论文部分内容阅读
背景:由于胰岛细胞分离技术难度较大,目前糖尿病细胞移植治疗尚缺乏理想的细胞来源。目的:构建具有生理性胰岛素分泌能力胰岛代理细胞系,为糖尿病基因治疗提供有效手段。设计:随机对照的实验研究。地点、材料和干预:本研究在第三军医大学附属新桥医院中心实验室进行。以携葡萄糖激酶(glucokinase,GK)基因的逆转录病毒及携人胰岛素原基因突变体(mutantproinsulingene,MPIN)的逆转录病毒共同感染人肝癌细胞系HepG2细胞,G418筛选,放免法、Western-blotting及RT-PCR鉴定;挑选联合表达GK及成熟胰岛素的HepG2细胞进行葡萄糖反应性胰岛素分泌反应的检测,以单独表达成熟胰岛素的HepG2细胞作对照。主要观察指标:不同葡萄糖浓度条件下,联合表达GK及成熟胰岛素的HepG2细胞培养上清中的胰岛素浓度。结果:G418筛选3周获阳性细胞克隆,放免法、Western-blotting挑选出两个目的基因表达均较强的HepG2细胞1株,命名为细胞克隆“β”;RT-PCR证实细胞“β”中已有两个目的基因的转录;在细胞“β”中获得葡萄糖反应性胰岛素分泌,葡萄糖浓度约1.75～2.00mmol/L时出现胰岛素分泌高峰;而在单独表达成熟胰岛素的HepG2细胞中,各种葡萄糖浓度引起的胰岛素分泌差异无显著性意义(P>0.05)。结论:成功构建了具有生理性胰岛素分泌能力的?
BACKGROUND: Due to the difficulty of pancreatic islet cell isolation technology, the ideal cell source is still lacking in current diabetic cell transplantation. OBJECTIVE: To construct islet cell line with physiological insulin secretion capacity and to provide an effective method for gene therapy of diabetes. Design: Randomized controlled experimental study. Location, Materials and Interventions: This study was conducted at the Xinqiao Hospital Central Laboratory Affiliated to the Third Military Medical University. HepG2 cells were co-infected with retroviruses carrying glucokinase (GK) gene and retroviral vectors carrying human proinsulin gene (MPIN), G418 screening, radioimmunoassay, Western-blotting and The HepG2 cells co-expressing GK and mature insulin were selected for detection of glucose-responsive insulin secretion. HepG2 cells expressing mature insulin alone were used as controls. MAIN OUTCOME MEASURES: Insulin concentrations in culture supernatants of HepG2 cells co-expressing GK and mature insulin at different glucose concentrations. RESULTS: Positive clones were screened by G418 for 3 weeks. One of the two HepG2 cells with strong gene expression was selected by Western-blotting and named as “β”. RT-PCR confirmed Two target genes have been transcribed; Glucose-responsive insulin secretion is obtained in cell “β”, and peak insulin secretion occurs at a glucose concentration of about 1.75-2.00 mmol / L; in HepG2 cells expressing mature insulin alone, various There was no significant difference in insulin secretion caused by glucose concentration (P> 0.05). Conclusion: The successful construction of a physiological ability of insulin secretion?