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目的:探讨细胞因子和诱导型一氧化氮合酶(iN-OS)对体外培养的大鼠胰岛细胞凋亡和功能的影响及其机制。方法:大鼠胰岛细胞体外培养,随机分组,培养液中分别加入细胞因子IL-1β、TNF-α和/或iNOS抑制剂氨基胍,分为空白对照组、细胞因子组、氨基胍组、氨基胍+细胞因子组。检测指标包括:培养液NO水平和组织iNOS、结构型NOS活性,RT-PCR检测胰岛组织中iNOS基因和凋亡相关基因Bax、Bcl-2的表达情况,AO/EB染色检测胰岛存活,胰岛素释放实验检测胰岛功能。结果:与细胞因子IL-1β和TNF-α共同培养后,大鼠胰岛组织中iNOS的表达增强,活性显著提高(38.93±4.72)U/mL,NO水平上升(313.0±35.4)mol/L,而结构型NOS没有变化;同时胰岛促凋亡基因表达上调,抗凋亡基因表达下降,细胞的存活率下降,胰岛素分泌大大减少。加入氨基胍后,随着胰岛组织中iNOS的活性明显受到抑制,胰岛的凋亡程度减轻,存活和胰岛素分泌情况都明显改善。结论:iNOS在细胞因子诱导的胰岛细胞凋亡中起到十分关键的作用,而氨基胍通过抑制iNOS活性,减轻了细胞因子的损害,降低了胰岛凋亡水平,改善胰岛的存活与功能。
Objective: To investigate the effects and mechanisms of cytokines and inducible nitric oxide synthase (iN-OS) on the apoptosis and function of rat islet cells cultured in vitro. Methods: Rat pancreatic islet cells were cultured in vitro and randomly divided into four groups: control group, cytokine group, aminoguanidine group, and aminoguanidine group, respectively. The cytokines IL-1β, TNF-α and / or iNOS inhibitor aminoguanidine were added into the culture medium. Guanidine + cytokine group. The detection indexes included the level of nitric oxide, the activity of iNOS and the activity of constitutive NOS in culture medium, the expression of iNOS gene and apoptosis related genes Bax and Bcl-2 in pancreatic islets by RT-PCR, the survival of islets and the release of insulin Experiment test islet function. RESULTS: After co-cultured with IL-1β and TNF-α, the expression of iNOS in pancreatic islets increased significantly (38.93 ± 4.72 U / mL) and NO level increased (313.0 ± 35.4) mol / L, While the structural NOS did not change; at the same time, the expression of pro-apoptotic genes was up-regulated, the expression of anti-apoptotic genes was decreased, the survival rate of cells was decreased, and the insulin secretion was greatly reduced. With the addition of aminoguanidine, the iNOS activity in pancreatic islets was significantly inhibited, and the degree of apoptosis in islets was reduced. The survival and insulin secretion were significantly improved. CONCLUSION: iNOS plays a crucial role in the cytokine-induced apoptosis of islet cells. However, aminoguanidine can reduce the damage of cytokines, reduce the apoptosis of pancreatic islets and improve the survival and function of islets by inhibiting iNOS activity.