中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2010年
1期
105-108
,共4页
刘志恒%桂伶俐%祝畅%姚文龙%张传汉
劉誌恆%桂伶俐%祝暢%姚文龍%張傳漢
류지항%계령리%축창%요문룡%장전한
NF-κB%干细胞%细胞凋亡
NF-κB%榦細胞%細胞凋亡
NF-κB%간세포%세포조망
NF-kappa B%Stem cells%Apoptosis
目的 探讨NF-κB在永生化神经前体细胞凋亡中的作用.方法 永生化神经前体细胞接种于6孔板,随机分为5组,每组6孔,INPC组不进行基因转染,INPC/CMV组转染表达质粒ReCMV;INPC/p50组、INPC/p65组分别转染含p50基因和p65基因的表达质粒RcCMV,转染后2 d,加入200 μg/ml G418筛选12～14 d,进行阳性克隆扩大培养3～4周,检测p50 mRNA、p65 mRNA表达、NF-κB活性和细胞凋亡情况.INPC/p50p65组转染含p65基因的表达质粒ReCMV,加入200 μg/ml G418筛选12～14 d,进行阳性克隆扩大培养3～4周,转染含p50基因的表达质粒ReCMV,2 d后进行上述指标的检测.结果 INPC/p50组和INPC/p50p65组p50 mRNA表达高于其他组(P<0.05),INPC/p65组和INPC/p50p65组p65 mRNA表达、NF-κB活性及细胞凋亡率高于其他组(P<0.05).结论 NF-κB活性升高可促进永生化神经前体细胞的凋亡.
目的 探討NF-κB在永生化神經前體細胞凋亡中的作用.方法 永生化神經前體細胞接種于6孔闆,隨機分為5組,每組6孔,INPC組不進行基因轉染,INPC/CMV組轉染錶達質粒ReCMV;INPC/p50組、INPC/p65組分彆轉染含p50基因和p65基因的錶達質粒RcCMV,轉染後2 d,加入200 μg/ml G418篩選12～14 d,進行暘性剋隆擴大培養3～4週,檢測p50 mRNA、p65 mRNA錶達、NF-κB活性和細胞凋亡情況.INPC/p50p65組轉染含p65基因的錶達質粒ReCMV,加入200 μg/ml G418篩選12～14 d,進行暘性剋隆擴大培養3～4週,轉染含p50基因的錶達質粒ReCMV,2 d後進行上述指標的檢測.結果 INPC/p50組和INPC/p50p65組p50 mRNA錶達高于其他組(P<0.05),INPC/p65組和INPC/p50p65組p65 mRNA錶達、NF-κB活性及細胞凋亡率高于其他組(P<0.05).結論 NF-κB活性升高可促進永生化神經前體細胞的凋亡.
목적 탐토NF-κB재영생화신경전체세포조망중적작용.방법 영생화신경전체세포접충우6공판,수궤분위5조,매조6공,INPC조불진행기인전염,INPC/CMV조전염표체질립ReCMV;INPC/p50조、INPC/p65조분별전염함p50기인화p65기인적표체질립RcCMV,전염후2 d,가입200 μg/ml G418사선12～14 d,진행양성극륭확대배양3～4주,검측p50 mRNA、p65 mRNA표체、NF-κB활성화세포조망정황.INPC/p50p65조전염함p65기인적표체질립ReCMV,가입200 μg/ml G418사선12～14 d,진행양성극륭확대배양3～4주,전염함p50기인적표체질립ReCMV,2 d후진행상술지표적검측.결과 INPC/p50조화INPC/p50p65조p50 mRNA표체고우기타조(P<0.05),INPC/p65조화INPC/p50p65조p65 mRNA표체、NF-κB활성급세포조망솔고우기타조(P<0.05).결론 NF-κB활성승고가촉진영생화신경전체세포적조망.
Objective To investigate the role of NF-κB in apoptosis of immortalized neural progenitor cells (INPCs) . Methods INPCs were cultured in 6-well plates and were randomly divided into 5 groups ( n = 6 each) : group I was not transfected with any plasmid (group INPC); group Ⅱ was transfected with control plasmid (group INPC/CMV); group Ⅲ was transfected with plasmid RcCMV-p50 (group INPC/p50); group Ⅳ was transfected with plasmid RcCMV-p65 (group INPC/p65) and group V was transfected with plasmid RcCMV-p50 and RcCMV-p65 (group INPC/p50p65). Group INPC/CMV ( H ), INPC/p50 (Ⅲ) and INPC/p65 (Ⅳ) were screened by G418, and the positive clones were then cultured for 3-4 weeks. The transcription of p50 mRNA or p6S mRNA was detected by RT-PCR. The NF-κB activity was measured by luciferase reporter gene assay. The cell apoptosis was measured by annexin V/PI staining. In group INPC/p50p65 and group INPC/p65, the cultured positive clone was transiently transfected with plasmid RcCMV-p50. Two days after transfection, the same measurement was performed in group INPC/pS0p65 and the other groups. Results The expression of p50 mRNA was significantly increased in group INPC/p50 and INPC/p50p65 as compared with the other groups ( P < 0.05) . The expression of p65 mRNA, the NF-κB activity and the apoptotic rate were significantly increased in group INPC/p65 and INPC/p50p65 as compared with the other groups ( P < 0.05). Conclusion Enhanced NF-κB activity can increase immortalized neural progenitor cell apoptosis.
