【摘要】 目的:观察硫化氢（H₂S）供体硫氢化钠（Na HS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X₇受体表达的影响。方法:实验取对数期形态结构及生长分化良好的大鼠小胶质细胞,随机分4组,每组设3个复孔。（1）正常对照组:常规培养,不进行ATP处理。（2）ATP组:接种细胞24 h后ATP处理。（3）NaHS+ATP组:Na HS预先孵育30 min后再用ATP处理,并且Na HS始终存在于反应体系中。（4）KN-62（P2X₇受体阻断剂）+ATP组:KN-62预先孵育30 min,其余同Na HS+ATP组。MTT检测各组细胞活力,荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性,Western blot检测各组P2X₇受体表达水平。结果:（1）与对照组相比,不同浓度的ATP（1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力,Na HS（200μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01）,但Na HS达400μmol/L浓度时,其保护作用未进一步增加。（2）随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,Na HS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。（3）ATP（3 mmol/L）能上调P2X₇受体蛋白表达水平,而Na HS（200μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论:Na HS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力,提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。更多还原

【Abstract】 Objective: To observed the effect of sodium hydrosulphide（ Na HS）,a donor of H₂S on the cell viability,the membrane permeability and the expression of P2X₇ receptor induced by adenosine triphosphate（ ATP） in rat microglia. Methods: Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group,the cells were cultured without ATP treatment. In ATP group,the cells were treatment with ATP after cultured for 24 hours. In Na HS + ATP group,the cells were incubated with Na HS for 30 min before ATP,and Na HS always existed in the reaction system. In KN-62 + ATP group,the cells were pretreated with KN-62 for 30 min,the others were as the same as Na HS + ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X₇ receptor was examined by immunofluorescence staining. Results:（1） Compared with control group,the cell viability dropped after treatment with ATP（ 1、3、5、10 mmol/L） for 3 hours. When pre-incubation with Na HS（ 200 μmol/L）,the cell viability was apparently higher than that of ATP alone group（ P < 0.01）,while 400 μmol/L had no further beneficial.（2）The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia,but this effect was counteracted by Na HS pretreatment（ P < 0. 01）.（3） The expression of P2X₇ receptor protein was significantly increased after ATP（ 3 mmol/L） for 3 h. While the expression upregulation of P2X₇ receptor protein induced by ATP was significantly counteracted by pretreating with Na HS（ 200 μmol/L）（ P < 0. 01）. Conclusion: Na HS could reduce the expression of P2X₇ receptor,decrease membrane permeability,and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X₇ receptor.更多还原