素信号肽(SS)及条件结合/解聚区域(CAD)的多聚核苷酸链，将其与人胰岛素原基因相连，构建SS-CAD-人胰岛素原表达载体(pSS-CAD-Proins)，将其经脂质体转染HepG2细胞，于不同浓度葡萄糖的培养液中培养, 检测培养液中的胰岛素浓度并提取细胞总RNA。 结果 成功构建pSS-CAD-Proins表达质粒。转染HepG2细胞后48 h，在葡萄糖浓度分别为5、15及25 mmol/L的培养基中，胰岛素分泌量分别为(4.73±0.52)、(8.84±0.43)及(14.15±0.32) mU/L，不同葡萄糖浓度诱导的胰岛素mRNA表达量明显不同。 结论 pSS-CAD-Proins载体能够在HepG2细胞中成功表达,并且胰岛素的分泌及转录受葡萄糖的调控。

　　【关键词】胰岛素基因治疗;葡萄糖反应蛋白78;条件结合/解离区域;内质网

　　doi:10.3969/j.issn.1006-6187.2011.03.004

　　The construction and function identification of the pSS-CAD-Proins expression plasmid in HepG2 cells

　　CHEN Cun-ren, LIU Yan-xia, HU ping, et al. Department of Endocrinology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052,China

　　Corresponding author: LI Xia-lian, E-mail: xialianli@hotmail.com

　　【Abstract】 Objective To construct and identify the pSS-CAD-Proins expression plasmid which can integrate and dissociate with GRP78 in the endoplasmic reticulum in ATP dependent way. Methods A fragment of oligonucleotide carrying insulin signal sequence (SS) and conditional aggregation domain (CAD) was synthesized and cloned into the pEGFP-N1 expression plasmid which was connected by human mutated proinsulin gene with furin site . The plasmid was transfected into HepG2 cells by lipofectamine. The insulin concentration in supernatant of cells cultured in different glucose medium was assayed by RIA. The expression levels of recombinant insulin mRNA at three glucose concentrations were determined by RT-PCR. Results The expression plasmid of pSS-CAD-Proins was successfully constructed. At the glucose concentration of 5.00 mmol/L, 15.00 mmol/L and 25.00 mmol/L, the amount of secreted insulin was (4.73±0.52) mU/L, (8.84±0.43) mU/L and (14.15±0.32) mU/L separately (n=3).The mRNA expression of insulin gene was positively correlated to glucose concentration. Conclusions The plasmid vector carrying glucose-regulating human proinsulin gene is successfully constructed and transfected in HepG2 cells, and the secretion and expression of insulin are regulated by glucose concentration.

　　【Key words】Insulin gene therapy; Glucose-regulated protein 78; Conditional binding domain / conditional aggregation domain; Endoplasmic reticulum