ATP通过P2Y_1受体引发脊髓背角星形胶质细胞[Ca^(2+)]i升高和GFAP表达上调

P2Y_1 receptor mediates the increase of [Ca^(2+)]i and the expression of GFAP in dorsal horn astrocyte

目的观察体外培养的背角星形胶质细胞P2Y1受体激活对其[Ca2+]i的变化和GFAP表达的影响。方法培养并纯化脊髓背角星形胶质细胞,采用免疫组织化学染色观察背角星形胶质细胞P2Y1受体及GFAP的表达,激光共聚焦技术观察星形胶质细胞[Ca2+]i的变化。结果体外培养的大鼠脊髓背角星形胶质细胞大多表达P2Y1受体;P2Y受体激动剂ATP、ADP、ADP-βs剂量依赖性促进星形胶质细胞[Ca2+]i升高;10μmol/L的ATP、ADP和ADP-βs显著增加胞内[Ca2+]i,此作用可被特异性P2Y1受体拮抗剂MRS2179所阻断,并具量效关系。免疫组织化学染色结果显示,100μmol/L的ATP、ADP和ADP-βs作用下,星形胶质细胞GFAP表达上升,此效应可被100μmol/L的MRS2179所抑制。结论体外培养的大鼠脊髓背角星形胶质细胞表达P2Y1受体;P2Y1受体介导了ATP、ADP及ADP-βs促进星形胶质细胞[Ca2+]i升高和GFAP表达增强的过程。

Objective To investigate the expression of P2Y1 receptor in cultured rat dorsal horn astmcytes, and the effects of ATP, ADP, and ADP-βs （P2Y receptor agonists） on [Ca^2＋]i and GFAP expression in dorsal horn astmcytes. Methods The expression of GFAP and P2Y1 receptor in cultured dorsal horn astrecytes were observed by using immunohistochemical staining. After addition of ATP, ADP, and ADP-βs respectively, the changes of intracellular calcium fluorescence intensity of astrocytes were measured by laser scanning confocal microscopy. Results Immunofluorescence labeling showed that most of GFAP positive astrocytes also stained for the P2Y1 receptor protein in cultured dorsal horn astrecyte. ATP, ADP, and ADP-βs increased the Ca^2＋ fluorescent intensity in cultured dorsal horn astrecytes in a dose-dependent manner. T he Ca^2＋ fluorescent intensity significantly increased by adding 10 μmol/L ATP, ADP, and ADP-βs, MRS2179, a P2Y1 receptor antagonist, could dose-dependently inhibit the [Ca^2＋]i mobilization induced by 10 μmol/L ATP, ADP, and ADP-βs. The expression of GFAP was increased when astrecytes treated with 100 μmol/L ATP, ADP, and ADP-βs, but MRS2179 （ 100 μmol/L） could inhibit these effects. Conclusion The cultured dorsal horn astrocytes express P2Y1 purinoceptors, which participate in the ATP-, ADP-, and ADP-βs-evoked Ca^2＋ mobilization and astrocytic activation.