【作者】 贾志敏；

【导师】 吴曙光；

【作者基本信息】 第一军医大学 ， 药理学， 2004， 博士

【摘要】 环孢酶素A(cyclosporin A，CsA)是一种强效的免疫抑制剂，广泛地应用于器官、细胞移植后排斥反应的防治以及自身性免疫疾病的治疗。但是，不少临床观察显示CsA有影响胰岛β细胞功能、破坏糖耐量甚至导致糖尿病的副作用。研究分析CsA对胰岛功能的影响及其作用机制，对于指导临床合理用药及防治CsA的副作用具有较重要的现实意义。 目的：通过体外实验，观察CsA对NIT-1胰岛β细胞胰岛素释放的影响；同时，应用DNA芯片技术检测CsA作用于NIT-1细胞不同时间的基因差异表达，并结合生物信息学方法从基因水平上探讨CsA对胰岛素分泌影响的作用机制。 方法：以转基因NOD小鼠来源的NIT-1胰岛β细胞作为细胞模型，用不同浓度的CsA(0.05、0.1、0.5、1、5、10 Mm／L)分别处理24h、48h和72h后，分别检测胰岛素的累积释放；应用含有4096个小鼠基因的cDNA芯片分别检测以10μM／L的CsA作用于NIT-1细胞24h及48h后基因的差异表达，并应用RT-PCR方法对出现差异表达的6个基因进行了检测，验证了芯片检测结果；结合生物信息学方法对差异表达基因进行分析，筛选出20个与CsA对NIT-1细胞作用机制密切相关的基因，并对其有关分子机制进行了探讨。 结果： 1、CsA对胰岛素的累计释放具有抑制作用，且其抑制作用具有一定的剂量依赖性和时效性； 2、应用DNA芯片检测，在CsA作用于NIT-1细胞24h及48h后，分别有84个基因(其中表达下调46个，表达上调38个)及710个基因(其中表达下调366个，表达上调344个)出现差异表达; 3、结合生物信息学方法，从差异表达基因中筛选出20个与CsA对N工T一1细胞作用机制密切相关的已知功能基因，它们都表达下调，其中包括氧化磷酸化过程相关基因(共15个，eox7al、Cox7e、Coxsa;Atp6voe、Atp6vlgl;Atplb3;AtP5k、Atpso;Mrps21、Mrpl23、MrP136、Mrp64、MrP13、MrPll3、论pl一9)、SNAI花蛋白有关基因(共2个，Stxbpl、vamp4)、CaZ‘靶蛋白有关基因(C aM kinasen一p亚单位类似蛋白)、G蛋白有关基因(Ar14因子)、Pax6基因等。我们对其参与CSA抑制胰岛素分泌的有关分子机制进行了分析、探讨。 结论:csA可抑制Nrr一1胰岛p细胞胰岛素的释放，其抑制作用可能与抑制细胞氧化磷酸化、胰岛素胞吐分泌等代谢一分泌偶联过程中有关功能性酶类或蛋白质mRNA的表达有关。更多还原

【Abstract】 Cycloporin A (CsA) is widely used for therapeutic purposes for rejection after organ and cell transplantations and autoimmune diseases as a kind of potent immunosurpressant. But recently, quite a lot of clinical observations revealed that CsA has some side-effects on pancreatic ?cells such as impaired glucose tolerance and can even lead to diabetes mellitus. So, to study and analyze the effects and mechanism of the effect of CsA on pancreatic ? cells is of great importance for the rational drug usage and prevention of the side-effects of CsA.Objective: To observe the impact of CsA on the insulin release of pancreatic ? cells. And to investigate the gene differential expression of NIT-1 cells treated by CsA at different time intervals. Then to explore the possible mechanism of the effects of CsA on insulin release combined with Bioinformatics.Methods: NIT-1 cells were exposed to CsA at various concentrations(0.05.. 0.1. 0.5. 1. 5.10 礛/L) at different time intervals(24h, 48h and 72h). And the insulin release was investigated. Differential gene expression was observed and analyzed using DNA microarrays after NIT-1 cells was treated with CsA (lOMm/L) for 24h and 48h respectively. The DNA microarray results were conformed by RT-PCR with 6 differentially expressed genes. We analyzed the differentially expressed genes by bioinformatic analysis and finally screened out 20 genes that have close relationships with CsA’s effects on NIT-1 cells, and explored their related molecular mechanisms.Results:1. CsA can inhibit insulin release, and its inhibitory effects are concentration-dependent and time-dependent2. By DNA microarray hibrization and analysis, 84 genes were differentially expressed at 24h. Among them, 46 genes were down-regulated and 38 genes wereup-regulated. 710 genes were differentially expressed at 48h. Among them, 366 genes were down-regulated and 344 genes were up-regulated.3. We screened out 20 genes (functions already known) that exhibit close relationship to CsA’s effects on NIT-1 cells. All of them were down-regulated. These genes include: genes related to oxidative phosphorylation (15 genes, Cox7al . Cox7c. Cox8a, Atp6vOe. Atp6vlgl. Atplb3, Atp5k. Atp5o, Mrps21 . Mrpl23, Mrpl36. Mrp64. Mrpl3. Mrpll3. Mrpll9), SNARE protein related genes (Stxbpl. Vamp4), Ca target protein (similar to CaM kinase II beta) gene, G protein related gene (Arl4), and Pax6. We explored their related molecular mechanisms with respect of CsA’s inhibitory effects.onclusion: CsA can inhibit insulin release of NIT-1 pancreatic P cells. Its inhibitory effects are probably related to its inhibition of genes expression of some functional enzymes and proteins in the process of metabolization-secretion couplet mechanism such as oxidative phosphorylation and insulin exocytosis.更多还原