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目的研究在人类胰岛nephrin的表达及糖尿病是否调控nephrin的表达,同时初步探索nephrin在β细胞中的功能。方法试验分糖尿病患者(n=5)和非糖尿病患者(n=7)组。采用western blot、PCR、共聚焦显微镜、亚细胞分离和免疫金标等方法研究人类胰岛和MIN6胰岛素瘤细胞中nephrin的表达。在MIN-6细胞或人类胰岛细胞中稳定转染nephrin和以siRNA技术沉默nephrin,然后研究体外nephrin的功能。采用GFP-nephrin转染细胞活动图像来研究nephrin的胞吞作用。结果 nephrin表达于细胞膜和胰岛素小囊泡中。糖尿病患者胰岛中nephrin表达低于非糖尿病患者组。转染nephrin的MIN-6细胞/假胰岛细胞进行体外实验,在葡萄糖刺激下胰岛素分泌更多,而基因沉默组不能引起胰岛素分泌。激光共聚焦图像显示GFP-nephrin转染细胞在葡萄糖刺激下,nephrin内吞。如果稳定肌动蛋白,可以防止nephrin迁移也可以防止nephrin对胰岛素释放中的正向效应。结论 nephrin是胰岛素囊泡中的一个重要活性组分,可以影响囊泡和肌动蛋白的相互作用从而促使囊泡迁移至细胞膜。以nephrin作为靶目标可以作为治疗糖尿病的一种新策略。
Objective To study the expression of nephrin in human islets and the regulation of nephrin in diabetes mellitus, and to explore the function of nephrin in β cells. Methods Patients with diabetes mellitus (n = 5) and non-diabetic patients (n = 7) were included. The expression of nephrin in human islets and MIN6 insulinoma cells was studied by western blot, PCR, confocal microscopy, subcellular separation and immunogold labeling. Stably transfecting nephrin in MIN-6 cells or human islet cells and silencing of nephrin with siRNA techniques were performed to investigate the function of nephrin in vitro. GFP-nephrin transfected cells were used to study nephrin endocytosis. Results nephrin was expressed in the cell membrane and in the insulin vesicles. Nephrin expression in the islets of diabetic patients was lower than in non-diabetic patients. In vitro experiments in nephrin-transfected MIN-6 cells / pseudoumpathetic islets revealed more insulin secretion under glucose stimulation, whereas gene silencing did not induce insulin secretion. Laser confocal images showed that nephrin was endocytosed by glucose-stimulated GFP-nephrin transfected cells. If actin is stabilized, it prevents the migration of nephrin and prevents the nephrin’s positive effect on insulin release. Conclusion Nephrin is an important active component in insulin vesicles and can affect the interaction between vesicles and actin to promote the migration of vesicles to the cell membrane. Using nephrin as a target can be a new strategy for the treatment of diabetes.