REST／CoREST对TBI后皮层神经元轴突修复作用的体外研究

Role of repressor element 1-silencing transcription factor/repressor element 1-silencing transcription factor coinhibitory factor in reparation of cortical neural axons after traumatic brain injury： an in vitro study

目的探讨抑制元素-1沉默转录因子（REST1、REST辅助抑制因子（CoREST）对创伤性脑损伤（TBI）后皮层神经元轴突再生及修复的影响。方法（1）体外原代培养C57BL／6胎鼠大脑皮层神经元，实时定量PCR（q．PCR）检测神经元培养1d、3d、5d、7d、9d、11d后微小RNA．124（miR-124）的表达；（2）将培养5d后神经元分为miR-124模拟物组、空白对照组、miR-124抑制物组，分别转染miR-124模拟物、无义对照序列、miR．124抑制物。转染后0h、6h、12h、24h、48h、72h采用q-PCR检测神经元miR-124的表达；（3）将培养7d后神经元分为空白对照组、氧糖剥夺（OGD）模型组、上调miR-124＋OGD模型组、下调miR-124＋OGD模型组，后2组细胞分别转染miR-124模拟物和miR．124抑制物。转染48h后3组细胞均制备OGD模型，q-PCR检测0GD处理后0h、6h、12h、24h、48h、72h各组神经元miR．124的表达：Westernblotting检测OGD处理后48h神经元生长相关蛋白43（GAP．431、REST、CoREST的表达；免疫荧光染色检测OGD处理后48h神经元REST、CoREST的表达。结果（1）皮层神经元培养1、3、5、7d后miR-124的表达逐渐增高，皮层神经元培养7、9、11d后miR．124的表达逐渐降低，差异均有统计学意义（p〈0．05）；（2）转染后24、48hmiR-124抑制物组神经元miR-124的表达低于空白对照组。转染后12h、24h、48h、72hmiR-124模拟物组神经元miR-124的表达高于空白对照组，差异均有统计学意义（氏0．05）。其中转染后48h神经元miR-124的表达最高。（3）q．PCR检测显示，0GD处理后12h、24h、48h、72hOGD模型组神经元miR-124的表达高于空白对照组，差异有统计学意义（p〈0．05），其中OGD处理后48h最高；OGD处理后0h、6h、12h、24h、48h、72h．上调miR-124＋OGD模型组神经元miR．124的表达高于空白对照组和0GD模型组，差异有统计学意义（P〈0．05），其中OGD处理后48h最高；Westemblotting检测显示空白对照组，OGD模型组、下调miR-124＋OGD模型组皮层神经元GAP-43、CoREST的表达依次增高，REST的表达依次降低，差异有统计学意义（P〈0．05）。上调miR-124＋OGD模型组神经元GAP．43、CoREST的表达低于0GD模型组，REST的表达高于OGD模型组，差异均有统计学意义（P〈0．05）。免疫荧光染色检测结果与Westernblotting检测结果一致。结论REST、CoREST作为-对调节子．可能是TBI后神经元轴突修复及再生的关键因素。

Objective To explore the effects of repressor element 1-silencing transcription factor （REST）/REST coinhibitory factor （CoREST） on axonal regeneration and repairmen of mouse cortical neurons after traumatic brain injury （TBI）. Methods （1） The primary cortical neurons were obtained from fetal C57BL/6 mice; one, three, 5, 7, 9, and 11 d after cultivation, miR-124 expression was detected by quantitative- （q-） PCR. （2） Neurons cultured for 5 d were divided into miR-124 mimics group, blank control group, and miR-124 inhibitor group, and miR-124 mimics, nonsense control sequences and miR-124 inhibitor were transfected, respectively; 0, 6, 12, 24, 48, and 72 h after transfection, miR-124 expression was detected by q-PCR. （3） Neurons cultured for 7 d were divided into blank control group I, oxygen glucose deprivation （OGD） model group, up-regulated miR-124＋OGD model group, and down-regulated miR-124＋OGD model group, and neurons in the later two groups were transfected with miR-124 mimics and miR-124 inhibitor; 48 h after transfection, OGD models in the later three groups were prepared; 0, 6, 12, 24, 48, and 72 h after OGD, miR-124 expression was detected by q-PCR; GAP-43, REST and CoREST expressions were detected by Western blotting 48 h after OGD; the REST and CoREST expressions were measured by immunoftuorescent staining 48 h after OGD. Results （1） One, three, 5, and 7 d after cultivation, miR-124 expression gradually increased, and 7, 9, and 11 d after cultivation, miR-124 expression gradually decreased, with significant differences （P〈0.05）. （2） Twenty-four and 48 h after transfection, miR-124 expression in the miR-124 inhibitor group was significantly lower than that in the blank control group （P〈0.05）; 12, 24, 48 and 72 h after transfection, miR-124 expression in the miR-124 mimics group was significantly higher than that in the blank control group （P〈0.05）, and peak level was noted at 48 h. （3） The miR-124 expression in the OGD model group was significantly higher than that in the blank control group I at 12, 24, 48 and 72 h after OGD （P〈0.05）, and peak level was noted at 48 h; 0, 6, 12, 24, 48, and 72 h after OGD, the miR-124 expression in the up-regulated miR-124＋OGD model group was significantly higher than that in the blank control group I （P〈0.05）, and peak level was noted at 48 h; Western blotting indicated that GAP-43 and CoREST gradually increased, and REST gradually decreased in blank control group I, OGD model group and down-regulated miR-124＋OGD model group, with significant differences （P〈0.05）; neurons in the up-regulated miR-124＋OGD model group had significantly lower GAP-43 and CoREST expressions, and significantly higher REST expression than those in the OGD model group （P〈0.05）; the results of immunofluorescence staining were consistent with those of Western blotting. Conclusion REST/CoREST, as a pair regulator, may play a key role in the repairment and regeneration of neuron axons after TBI.