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目的通过应用基因转染及细胞融合技术构建永生化细胞,建立1种功能良好、无致瘤性、增殖可控的杂交胰岛β细胞系。方法通过脂质体介导逆转录病毒载体SSR69转染至原代成纤维细胞中,筛选稳定表达SV40 LTag的永生化细胞。提取大鼠胰岛,通过胰蛋白酶及DNA酶裂解成单胰岛细胞。对永生化成纤维细胞及单个胰岛β细胞进行电融合,通过有限稀释法获得杂交胰岛β细胞克隆。比较杂交及正常β细胞内胰岛素的含量及对葡萄糖刺激胰岛素释放反应。通过RT-PCR及Westernblot分析杂交胰岛β细胞中SV40 LTag、insulin、葡萄糖激酶(GK)及葡萄糖转运体-2(Glut-2)基因的表达。结果建立的永生化成纤维细胞系的增殖能力明显优于原代成纤维细胞(P<0.05)。成功获得杂交胰岛β细胞克隆,其细胞呈短梭形或扁圆形,且具有贴壁生长等特性。透射电子显微镜下可见细胞内胰岛素分泌颗粒,杂交与正常胰岛β细胞之间细胞内胰岛素含量、胰岛素释放指数及insulin、GK、Glut-2蛋白及mRNA表达水平无统计学差异(P>0.05),但2种细胞内insulin、GK、Glut-2的表达水平显著高于RIN-m5F细胞(P<0.05)。结论可通过永生化成纤维细胞及单个胰岛β细胞电融合诱导生成杂交胰岛β细胞,且杂交细胞能够对葡萄糖的变化产生应答反应。
Objective To construct immortalized cells by gene transfection and cell fusion technology and establish a hybrid islet β cell line with good function, non-tumorigenicity and proliferation control. METHODS: Primary fibroblasts were transfected with liposome-mediated retroviral vector SSR69 and immortalized cells stably expressing SV40 LTag were screened. Rat islets were isolated and lysed by trypsin and DNase into single islet cells. Immortalized fibroblasts and single islet β-cells were electrofused, and the hybrid islet β cell clones were obtained by limiting dilution. The content of insulin in hybridization and normal beta cells and the response to glucose stimulated insulin release were compared. The expression of SV40 LTag, insulin, GK and Glut-2 genes in hybrid islet β cells were analyzed by RT-PCR and Western blotting. Results The immortalized fibroblast cell line established was significantly superior to primary fibroblasts (P <0.05). Successfully obtained hybrid islet β cell clones, the cells were short spindle or oblate, and has the characteristics of adherent growth. There was no significant difference in intracellular insulin content, insulin release index, insulin, GK, Glut-2 protein and mRNA expression between the hybridization and normal pancreatic βcells under transmission electron microscope (TEM) However, the expressions of insulin, GK and Glut-2 in the two kinds of cells were significantly higher than those in RIN-m5F cells (P <0.05). Conclusion The hybrid islet β cells can be induced by immortalized fibroblasts and single islet β cell electrofusion, and the hybrid cells can respond to the changes of glucose.