Visfatin转基因对胰岛β细胞功能和活性的影响

Effects of Visfatin gene on activity and function of islet beta-cell

目的 探讨Visfatin基因过表达对胰岛β细胞增殖、凋亡、细胞周期和胰岛素释放的影响.方法 将MIN6细胞分为对照组、绿色荧光蛋白转染组和Visfafin转染组,对照组以空质粒、绿色荧光蛋白转染组以荧光蛋白质粒、Visfatin转染组分别以2、5、10 mg/L Visfatin质粒转染细胞,40、60 h后收集细胞,采用实时聚合酶链反应和Western blot法检测Visfatin mRNA和蛋白表达,应用噻唑蓝法及流式细胞仪检测细胞增殖、凋亡和周期,运用酶联免疫法检测葡萄糖刺激的胰岛素释放.采用单因素方差分析进行统计学分析.结果 与对照组相比,2、5、10 ms/L Visfatin质粒转染40 h及5 mg/L Visfatin质粒转染60 h后细胞增殖率分别升高1.60、1.87、1.75、1.51倍(t值分别为4.98、13.52、11.02、6.14,均P<0.05).棕榈酸诱导的细胞凋亡率降低,与对照组和绿色荧光蛋白转染组相比,质粒转染组细胞凋亡率分别降低42%、48%(F值分别为4.58、6.12,均P<0.05).与对照组相比,Visfatin质粒转染40 h后G_0/G_1期细胞减少12.3%,S期细胞增加13.2%(F值分别为5.25、6.23,均P<0.05).与对照组相比,质粒转染组细胞基础胰岛素分泌无改变,葡萄糖刺激后胰岛素释放有所增加,差异无统计学意义(P>0.05).结论 Visfatin可促进胰岛β细胞增殖,抑制胰岛β细胞凋亡,对细胞周期有调节作用.

Objective To explore the effects of Visfatin on islet beta-cell proliferation, apoptosis and insulin secretion. Methods MIN6 cells were divided into three groups:control group, GFP group, and Visfatin transfection group, which were transfected by empty plasmid, GFP plasmid and 2, 5, or 10 mg/L Visfatin plasmid respectively and collected at 40 or 60 h after transfection. Visfatin mRNA and protein expression were detected by real time PCR and Western blot. Cell proliferation, apoptosis and cell cycle were detected by MTT or flow cytometry, and glucose-stimulated insulin release was detected by ELISA. ANOVA test was used for data analysis. Results The proliferation of Visfatin transfected cells was significantly increased. Compared with the controls, proliferation rate of 2, 5, and 10 mg/L Visfatin plasmid at 40 h and 5 mg/L Visfatin plasmid at 60 h was increased by 1.60, 1.87, 1.75 and 1.51, respectively (t values were 4.98, 13.52, 11.02, and 6. 14; all P <0.05). Palmitate-induced islet cell apoptosis decreased. Compared with the controls and GPF group, the apoptosis rate of Visfatin transfected cell was 42% and 48% lower (F values were 4.58 and 6. 12; both P <0. 05). Compare with the controls, the percentage of cells at G_0/G_1 phase was decreased and S phase increased in Visfatin transfected cells (F values were 5.25 and 6.23 ; both P < 0.05 ). Compared with the controls, the basal insulin secretion was not changed. Glucose stimulated insulin release was increased in Visfatin transfected cells, although no significant difference was found (P>0.05). ConclusionVisfatin may promote islet beta-cell proliferation, inhibits cell apoptosis, and regulates the cell cycle.