CAJ | 학술논문

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.
목적 이PC12세포건입체외뇌결혈예처리세포모형,탐토NO재예처리뇌보호중적작용.방법 건립결혈예괄응적세포모형,수궤분위사조,매조오설세포.정상대조조:상양、저당(<1g/L)2%태우혈청DMEM배양;결혈예괄응조(IPC):예선양당박탈(OGD)6 h,후경복관화OGD처리;비결혈예괄응조(NIPC):상양、저당저혈청배양6 h,후경복관화OCD처리;일양화담합매억제제조(L-NAME):OGD예처리전30 min가L-NAME,OGD6 h,후경복관화OGD처리.통과세포MTT대사솔、유산탈경매(LDH)석방량、세포조망솔래평판IPC모형시부건립,생화법관찰각조일양화담합매(NOS)활성변화.통계분석이용단인소방차분석,량량조간균수적비교용LSD법,이P<0.05위차이구유통계학의의.결과 여NIPC조비교,IPC조MTT대사솔명현증고(94.9%±15.1%,P<0.05),LDH석방량감소(279.1%±28.1%,P<0.01),세포조망솔강저;여대조조(100.0%±13.5%)비교,NIPC조급IPC조NOS활성균승고(190.0%±14.6%,P<0.01;126.10k±10.6%,P<0.01);류식검측결과현시,대조조、IPC조、NIPC조화L-NANE조인기적세포조망솔분별위5.90%,8.71%,18.62%급11.73%.결론 IPC감소PC12세포결당결양손상후세포적사망여조망,차NO삼여료차보호궤제,단병비유일인소.
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.