【摘要】 目的构建稳定表达人胰岛素原突变(mutated human proinsulin,mhPINS)基因的HepG2细胞,将HepG2细胞改建为具有胰岛素分泌能力的"胰岛代理细胞"(artificial beta cell)。方法重组逆转录病毒载体pL-mhPINS-SN转包装细胞PA317,经G418筛选,NIH3T3细胞测定病毒滴度,获取高滴度的稳定产毒细胞克隆,以PCR进行鉴定。以mhPINS病毒感染HepG2细胞,经G418筛选,对HepG2/mhPINS细胞进行RT-PCR、Western blot、放免法鉴定,并行葡萄糖刺激的胰岛素分泌检测。结果所获HepG2/mhPINS细胞生长良好,RT-PCR获得286bp目的条带,Western blot检测可见34×103的特异性条带,放免法在其培养上清中检测到胰岛素与C肽,而HepG2/pLXSN细胞上述各项检测均为阴性。葡萄糖刺激的胰岛素分泌检测显示,HepG2/mhPINS细胞在不同葡萄糖浓度(0.1、10、20mmol/L)条件下引起的胰岛素分泌无显著性差异(P>0.05)。结论成功构建稳定表达mhPINS基因的HepG2细胞,其内获得成熟胰岛素的表达与分泌,但其胰岛素分泌对葡萄糖刺激缺乏反应。更多还原

【Abstract】 Objective To construct an "artificial beta cell" that possesses mature insulin secretion from a human hepatoma cell line HepG2.Methods The recombinant retroviral vector pL-mhPINS-SN was transfected into PA317 packaging cells and selected with G418 selective medium.Virus titer was detected with NIH3T3 cells,and PA317/mhPINS cells were assessed with PCR.HepG2 cells were infected by mhPINS virus and screened with G418.HepG2/mhPINS cells were tested with RT-PCR,Western blot and radioimmunoassay,respectively.The insulin secretion stimulated by glucose in HepG2/mhPINS cells was also detected.Results HepG2/mhPINS cells grew well.The target fragment of 286 bp was acquired in RT-PCR.A special strap of 34×103 was exhibited in Western blot.Insulin and C peptide were detected in the culture supernatant of HepG2/mhPINS cells by radioimmunoassay.The results of HepG2/pLXSN cells in the detections above were all negative.As shown in detection of glucose-stimulating insulin secretion in HepG2/mhPINS cells,there were no significant differences in the insulin secretion at 0.1,10,20 mmol/L glucose concentration(P>0.05).Conclusion HepG2 cells expressing mhPINS gene were successfully constructed,and the mature insulin secretion was made in HepG2/mhPINS cells.更多还原