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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-CADProins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。
OBJECTIVE: To construct human proinsulin gene expression vector that reversibly binds to and depolymerizes with endoplasmic reticulum (ER) and glucose responsive protein 78 (GRP78) with the change of ATP concentration, and its expression in HepG2 cells and the effect of glucose on its secretion . Methods The polynucleotide sequence of insulin signal peptide (SS) and conditional binding / disassembly region (CAD) was synthesized and ligated with human proinsulin gene to construct SS-CAD-proinsulin expression vector (pSS- CAD-Proins). The recombinant plasmid was transfected into HepG2 cells by lipofectamine. The cells were cultured in different concentrations of glucose. Insulin concentrations in the medium were measured and total cellular RNA was extracted. Results The pSS-CADProins expression plasmid was successfully constructed. At 48 h after transfection, the insulin secretion of HepG2 cells were (4.73 ± 0.52), (8.84 ± 0.43) and (14.15 ± 0.32) mU / L in medium with glucose concentrations of 5, 15 and 25 mmol / L, different glucose concentrations induced significantly different levels of insulin mRNA expression. Conclusion pSS-CAD-Proins vector can be successfully expressed in HepG2 cells, and insulin secretion and transcription are regulated by glucose.