PC12细胞缺血预处理模型的建立及一氧化氮的保护作用

目的 以PC12细胞建立体外脑缺血预处理细胞模型,探讨NO在预处理脑保护中的作用.方法 建立缺血预适应的细胞模型,随机分为四组,每组五碟细胞.正常对照组:常氧、低糖(<1g/L)2%胎牛血清DMEM培养;缺血预适应组(IPC):预先氧糖剥夺(OGD)6 h,后经复灌和OGD处理;非缺血预适应组(NIPC):常氧、低糖低血清培养6 h,后经复灌和OCD处理;一氧化氮合酶抑制剂组(L-NAME):OGD预处理前30 min加L-NAME,OGD6 h,后经复灌和OGD处理.通过细胞MTT代谢率、乳酸脱氢酶(LDH)释放量、细胞凋亡率来评判IPC模型是否建立,生化法观察各组一氧化氮合酶(NOS)活性变化.统计分析利用单因素方差分析,两两组间均数的比较用LSD法,以P<0.05为差异具有统计学意义.结果 与NIPC组比较,IPC组MTT代谢率明显增高(94.9%±15.1%,P<0.05),LDH释放量减少(279.1%±28.1%,P<0.01),细胞凋亡率降低;与对照组(100.0%±13.5%)比较,NIPC组及IPC组NOS活性均升高(190.0%±14.6%,P<0.01;126.10k±10.6%,P<0.01);流式检测结果显示,对照组、IPC组、NIPC组和L-NANE组引起的细胞凋亡率分别为5.90%,8.71%,18.62%及11.73%.结论 IPC减少PC12细胞缺糖缺氧损伤后细胞的死亡与凋亡,且NO参与了此保护机制,但并非唯一因素.

Establishment of ischemic precondition model and the protective effect of nitric oxide on PC12 cell line

Objective To establish the ischemic precondition (PC) model of PC12 cell line in vitro, and to explore the effect of nitric oxide (NO) on the IPC cerebral protection. Method PC12 cells were cultured and used for producing the model of ischemie precondition by the way of oxygen-glucose deprivation. Twenty dishes of cells were randomly divided into four groups (5 dishes for each group): control group, ischemic precondition group (IPC),non-ischemic precondition group (NIPC) and L-NAME treatment group (L-NAME). In control group, the cells were in-cubated with low glucose (<1 g/L) and2% FBS medium in normal oxygen; in IPC group, the cells were administrated with oxygen-glucose deprivation (OGD) for 6 hours, and then subjected with reperfuaion before OGD 15 hours; in NIPC group, the cells were treated the same as control group for 6 hours, and then subjected with reperfusion before OGD 15 hours; in L-NAME group, the cells received L-NAME (1 mmol/L) and cocultured for 30 minutes before OGD 6 hours, and then received the same treatment as the IPC group. To test whether the model was established, metabolic rate of MIT, LDH release were measured and the apoptosis rate was detected by flow cytometry following oxygen-glucose deprivation 15 hours. The activity of nitric oxide synthases (NOS) was as-sessed by biochemical assay. One-way ANOVA and LSD multiple comparison test were used to analyze differences among different groups, and P<0.05 was considered different. Results Compared with NIPC group, the metabolic rate of MTT increased (94.9%±35.1%, P<0.05), while LDH release and the cell apoptotic rate decreased significantly in IPC group (279.1%±28.1%, P<0.03). Compared with control group(100.0%± 13.5%),the activities of NOS increased both in NIPC and IPC groups (390.0%±14.6%, P<0.01;126.3% ±10.6%, P<0.01). Moreover, the apoptosis rates in each group (control group, IPC group, NIPC group and L-NAME group) were 5.90, 8.73, 38.62 and 11.73%,respectively. Conclusions IPC reduces the death and apoptosis rate of PC12 cell after oxygen-glucose deprivation injury. NO might be involved, but it is not the only factor.