PC12细胞转染nNOSα和nNOSβ对NF-kappa B转录活性和细胞凋亡的影响

Effect of nNOSα and nNOSβ gene transfections into PC12 cells on transcription activity of NF-kappa B and apoptosis

目的:比较转染nNOSα和nNOSβ的PC12细胞NF-kappaB转录活性和细胞凋亡百分数,探讨nNOSα和nNOSβ在功能上的差异性.方法:将携带nNOSα和nNOSβ基因的pcDNA3.0表达质粒转化感受态细胞,在大肠杆菌中扩增后,提纯质粒酶切鉴定.nNOSα和nNOSβ基因的pcDNA3.0表达质粒通过脂质体转染PC12细胞,G418筛选,使用抗nNOS羧基端的抗体,通过免疫组织化学和WesternBlot方法检测nNOS的表达效率.提取经G418筛选PC12细胞核蛋白,采用凝胶电泳阻滞实验(EMSA)检测细胞核NF-kappa B转录活性.不同PC12细胞经PI染色后流式细胞仪分选,测定不同增殖周期的细胞数.结果:质粒酶切鉴定结果与携带nNOSα和nNOSβ基因的pcDNA3.0质粒结构相符;免疫组织化学和Western Blot方法检测转染nNOS基因后表达的nNOSα和nNOSβ均高于未转染的PC12细胞(P<0.05),PC12细胞转染nNOSα增加NF-kappa B转录活性(P<0.05),而nNOSβ降低NF-kappaB转录活性(P<0.05);转染nNOSα和nNOSβ及未转染的PC12细胞均不改变细胞凋亡百分数(P>0.05).结论:nNOSα增加PC12细胞NF-kappaB转录活性,nNOSβ降低C12细胞NF-kappa B转录活性,但均不改变细胞凋亡百分数,nNOSα和nNOSβ在功能上具有差异性.

AIM： To determine the effects of overexpression of nNOSα and nNOSβ on NF-kappa B activity and apoptosis in PC12 ceils. METHODS： The expression vectors of pcDNA3-nNOSα and pcDNA3-nNOSβ were replicated in HSα cell through transformarion process. The vectors were harvested and digested with HindⅢ to confirm it. Then PC12 cells were transfected with pcD- NA3-nNOSα and pcDNA3-nNOSⅢ, respectively. The expression efficiency of nNOS was detected by immunohistochemistry and Western Blot analysis with an antibody specific for the COOH-terminal domain of nNOS. The nuclear translocation of NF-kappa B was assayed by electrophoretic mobility shift assay （EMSA）. The percentage of sub-G1/G0 was determined by flow cytometry （ FCM ） with propidium iodide （ PI ）. RESULTS： The number and size of digested products in gel eletrophoresis were consistent with the structure of nNOS gene and peDNA3 vector. Immunohis-tochemistry and Western Blot analysis indicated that pcDNA3- nNOSα and peDNA3-nNOSβ were respectively transfected into PC12 ceils successfully. PC12 cells transfected with nNOSα gene increased nuclear translocation of NF-kappa B compared with non-transfected PC12 ceils. But overexpression of nNOSβ in PC12 ceils decreased nuclear translocation of NF-kappa B when compared with non-transfectants ceils. There was no difference in apoptosis rate in PC12 transfected with null, nNOSα and nNOSβ gene. CONCLUSION： nNOSα increase nuclear translocation of NF-kappa B in PC12 cells, but nNOSβ decreased it; all have no effect on apoptosis rate of PC12 cells.