小干扰质粒干扰肌腱膜纤维肉瘤癌基因同源物A对小鼠胰岛β细胞增殖及胰岛素基因表达的影响

摘要

Objective To investigate the effect of v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA)on the expression of insulin gene and the proliferation of the pancreatic β-cell of mouse. Methods The expression of MafA mRNA and protein respectively was detected by the method of real time PCR and Western blot in order to obtain the best effective small interfering siRNA(siRNA). The MIN6 cells in different concentration of glucose (5.6 mmol/L,10 mmol/L and 25 mmol/L were divided into the control group and the experimental group;The effect of siRNA interfering MafA mRNA on the proliferation of MIN6 cells,the expressions of insulin1 and insulin2 mRNA,the expression of MafA protein and the concentration of insulin in the supernatant were examined. Results The effect of interference in siMafA-2 group was the best. The expressions of MafA protein were significantly decreased in the C1 group compared with the C0 group(P<0.05), and the proliferation of MIN6 cells ,the expression of insulin2 mRNA, and the concentration of insulin in the supernatant were significantly lower(P<0.05).Compared with the A0 group, there was no significant difference in the expression of MafA protein in the A1 group(P>0.05),and the proliferation of MIN6 cells ,the expression of insulin2 mRNA and the concentration of insulin in the supernatant had no significant changes(P>0.05). The expression of insulin1 mRNA was not changed in the siRNA group compared with the control group in different concentrations of glucose(P>0.05). Conclusion The siRNA-2 in siRNA group is ideal. Down-regulation of MafA can inhibit the proliferation of pancreatic β-cells and inhibit the expression of insulin2 mRNA and secretion of insulin in mouse.%目的 探讨肌腱膜纤维肉瘤癌基因同源物A (MafA)对胰岛β细胞增殖及胰岛素基因表达的影响. 方法 采用RT-PCR和Western blot分别测MafA mRNA 和蛋白的表达,筛选最佳小干扰质粒(siRNA),分为转染对照组(Cy3组)、阴性对照组(Scramble组)、阳性对照组(siβ-actin组)及实验组(即siMafA-1、siMafA-2和siMafA-3组).按不同葡萄糖浓度分为5.6 mmol/L、10 mmol/L、25 mmol/L.根据不同葡萄糖浓度是否加入siRNA分为未加siRNA的对照组:A0组5.6 mmol/L、B0组10 mmol/L、C0组25 mmol/L;加入siRNA的实验组:A1组5.6 mmol/L、B1组10 mmol/L、C1组25 mmol/L.测MafA基因沉默后MIN6细胞增殖、胰岛素(Insulin)-1和Insulin-2 mRNA表达、MafA蛋白表达及上清液胰岛素浓度的变化. 结果 siMafA-2组干扰效果最好.C1组MafA蛋白表达较C0组下降(P<0.05);MIN6细胞增殖、Insulin-2 mRNA表达和上清液胰岛素浓度均降低(P<0.05).A1组与A0组比较,MafA蛋白表达比较,差异无统计学意义(P>0.05);MIN6细胞增殖、Insulin-2 mRNA表达和上清液胰岛素浓度比较,差异无统计学意义(P>0.05).不同糖浓度各亚组与对照组Insulin-1 mRNA表达比较,差异均无统计学意义(P>0.05). 结论 siRNA-2组是理想的siRNA.MafA下调可抑制小鼠胰岛β细胞增殖.MafA下调可抑制小鼠Insulin2 mRNA表达和胰岛素分泌.