炎症因子诱导星形胶质细胞凋亡及机制探讨

目的：探讨LPS，IL-1β，TNF-α所致星形胶质细胞(AC)凋 亡的机制及途径。方法：原代培养的新生SD大鼠前脑AC分为正常对照组、 炎症组(LPS+IL 1β+TNF α)与预处理组(L-NMMA预处理30 min后加入LPS+TNF-α+IL-1 β)。培养72h后，MTT测定AC活力；分光光度仪测定细胞培养上清中NO2-/NO3-含量；电镜、流式细胞术检测细胞凋亡；Western Blot检测胞浆内细胞色素C的表达；原位杂 交检测AC Caspase-3 mRNA表达。结果：预处理组细胞活力明显高于炎症 组(0.81±0.29 vs 0.46±0.15)，差异有显著性(P<0.01)。炎症 组AC活力与其分泌的一氧化氮(NO)呈负相关(r=-0.604，P<0.05)。预处理 组凋亡率［(15.2±7.9)%］低于炎症组［(29.7±10.4)%］，P<0.0 5。预处理组胞浆内细胞色素C的含量较炎症组明显减少(14 784±2 096 vs 31 049± 3 784)，P<0.01。炎症组的Caspase 3 mRNA表达强于预处理组。结论：①LPS，IL-1β，TNF-α可导致细胞活力下降。其细胞活力的下降与其分泌的NO增多有关。②增多的NO可导致AC凋亡；NO导致的AC凋亡可能与细胞色素C凋亡途径有关。

Mechanism of Cytokine Induced Apoptosis of Astrocytes in Rats

OBJECTIVE: To study the mechanism of LPS, IL 1β and TNF α induced apoptosis of astrocytes in rats. METHODS: The astrocytes of the neonatal rat were cultured in vitro and randomely assigned into the inflammation group (treated with LPS, IL 1β and TNF α), pre treatment group (L NMMA was administrated half an hour before treatment with LPS, IL 1β and TNF α) and normal control group. The cell viability was assayed by MTT; apoptosis was evaluated by electron microscope and flow cytometry; nitric oxide (NO) content was measured by spectrophotometer; cytochrome C content in cytoplasm was measured by Western Blot and Caspase 3 mRNA expression was detected by hybridization in situ. RESULTS: The cell viability in the pre treatment group was significantly higher than that of the inflammation group ( 0.81 ± 0.29 vs 0.46 ± 0.15 ), P< 0.01 . There was a negative correlation between the cell viability and NO content produced by astrocytes in the inflammation group (r= -0.604 , P< 0.05 ). The apoptosis rate of the pre treatment group was lower than that of the inflammation group ( 15.2 ± 7.9 ) % vs ( 29.7 ± 10.4 )%; P< 0.05 ]. The cytochrome C content in cytoplasm of astrocytes of the pre treatment group (14 784±2 096) decreased compared with that of the inflammation group (31 049±3 784) (P< 0.01 ). The expression of Caspase 3 mRNA in the inflammation group was intensified compared with that of the pre treatment group. CONCLUSIONS: Cytokines may lead to decrease of viability of astrocytes through increasing NO content produced by astrocytes. The increase of NO content can induce astrocyte apoptosis, which is related to the release of the cytochrome C from mitochondria to cell plasma, and activating the expression of Caspase 3 mRNA.