全新的杂交胰岛β细胞系的建立及其移植治疗糖尿病的实验研究

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.

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。

我国是世界上糖尿病患病人数最多的国家。目前对糖尿病的治疗方法有限，且效果均不够理想，尤其是1 型糖尿病的治疗，仍需依靠终生注射胰岛素。临床沿用的胰岛素替代治疗难以保持糖代谢的稳定，不能有效地阻止慢性血管病变的发展，且有发生低血糖的风险。目前，胰岛移植仍是治疗1型糖尿病最有潜力的方法。然而胰岛移植仍然面临着一些问题和挑战：如成人胰腺供者数量有限，大规模胰岛细胞分离纯化技术极其复杂，费用昂贵；而采用异种胰岛移植可能导致感染性病原体在人类的传播和扩散以及目前尚难以解决的跨越种族间免疫排斥等。其中最主要的障碍是人供胰短缺，已严重地制约了胰岛移植的广泛开展。利用现代分子生物学和基因工程等技术建立可供移植并分泌胰岛素的细胞或细胞系是解决目前供胰短缺最有前景的策略和途径。本项研究首次尝试整合最新的基因编辑技术及细胞电融合技术构建可供移植并分泌胰岛素的细胞系。研究结果如下：a) 成功地建立了可逆性“永生化”大鼠肝细胞系；b) 明确mPdx1, mNgn3, mMafA 三转录因子不足以使SV40T介导的永生化肝细胞向胰岛β样细胞转分化；c) 成功地建立永生化肝细胞与胰岛β细胞电融合而成的杂交四倍体细胞NIH-β，具有一定胰岛素分泌功能；d) mPdx1, mNgn3,mMafA三转录因子不足以使NIH-β细胞进一步分化为功能更强的胰岛素分泌细胞。 综上，本项目研究结果提示：(1) mPdx1, mNgn3, mMafA 三转录因子的转分化作用不适用于SV40T介导的永生化肝细胞；(2) 永生化肝细胞可与胰岛β细胞融合形成可增殖的四倍体细胞，并成胰岛样生长，但最重要的胰岛素分泌功能却逐渐降低，有待今后进一步研究。