氧葡萄糖血清剥夺/再恢复诱导 PC12细胞凋亡模型的建立

摘要

目的：明确氧葡萄糖血清剥夺/再恢复（oxygen-glucose-serum deprivation/restoration，OGSD/R）诱导 PC12细胞凋亡的变化趋势，建立稳定的细胞凋亡模型，为体外模拟脊髓缺血再灌注损伤导致神经细胞凋亡的研究提供实验工具。方法常规培养高分化 PC12细胞，取对数生长期的细胞进行实验。以低糖不含血清的 DMEM 培养基于三气培养瓶（体积分数：95%N2+5%CO2，O2<1%）培养12 h，然后换成高糖含血清DMEM 培养基和普通培养箱继续培养，诱导PC12细胞凋亡。于复氧复糖复血清后0~6 h进行DAPI染色、CCK-8细胞活性检测及流式细胞术分析并检测相关凋亡蛋白caspase-3、caspase-12水平，比较不同时间点细胞凋亡的差异。结果DAPI染色显示，OGSD 12 h/R 1 h细胞凋亡最重，凋亡细胞核固缩，形成较多颗粒光斑，凋亡严重的细胞破裂形成碎片，核解体。CCK-8活性检测表明在OGSD 12 h/R 1 h细胞活力最低。流式细胞术分析表明 OGSD 12 h/R 1 h 细胞凋亡率达到最大。凋亡蛋白检测结果提示在正常的PC12细胞中 caspase-3、caspase-12含量极低，而在给予 OGSD 刺激后细胞内 caspase-3、caspase-12的表达增强，复氧复糖复血清后2种凋亡蛋白表达进一步增强，caspase-12于 OGSD 12 h/R 1 h 时表达最强，caspase-3于 OGSD 12 h/R 2 h 表达最强。结论OGSD 可以诱导 PC12细胞凋亡，且在复氧复糖复血清后细胞凋亡进一步加重；OGSD/R 诱导 PC12细胞凋亡具有时间依赖性，能有效模拟神经细胞缺血再灌注损伤病理生理过程，为脊髓缺血再灌注损伤的进一步研究提供实验模型。%Objective To observe the apoptosis of PC12 cells induced by oxygen-glucose-serum deprivation/restoration (OGSD/R) and establish a stable cell apoptosis model to provide an experimental tool for spinal cord ischemia-reperfusion injury in vitro. Methods Well-differentiated PC12 cells were culturedand the cells in logarithmic phase were used to experiment. The PC12 cells were cultured for 12 h in low glucose free serum DMEM under 95% N2 + 5%CO2 and O2<1% culture condition,and then cultured in high glucose serum DMEM to induce apoptosis. DAPI staining,CCK-8 cell viability assay and flow cytometry were performed to test the cell apoptosis at OGSD 12 h/R 0-6 h. Caspase-3 and caspase-12 levels were tested at OGSD 12 h/R 0-6 h. The differences in cell apoptosis between different time points were analyzed compared in each group . Results At OGSD 12 h/R 1 h, the apoptosis was severe in experimental group by DAPI staining. Karyopyknosis and innumerable bright faculae were observed in the apoptotic cells. Furthermore,plasmatorrhexis and nuclear disintegration were observed in some apoptosis. CCK8 assay result reminded that the cells viability were the worst at OGSD 12 h/R1 h via statistical analysis. The maximum apoptosis rate was observed at OGSD 12 h/R 1 h via flow cytometric method. Caspase-3 and caspase-12 were almost none in normal PC12 cells. When the cells were stimulated by OGSD,the enhanced expression of caspase-3 and caspase-12 were observed in experiment group and further expression at restoration(1-2 h). Conclusion PC12 cells apoptosis could be induced by OGSD and be exacerbated at restoration. OGSD/R-induced cell apoptosis is time dependent and could effectively simulate the pathophysiological process of neural cell ischemia-reperfusion injury,thus providing an experimental model for further research of spinal cord ischemia-reperfusion injury.