细胞因子对大鼠胰岛细胞凋亡影响和机制的实验研究

Effect and mechanism of cytokines on the pancreatic islet cell apoptosis in rats

大鼠胰岛体外培养，根据是否加入细胞因子[肿瘤坏死因子仅（TNF—α）和白细胞介素1β（IL-1β）]及是否使用RNA干扰（RNAi）诱导型一氧化氮合酶（iNOS）基因，将胰岛分为5组：空白对照组（A），阴性RNAi对照组（B），RNAi组（C），RNAi＋细胞因子组（D）和细胞因子组（E）。检测胰岛iNOS表达、培养液NO浓度和组织iNOS活性、胰岛细胞凋亡和胰岛素分泌。E组组织iNOS活性和培养液NO水平显著升高，凋亡细胞明显增多，胰岛存活率降低，胰岛素分泌减少（P〈0．05或P〈0．01）。经RNAi沉默iNOS后，胰岛组织iNOS活性和培养液NO水平显著降低，凋亡细胞明显减少，胰岛功能均得到改善（P〈0．05或P〈0．01）。本研究提示，iNOS／NO通路是细胞因子IL-1β和TNF—α对胰岛损害的重要机制，是促使细胞凋亡，影响胰岛存活与功能的重要因素。

Islets obtained from 30 Wistar rats were divided into 5 groups according to the uses of cytokines and RNA interference （RNAi）. Islets cultured solely were taken as blank control （group A）, and cultured with interleukin-1β （IL-1β, 50 U/ml） and tumor necrosis factor-α （TNF-α, 250 U/ml） as group E. Islets transfected with negative control gene or inducible NO synthase （iNOS） small interfering RNA （siRNA） were taken as negative transfecion control （ group B ） or group C. Islets both transfected with iNOS siRNA and cytokines served as group D. Expressions of iNOS were evaluated by RT-PCR and Western-blot. NO level, tissue iNOS activity, islet cell apoptosis, and insulin secretion were also determined. 500-600 IEQ islets could be extracted from every rat by isolation and purification. In group E, the activity of iNOS in islets tissue and concentration of NO in medium increased remarkably, the apoptosis ceils were increased, and insulin secretion was decreased strikingly （P〈0.05 or P〈0.01 ）. After iNOS RNA interference, iNOS activity, NO levels, apoptosis status, and insulin secretion were all improved （P〈0. 05 or P〈0. 01 ）. The results suggest that the iNOS/NO pathway may mediate the damage from cytokines, which leads to islet cell apoptosis and islet dysfunction.