摘要
目的:观察低氧预适应(HPC)对氧糖剥夺(OGD)损伤人神经母细胞瘤细胞(SH-SY5Y)的保护作用,并探讨其可能机制。方法:SH-SY5Y细胞随机分为4组:正常对照组:常规培养,不进行OGD处理;HPC处理组:将神经元放入低氧培养箱内(2%O_2),30 min后立即取出,再恢复常氧培养,反复5次;OGD组:无糖培养基、低氧培养箱内(1%O_2)处理细胞10 h,然后复氧复糖培养24 h;HPC+OGD处理组:细胞HPC后,行OGD处理。通过形态学观察,MTT比色法检测细胞存活率,乳酸脱氢酶(LDH)漏出量判断细胞损伤的程度,原位末端标记(TUNEL)法检测凋亡水平,Western blot检测Caspase 3、低氧诱导因子1α(HIF-1α)的蛋白表达。结果:HPC可减轻OGD引起的SH-SY5Y细胞凋亡,降低LDH漏出量,明显增加OGD组SH-SY5Y细胞的活力(P<0.05)。Western blot显示HPC+OGD组Caspase 3蛋白的表达明显低于OGD组(P<0.05);HIF-1α蛋白的表达明显高于OGD组(P<0.05)。结论:HPC对体外培养的SH-SY5Y细胞OGD损伤具有保护作用,其机制可能与上调HIF-1α蛋白有关。
Objective： To investigate the protection effects of hypoxic preconditioning（HPC） on the SH-SY5Y cell injured by oxygen-glucose deprivation（OGD）,and to discuss the possible mechanism. Methods： SH-SY5Y cells were randomly divided into 4 groups. In normal group, the cells were cultured without OGD treatment. In HPC group, the cultured SH-SY5Y cells were treated for 5 days by intermittently exposing to hypoxic gas mixture （2% 02,5% CO2）for 30 rain in every day. In OGD group,the culture medium was replaced by glucose - free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O2 and 5% CO2 for 10 h. After that, the cells were fed with glucose - supplemented medium and cultured under normoxic condition for 24 h. In HPC ＋ OGD group, the cultured SH-SY5Y cells were treated for 5 days by intermittently exposing to hypoxic gas mixture for 30 rain in each day, then the cells were given the same treatments as those in OGD group. The cell viability was assessed by MTF assay. The degree of the cell damage was evaluated by determining lactate dehydrogenase （LDH） leakage. TUNEL staining were used to detect the variation of cell apoptosis. The expression of Caspase 3 and hypoxia inducible factor-1α（HIF-1α） at protein levels was examined by Western blot. Results： Hypoxic preconditioning relieved the cells apoptosis, decreased the amount of LDH leakage and improved the viability of SH-SY5Y cells injured by OGD （ P 〈 0.05）. Western blot showed that the expression of Caspase 3 protein in HPC ＋ OGD group was significantly lower than that in OGD group （ P 〈 0.05） ; HIF-1α protein expression was significantly higher than that of OGD group （ P 〈 0.05）. Conclusion： Hypoxic preconditioning has protective effect on in vitro cuhured SH-SY5Y cells injured by OGD. The mechanism may be related to the increase of HIF-1α protein.
出处
《中国应用生理学杂志》
CAS
CSCD
2016年第4期319-323,共5页
Chinese Journal of Applied Physiology
基金
河南省教育厅科学技术重点研究项目(14A310009)
