CAJ | 학술논문

目的观察干预细胞核因子-κB(NF-κB)信号通路对星形胶质细胞(AST)自噬、凋亡和坏死的影响. 方法取纯化AST分成3组:(1)空白组:加入2μL PBS液1h后,再加入2μLPBS液;(2)激活组:加入2μL PBS液1h后,再加入2μL NF-κB激活剂PMA(终浓度1μmol/L);(3)抑制组:先加入2 μL NF-κB阻断剂PDTC(终浓度20 μmol/L)预处理lh,再加入2μL PMA(终浓度1μmol/L).各组细胞分别处理1、6、12、24、48 h,应用免疫细胞化学荧光法测定NF-κB蛋白的表达,应用AnnexinV/PI染色法检测凋亡和坏死,应用Western blotting检测自噬蛋白Beclin 1表达情况. 结果 (1)随着作用时间增加,NF-κB蛋白表达量也逐渐增高,24 h时空白组、激活组及抑制组表达量分别为0.27±0.46、3.13±0.35、2.00±0.53,差异有统计学意义(P＜0.05).(2)各组细胞凋亡及坏死程度也随着作用时间增加而相应增高.激活组AST凋亡程度总体高于空白组与抑制组,12、24、48 h时间点3组细胞两两比较差异均有统计学意义(P＜0.05).激活组AST坏死程度总体高于空白组与抑制组,24 h、48 h时间点3组细胞两两比较差异均有统计学意义(P＜0.05).(3)激活组Beclin 1蛋白各时间点均较抑制组及空白组明显增加,6h时空白组、激活组及抑制组Beclin 1蛋白表达量分别为0.17±0.01、0.59±0.03、0.51 ±0.03,两两比较差异均有统计学意义(P＜0.05). 结论 PMA诱发的NF-κB活化能够引起AST不同程度的凋亡、坏死及自噬进程,早期以凋亡和自噬为主,晚期以坏死为主,而PDTC能够在一定程度上抑制NF-κB通路所诱导的这些死亡进程.
목적 관찰간예세포핵인자-κB(NF-κB)신호통로대성형효질세포(AST)자서、조망화배사적영향. 방법 취순화AST분성3조:(1)공백조:가입2μL PBS액1h후,재가입2μLPBS액;(2)격활조:가입2μL PBS액1h후,재가입2μL NF-κB격활제PMA(종농도1μmol/L);(3)억제조:선가입2 μL NF-κB조단제PDTC(종농도20 μmol/L)예처리lh,재가입2μL PMA(종농도1μmol/L).각조세포분별처리1、6、12、24、48 h,응용면역세포화학형광법측정NF-κB단백적표체,응용AnnexinV/PI염색법검측조망화배사,응용Western blotting검측자서단백Beclin 1표체정황. 결과 (1)수착작용시간증가,NF-κB단백표체량야축점증고,24 h시공백조、격활조급억제조표체량분별위0.27±0.46、3.13±0.35、2.00±0.53,차이유통계학의의(P＜0.05).(2)각조세포조망급배사정도야수착작용시간증가이상응증고.격활조AST조망정도총체고우공백조여억제조,12、24、48 h시간점3조세포량량비교차이균유통계학의의(P＜0.05).격활조AST배사정도총체고우공백조여억제조,24 h、48 h시간점3조세포량량비교차이균유통계학의의(P＜0.05).(3)격활조Beclin 1단백각시간점균교억제조급공백조명현증가,6h시공백조、격활조급억제조Beclin 1단백표체량분별위0.17±0.01、0.59±0.03、0.51 ±0.03,량량비교차이균유통계학의의(P＜0.05). 결론 PMA유발적NF-κB활화능구인기AST불동정도적조망、배사급자서진정,조기이조망화자서위주,만기이배사위주,이PDTC능구재일정정도상억제NF-κB통로소유도적저사사망진정.
Objective To study the effect of nuclear factor-κb (NF-κB) signaling pathway on autophagy, apoptosis and necrosis of astrocytes (ASTs).Methods Purified ASTs were divided into three groups: blank control group, adding 2 μL PBS at one h after 2 μL PBS;activator group, adding 2 μL NF-κB activator PMA (final concentration 1 μmol/L) at one h after 2 μL PBS;blocker group, adding 2μL NF-κB activator PMA (final concentration 1 μmol/L) at one h after 2 μL NF-κB blocker PDTC (final concentration 20 μmol/L);each treatment was given to the cells for one, 6, 12, 24 and 48 h.NF-κB protein expression was detected by immunocytochemistry assay, apoptosis and necrosis of the cells in each group were observed by Annexin V/PI staining, autophagy protein Beclin 1 expression was measured by Western blotting.Results (1) With time being prolonged, NF-κB protein expression gradually increased;its expression quantity at 24 h after treatment was 0.27±0.46, 3.13±0.35 and 2.00± 0.53 in the blank control group, activator group and blocker group, with significant differences (P＜0.05).(2) With time being prolonged, apoptosis and necrosis of the cells gradually became much severe;ASTs apoptosis in the activator group was significantly severer than that in the other two groups (P＜0.05), and at 12, 24 and 48 h after treatment, the ASTs apoptosis was significantly different between each two groups (P＜0.05);ASTs necrosis in the activator group was significantly severer than that in the other two groups (P＜0.05), and at 24 and 48 h after treatment, the ASTs necrosis was significantly different between each two groups (P＜0.05).(3) Beclinl protein expression in the activator group was obviously increased as compared with that in the other two groups;its expression quantity at 6 h after treatment was 0.17± 0.01, 0.59±0.03 and 0.51±0.03 in the blank control group, activator group and blocker group, with significant differences (P＜0.05).Conclusion NF-κB activation induced by PMA could cause apoptosis, necrosis and autophagy of ASTs at different levels correspondingly;apoptosis and autophagy appear mainly at early stage, and necrosis is mainly at late stage;PDTC could inhibit these programmed cell death process induced by NF-κB pathway to some extent.