Center for Molecular Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, 310013, Zhejiang Province, People's Republic of China.
Cytotechnology. 2014 Aug;66(4):687-97. doi: 10.1007/s10616-013-9622-y. Epub 2013 Aug 27.
Diabetes, a disease resulting from loss of functional β cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating β cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic β cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic β cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic β cell proliferation model was further validated by a proliferation assay using differentiated pancreatic β cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic β cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic β cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.
糖尿病是一种由于功能性β细胞丧失而导致的疾病，在全球范围内，这种疾病的发病率正日益增高。基于导管上皮细胞的胰岛分化能力和 Reg Iα 基因刺激β细胞增殖的能力，我们旨在建立一种体外胰腺β细胞增殖模型，用于未来筛选糖尿病的治疗药物。从雄性 Wistar 大鼠中分离胰腺导管上皮细胞，并诱导其分化为胰腺β细胞。采用免疫荧光染色、Western blot、RT-PCR 分析和二噻嗪染色来鉴定细胞。通过转染 PCMV6-entry-REG Ia 质粒，用瞬时转染方法在体外表达大鼠 Reg Iα 蛋白，并用 RT-PCR 分析、增殖试验和凋亡试验来验证表达情况。使用转染上清液处理分化的胰腺β细胞进行增殖试验，进一步验证胰腺β细胞增殖模型。最后，我们成功地建立了一种使用瞬时表达的大鼠 Reg Iα 蛋白和从胰腺导管上皮细胞分化而来的胰腺β细胞的体外胰腺β细胞增殖模型。该模型可作为筛选新的胰岛新生药物来治疗糖尿病的平台，尤其是未来的中药。
