糖氧剥夺再灌注对 PC12细胞自噬、凋亡的影响及意义

摘要

目的：观察糖氧剥夺再灌注对PC12细胞自噬、凋亡的影响，探讨激活自噬调控神经元损伤修复的有效时间窗。方法将高分化的大鼠肾上腺嗜铬细胞瘤PC12细胞随机分为四组，分别置于缺氧（1％ O2、5％ CO2、94％N2）环境中培养0 h（ OGD 0 h组）、1 h（ OGD 1 h组）、2 h（ OGD 2 h组）、3 h（ OGD 3 h组）；换用含10％灭活胎牛血清的高糖双抗DMEM培养液，正常条件下继续培养，分别培养0、4、8、12 h；采用CCK-8法检测四组各时间点细胞增殖率，采用 RT-PCR 法检测自噬基因微管相关蛋白2轻链3（ LC3-Ⅱ）、凋亡基因Caspase-3相对表达量。结果 OGD 1 h组灌注后各时间点细胞增殖率均高于同时间点OGD 2 h及OGD 3 h组，组间比较P均＜0．05；三组再灌注4、8、12 h时细胞增殖率均逐渐降低（P均＜0．05），0、4 h时细胞增殖率比较差异无统计学意义（P均＞0．05）。四组再灌注4 h时LC3-Ⅱ相对表达量均低于同组0 h，OGD 1 h、OGD 2 h组再灌注4 h时Caspase-3相对表达量均低于同组0 h，OGD 1 h、OGD 2 h组再灌注12 h时LC3-Ⅱ、Caspase-3相对表达量均高于同组4、8 h，组间及组内比较P均＜0．05。结论糖氧剥夺再灌注可诱导PC12细胞发生自噬及凋亡，半暗带修复时间窗以糖氧剥夺4 h内为宜。%Objective To observe the effects of oxygen and glucose deprivation-reperfusion ( OGD/OGD-R) on auto-phagy and apoptosis of PC12 cells, and to evaluate the effective time window of OGD/OGD-activated autophagy-regulated neuron injury repair .Methods The high differentiated PC12 cells were cultured under hypoxia (1%O2 , 5%CO2 , 94%N2) and then randomly divided into 4 groups which were respectively cultured for 0 h (OGD 0 h group), 1 h(OGD 1 h group), 2 h (OGD 2 h group), 3 h (OGD 3 h group), followed by persistent culture in high glucose Dulbecco′s modified Eagle′s medium (DMEM) which supplemented with 10 %fetal bovine serum for another 0, 2, 4, 8, 12 h, respectively. The cell viability was analyzed by CCK-8.The expression of microtubule-associated protein 2-light chain 3 ( LC3Ⅱ) and Caspase-3 gene of PC12 cells in each group at different time was detected RT-PCR.Results At each oxygen-glucose reperfusion time point , the cell viability of OGD 1 h group was higher than that of OGD 2 h and OGD 3 h group, the differ-ence was statistically significant between these groups (all P<0.05).The cell viability of each OGD group gradually de-creased as the reperfusion duration till to 4, 8 and 12 h, the difference was statistically significant between these time points (all P<0.05), except the comparison between 0 h and 4 h (all P>0.05).The relative expression of LC3Ⅱgene at reperfusion of 4 h ( R4H) was lower than that of 0 h in each reperfusion groups including the control group .The relative ex-pression of Caspase-3 gene at R4H was lower than that of R0 h in OGD 1 h and OGD 2 h groups respectively .The relative expression of LC3Ⅱand Caspase-3 gene at R12H in the OGD 1 h and OGD 2 h groups was higher than that at R 4H and R8H, and the difference between groups and within groups was statistically significant (all P<0.05).Conclusions OGD-R can induce the autophagy and apoptosis of PC 12 cells.The penumbra repair time window is advised to be less than 4 hours after oxygen and glucose deprivation .