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RNA干扰特异性阻断胰岛局部肾素-血管紧张素系统对胰升血糖素分泌功能的影响

来自:中国糖尿病杂志  编辑: 刘艳清 张珍 刘春燕 易秋艳 卢斌 邵|点击数:|2014-11-24

 糖尿病基础研究

  【摘要】目的 采用RNA干扰技术特异性阻断胰岛局部ATⅡ1型受体(AT1R)表达,观察其对小鼠离体胰岛胰升血糖素分泌功能的影响。 方法 分离培养db/db和db/m小鼠的胰岛细胞,采用RT-PCR、Western blot检测AT1R表达。构建小鼠AT1R RNA干扰基因重组腺病毒(Ad-siAT1R),将分离培养的db/db小鼠胰岛细胞分为Ad-siAT1R感染胰岛细胞组(Ad-siAT1R)组、空病毒感染胰岛细胞(Ad-siControl)组和空白对照(Mock)组,培养48 h后检测AT1R表达,利用胰岛灌流系统检测胰岛素和胰升血糖素动态分泌。 结果 db/db小鼠胰岛AT1R mRNA和蛋白的表达水平分别为db/m小鼠胰岛的3.2、3.1倍。腺病毒感染后,Ad-siAT1R组胰岛AT1R mRNA表达水平较Ad-siControl组下降70%~80%,蛋白表达水平下降60%~70%(P<0.05)。胰岛灌流显示,Ad-siAT1R组在高糖刺激1~2 min后胰岛素分泌达峰值140 mU/L,胰升血糖素分泌立即出现下降,达14 pmol/L,较基础值下降13 pmol/L。Ad-siControl组在高糖刺激1~2 min时胰岛素分泌也出现一定程度升高,峰值仅为90 mU/L,为基础值的1.8倍,其胰升血糖素分泌逐渐降至35 pmol/L,仅较基础值下降5 pmol/L,提示Ad-siAT1R组胰岛素第一时相分泌和胰升血糖素过度分泌情况均改善。 结论 RNA干扰特异性阻断胰岛局部RAS可减轻胰升血糖素过度分泌。

  【关键词】 RNA干扰;肾素-血管紧张素系统;血管紧张素Ⅱ1型受体;胰升血糖素

  【Abstract】Objective To investigate the effects of intra-islet inhibition of ATⅡtype 1 receptor (AT1R) expression through RNA interference strategy on glucagon secretory function of islets in vitro in db/db mice. Methods Islets of db/db and db/m mice were isolated and cultured. The expression of AT1R in islets was detected by RT-PCR and Western blot. To further evaluate the role of AT1R in α cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to AT1R (Ad-siAT1R). Isolated islets of db/db mice were divided into Ad-siAT1R group, Ad-siControl group and Mock group. Islets were cultured for 48 h. AT1R mRNA and protein levels were measured for each condition and islet perifusion was performed to evaluate kinetics of insulin and glucagon release in vitro. Results The expression level of AT1R mRNA and protein in isolated islets of db/db mice were 3.2 times and 3.1 times of db/m mice, respectively (P<0.05). After virus transduced, Ad-siAT1R treatment resulted in 70%~80% decrease in AT1R mRNA levels and 60%~70% decrease in AT1R protein compared with islets treated with Ad-siControl (P<0.05). In addition, the insulin secretion of Ad-siAT1R group immediately increased to the peak level of 140 mU/L at 1~2 min after administration of 16.7 mmol/L glucose. And its glucagon secretion immediately decreased to 14 pmol/L, lower 13 pmol/L than the basal level. There was also a little degree of rise in insulin secretion of Ad-siControl group at 1~2 min after administration of 16.7 mmol/L glucose, its peak only 1.8 times the height of the basal level. And its glucagon secretion slowly decreased to 35 pmol/L, only lower 5 pmol/L than the basal level. These suggested that the first-phase insulin secretion and excessive glucagon secretion of Ad-siAT1R group were markedly improved. Conclusion The siRNA specific to AT1R can ameliorate glucagon secretory function in islets of db/db mice.

  【Key words】RNA interference;Rennin-angiotensin system (RAS);AngiotensinⅡtype 1 receptor (AT1R);Glucagon

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