目的 观察缺血缺氧状态下不同低温对人神经母细胞瘤细胞(SHSY5Y)表达小泛素样修饰蛋白(SUMO)化相关蛋白的影响,探讨SUMO化在深低温停循环(DHCA)过程中对人神经系统的保护作用.方法 在18℃、26℃、30℃、37℃环境中进行4 h缺氧的细胞与正常培养细胞对比,观察细胞缺氧后形态,微板法检测再灌注24 h后培养液中乳酸脱氢酶(LDH)水平,WestemBlot检测缺氧后UBC9、SUMO1、SUMO2/3水平及再灌注24 h后caspase-3水平,qPCR检测缺氧后UBC9的mRNA表达情况.结果 LDH检测结果示,糖氧剥夺(OGD)4h使LDH漏出量显著增加,低温可减少LDH漏出(P＜0.05,n = 6). Western Blot结果显示,与37℃ OGD组相比,低温干预可上调结合态SUMO1表达,温度越低表达量越高,18℃组明显高于30℃组(P＜0.05,n = 6),但18℃组与26℃组差异无统计学意义(P＞0.05,n=6);低温干预可同时上调结合态及游离态SUMO2/3表达(P＜0.05,n = 6),但与37℃ OGD组相比,并不额外增加SUMO2/3蛋白结合游离比(P ＞0.05,n = 6);OGD和低温对UBC9蛋白表达影响均不显著(P＞0.05,n = 6);OGD损伤4h再灌注24h后Caspase-3蛋白水平明显上调,低温干预下调Caspase-3表达(P＜0.05,n = 6).qPCR结果显示,OGD和低温对UBC9蛋白表达影响均不显著(P＞0.05,n = 3).结论 细胞遭受缺血缺氧时,低温可增加SUMO蛋白整体表达水平,提高SUMO1蛋白结合能力,提高神经细胞抗缺血缺氧耐受能力.短时间缺血缺氧情况下,26℃和30℃的神经保护作用与18℃深低温效果相近.%Objective To investigate how temperature affects SHSY5Y cells expressing sumoylation related protein under hypoxic-ischemic stress. Methods The SHSY5Y cells were exposed to oxygen - glucose deprivation (OGD) with different temperature (18℃, 26℃, 30℃, 37℃). The morphological change was observed by inverted microscope. Expression of UBC9 was detected by qPCR and western blot, and sumoylation related protein SUMO1 and SUMO2/3 was detected by western blotting at the time we ended oxygen deficit. 24 hours after reperfusion, LDH and caspase-3 released in cell culture supernatant were detected by western blotting. Results LDH was significantly higher after 4 hours OGD, and hypothermia reduced LDH released in culture than that in 37℃ group. Western blotting results showed hypothermia could up-regulate SUMO1-conjugated expression, which was significantly different between 18℃ and 30℃ (P ＜0.05, n = 6), but not between 18℃ and 26℃ (P ＞0.05, n = 6). Hypothermia increased the ratio of conjugated and free SUMO1 (P ＜0.05, n = 6); on the other hand, hypothermia up-regulated expression of both SUMO2/3-conjugated and SUMO2/3-free. OGD increased the ratio of conjugated and free state of SUMO2/3 (P ＜0.05, n = 6). There was no significant difference in UBC9 expression between experimental group and control group (P ＞0.05, n = 6). After 4 hours of OGD and 24 hours reperfusion, caspase-3 protein level increased and hypothermia reduced the expression of caspase-3 (P ＜0.05, n = 6). OGD and hypothermia did not affect UBC9 mRNA expression (P ＞0.05, n=3). Conclusions Nervous cells express fewer SUMO1 and SUMO2/3 protein when lacking nutrition, low temperature could protect nervous cells possibly by global SUMOylation, especially through enhanced level of conjunctive capacity of SUMO1. No difference of protective effect was found in 26℃, 30℃ and 18℃ after 4 hours OGD.
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