三磷酸腺苷对N9小胶质细胞的损伤作用及其机制

Injury effect of adenosine triphosphate on N9 microglia

目的:研究三磷酸腺苷(ATP)对N9小胶质细胞的损伤作用及其机制。方法:取对数生长期N9小胶质细胞,随机分为3组:(1)正常对照组:常规培养,不进行ATP处理;(2)ATP组:接种24 h后行ATP处理;(3)KN-62(P2X7受体阻断剂)干预组:KN-62孵育30 min后行ATP处理,且KN-62存在于ATP作用整个过程。XTT法测各组N9小胶质细胞的活力;倒置相差显微镜观察细胞形态变化;流式细胞术检测细胞周期及凋亡;细胞免疫荧光检测P2X7受体表达;Western blotting检测细胞P2X7受体蛋白水平;ELISA检测细胞上清液中白细胞介素1β(IL-1β)含量。结果:500μmol/L ATP和1 mmol/L ATP对细胞活力损伤程度较小,作用24 h后,细胞活力仍可达88.5%±5.5%和88.2%±8.4%。当ATP浓度达到或高于2 mmol/L时,细胞活力迅速降低,细胞发生皱缩,且随ATP作用时间延长,细胞活力逐渐降低,细胞密度逐渐减少,细胞皱缩程度加重,而KN-62干预后细胞活力较ATP组明显增多,细胞密度及形态明显好于ATP组。细胞周期及凋亡检测结果显示,ATP可使细胞周期阻滞于S期,细胞凋亡比例明显较对照组增多(P<0.01),KN-62干预可显著减轻ATP引起的细胞周期阻滞,细胞凋亡比例显著减少(P<0.01)。免疫荧光显示,ATP及KN-62干预对N9小胶质细胞上P2X7受体的表达分布没有影响。Western blotting显示,正常对照组、ATP组及KN-62干预组间P2X7受体蛋白水平没有明显差异(P>0.05)。ELISA结果显示,ATP及KN-62干预对IL-1β的释放没有作用。结论:高剂量ATP可诱发N9小胶质细胞损伤,其机制可能与P2X7受体介导的细胞周期阻滞和细胞凋亡有关,而与小胶质细胞的炎症反应无关。

AIM： To investigate the injury effect of adenosine triphosphate （ATP） on N9 microglia. METHODS： N9 microglia in logarithmic growth phase was randomly divided into 3 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 h. In KN -62 intervention group, after pretreatment with KN - 62 for 30 min, ATP was added in the ceils. The cell viability was assessed by XTT assay. Cellular morphological changes were observed under phase - contrast microscope. The cell cycle and apoptosis were detected by flow cytometry. The expression of P2X7 receptor was examined by staining. The pro- tein levels of P2X7 receptor were measured by Western blotting. The concentration of IL - 1β in the culture supernatant was detected by ELISA. RESULTS： ATP at dose of 500 μmol/L and 1 mmol/L only caused small damage to the cell viability of N9 microglia. The cell viability was 88.5% ＋5.5% and 88.2% ＋8.4% after treated with ATP for 24 h,respectively. The cell viability dropped rapidly and cell shrinkage occurred when the concentration of ATP increased to 2 mmol/L or higher. With the extension of experiment time, the cell viability and cell density decreased further and cell shrinkage was getting worse. KN -62 intervention improved the viability of N9 microglia injured by ATP. The morphology and density of N9 microglia in KN -62 intervention group were much better than those in ATP group. ATP arrested N9 microglia at S phase and increased cell apoptosis significantly （P 〈0. 01 vs control group）. KN -62 intervention obviousl.y relieved the cell cycle arrest and decreased the cell apoptosis caused by ATP （P 〈0. 01 ）. ATP and KN -62 intervention had no effect on the distribution of P2X7 receptor. The protein levels of P2X7 receptor had no significant difference among the 3 groups（ P 〉 0. 05 ）. ATP and KN - 62 intervention had no effect on the release of IL - 1 β. CONCLUSION： High dose of ATP damages N9 mieroglia and its mechanism may be related to cell cycle arrest and apoptosis mediated by P2X7 receptor but not to inflammatory response caused by micreglia.