论文部分内容阅读
目的观察星形胶质细胞活化后胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)、肿瘤坏死因子α(tumor necrosis factor,TNF)α两种不同作用因子的分泌特点,探讨星形胶质细胞活化在神经退行性疾病后的神经再生和功能恢复的作用。方法体外分离培养星形胶质细胞,分为对照组、活化组、抑制组。通过光学显微镜及免疫荧光化学观察各组细胞的形态变化;应用半定量RT-PCR方法分析各组细胞间胶原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)mRNA及GDNF mRNA、TNF mRNA表达变化;用ELISA法检测各组细胞上清液中不同时间点(6、24、48、72 h)GDNF、TNF的含量。计量资料用x珋±s表示,采用t检验,多组数据间的比较采用单因素方差分析,P<0.05为差异有统计学意义。结果活化组与对照组比较,细胞数量增多,胞体变大,突起变长增多,交织成网状,GFAP荧光增强;RT-PCR示GFAP mRNA、GDNF mRNA、TNF mRNA表达均明显增高,与对照组比较差异有统计学意义(P<0.01);ELISA法检测示星形胶质细胞活化后GDNF分泌量在活化后48 h达到高峰,与对照组比较差异有统计学意义(P<0.01);活化后24 hTNF的含量达到高峰,与对照组比较差异有统计学意义(P<0.01);应用抑制剂Olomoucine干预后,与活化组相比,抑制组细胞胞体变小,数量减少,星形胶质细胞活化被抑制,GFAP mRNA表达下降,GDNF mRNA、TNF mMRA表达亦下降,与活化组比较差异有统计学意义(P<0.01)。结论星形胶质细胞活化后GDNF、TNF表达均上调,但TNF表达高峰时间早于GDNF表达时间;Olomoucine抑制星形胶质细胞活化,可使GDNF、TNF表达下调。表明星形胶质活化后TNF可能有促进GDNF分泌从而起到一定的神经功能恢复作用。
Objective To observe the secretory characteristics of glial cell derived neurotrophic factor (GDNF) and tumor necrosis factor (TNF) α after activation of astrocytes, Activation of astrocytes in neuroregeneration and functional recovery after neurodegenerative diseases. Methods Astrocytes were isolated and cultured in vitro and divided into control group, activation group and inhibition group. The morphological changes of the cells were observed by light microscopy and immunofluorescence staining. The changes of GFAP mRNA, GDNF mRNA and TNF mRNA expression in each group were analyzed by semi-quantitative RT-PCR. The contents of GDNF and TNF at different time points (6, 24, 48, 72 h) in supernatants were detected by ELISA. Measurement data with x 珋 ± s said, using t test, multiple sets of data between the use of single-factor analysis of variance, P <0.05 for the difference was statistically significant. Results Compared with control group, the number of cells increased, the number of cytoplasm became larger, the length of protuberances increased, the number of GFAP increased, and the fluorescence intensity of GFAP increased. The expression of GFAP mRNA, GDNF mRNA and TNF mRNA were significantly increased in activated group compared with control group (P <0.01). The amount of GDNF secreted by astrocytes reached a peak at 48 hours after activation, which was significantly different from the control group (P <0.01) Compared with the control group, the level of TNF peaked at 24 h (P <0.01). Compared with the control group, the inhibitory effect of Olomoucine group was less than that of the activated group Cell activation was inhibited, GFAP mRNA expression decreased, GDNF mRNA, TNF mMRA expression also decreased, compared with the activation group was statistically significant (P <0.01). Conclusion The expression of GDNF and TNF were up-regulated after activated astrocytes, but the peak time of TNF expression was earlier than that of GDNF. Olomoucine inhibited the activation of astrocytes and down-regulated the expression of GDNF and TNF. Indicating that astrocytes activated TNF may promote GDNF secretion and thus play a role in nerve function recovery.