在实验室建立可供移植的分泌胰岛素细胞是解决临床中胰岛移植供胰短缺最有效的途径。课题组前期采用电融合技术成功地将可复性表达SV40T和hTERT基因的永生化大鼠肝细胞与胰岛β细胞融合，筛选获得了杂交胰岛β细胞克隆。这些细胞虽能适时有效地分泌胰岛素，但细胞内胰岛素含量及对葡萄糖刺激胰岛素分泌量明显低于正常胰岛β细胞。为了克服和弥补上述不足，本研究在前期工作基础上，拟将Pdx1/Ngn3/MafA三种基因转染至永生化肝细胞中，促进其转分化成胰岛素分泌细胞，将其与胰岛β细胞融合，筛选获得杂交胰岛β细胞克隆，再将Nkx6.1基因转染至杂交胰岛β细胞中，经体内外功能鉴定，筛选获得全新的增殖活性可控、功能更加高效的杂交胰岛β细胞系。用Cre-recombinase处理细胞，SV40T和hTERT基因可被切除而成为回复性杂交胰岛β细胞，移植上述细胞治疗糖尿病小鼠，全面评价杂交β细胞移植治疗效果。

Islet transplantation has been considered as an ideal method to cure type I diabetes, however, the shortage of donor organ is a major obstacle to limit the development of clinical islet transplantation. The establishment and development of insulin-secreting cells available for transplantation in the laboratory is the most effective strategy to solve the shortage of islets in clinical.Before,we have smoothly screened and achieved hybrid islet β cells fused by reversible immortal hepatocytes expressing SV40T and hTERT and islet β cells via the technology of cell electrofusion.These cells could appropriately secrete insulin effectively, however, the cells showed a feeble secretory output and glucose-stimulated insulin secretion comparing with the normal islet β cells.In order to overcome and compensate for these shortcomings,we further design to transfect Pdx1/Ngn3/MafA to the reversible immortal hepatocytes for producing the transdifferentiation of insulin-secreting cells.Next,the insulin-secreting cells will be electro-fused with islet β cells and the fused hybrid islet β cells will be screened and acquired. Then the Nkx6.1 cDNA of promoting proliferation and glucose-stimulated insulin secretion is transfected to the hybrid islet β cells with the technology of gene transfection. So a completely new hybrid islet β cell line with more insulin secretory output, better glucose-stimulated insulin secretion and proliferation will be screened and established by means of a seires of vivo and vitro assays. As the Cre/LoxP site was located on the two flanks of the gene of SV40T and hTERT, the gene of SV40T and hTERT will be knockout with Cre-recombinase, therefore, the reversible immortal hybrid islet β cells will be reversed. Subsequently,the reversed hybrid islet β cells will be transplanted to control diabetes in SCID mice induced by STZ and the efficacy of transplantation will be evaluated comprehensively.