锤头状核酶降低小鼠胰岛细胞经CD95途径的凋亡

Hammerhead ribozyme reducing the apoptosis of mouse islet cells via CD95 pathway

目的研究锤头状核酶通过调控CD95的表达,对小鼠胰岛细胞凋亡的影响;探索提高胰岛移植物存活率的新途径。方法体外构建针对CD95mRNA93位点的锤头状核酶(pU6-Rz93)和突变型核酶(pU6-dRz93)。采用Ⅴ型胶原酶消化法分离小鼠胰岛细胞,通过干扰素-γ(IFN-γ)和白细胞介素1α(IL-1α)诱导其高表达CD95后,经钠米载体-Effectene将核酶转染至该细胞。通过逆转录-聚合酶链反应(RT-PCR)和免疫印迹检测(Westernblot)法检测胰岛细胞CD95的表达。胰岛细胞经抗CD95的抗体(JO2)作用后,以Caspase-3活性检测试剂盒检测转染前后细胞Caspase-3活性的变化;MTT法测细胞的增殖;Annexin-Ⅴ凋亡检测试剂盒检测细胞凋亡,并观察了各组细胞对细胞毒T淋巴细胞(CTL)杀伤的反应能力。结果细胞因子可诱导小鼠胰岛细胞高表达CD95分子。转染pU6-Rz93组的胰岛细胞CD95的表达与转染空载体组和转染pU6-dRz93组相比,明显下降。经JO2处理后,转染pU6-Rz93组胰岛细胞的Caspase-3活性和凋亡率明显降低,细胞增殖活性和抵抗CTL杀伤的能力显著增强。结论针对FasmRNA93位点的锤头状核酶能显著抑制小鼠胰岛细胞CD95的表达,使其免于CD95途径的凋亡;为提高胰岛移植物的存活率提供了实验基础。

Objective To investigate the effect of hammerhead ribozyme controlling CD95 on apoptosis of mouse islet cells, and explore a new way to improve the survival rate of islet grafts. Methods The ribozyme pU6-Rz93 targeting the position 93 of CD95 mRNA and mutant ribozyme pU6-dRz93 were constructed in vitro. Mouse islet cells were digested by collagenase Ⅴ and induced to express CD95 through treatment with IL-1α and IFN-γ, then respectively transfected with empty vector, pU6-Rz93 and pU6-dRz93 by Effectene reagent. CD95 expression in mouse islet cells was detected by using RT-PCR and Western blot. Treated with anti-CD95 antibody (JO_2), cell viability was measured via MTT assay, Caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to Annexin Ⅴ-FITC apoptosis detection kit. Cells viability against killing role of CTL in vitro was detected, too. Results After being treated with cytokines, primary islet cells could express CD95, which was remarkably decreased in pU6-Rz93-transfected cells. After treatment with JO_2 for 24 h, the Caspase-3 activity and apoptosis rate of pU6-Rz93-transfeced cells were significantly decreased, and cells viability against CTL were enhanced remarkably in pU6-dRz93-transfected group as compared with mock-transfected group. Conclusion Anti-CD95 ribozyme can obviously inhibit the CD95 expression in mouse islet cells and make them avoidance of CD95-mediated apoptosis, which may afford basis for improving survival rates of islet grafts and successful rates of islet transplantation.