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目的研究P2Y1受体对缺血时星形胶质细胞产生胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)及胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor, GDNF)的影响及其相关信号通路。方法分别利用右侧大脑中动脉线拴阻塞及培养细胞缺氧无营养后恢复正常培养,造成体内、外缺血再灌注模型。用免疫荧光标记、实时定量 RT-PCR、Western blotting、酶联免疫吸附试验观察 P2Y1 受体、GDNF 定位,检测 GFAP、GDNF及信号分子的表达变化。结果与单纯性缺血组比较,用选择性拮抗剂 MRS2179 阻断 P2Y1 受体后,可使体内、外星形胶质细胞产生的GFAP减少,同时使其产生GDNF增加。体外缺氧无营养并阻断P2Y1受体后:可使磷酸化蛋白激酶B(Akt)及cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)升高,而使磷酸化JAK2及STAT3 (Ser727)降低;JAK2的抑制剂AG490在降低磷酸化STAT3(Ser727)的同时也降低GFAP表达水平;PI3-K的抑制剂LY294002可降低磷酸化的Akt及CREB; MEK1/2抑制剂U0126可同时降低磷酸化的JAK2、STAT3 (Ser727)、Akt及CREB。结论 P2Y1受体参与短时性缺血时星形胶质细胞GFAP及GDNF的产生过程,相关信号途径分别为JAK2/STAT3 和 PI3-K/AKT/CREB,并且两条途径存在串话。
Objective To investigate the effects of P2Y1 receptor on the expression of glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor (GDNF) in astrocytes during ischemia Influence and its related signal pathways. Methods The right middle cerebral artery occlusion and occlusion of cultured cells were cultured without hypoxia and then returned to normal culture, resulting in in vivo and external ischemia-reperfusion models. Immunofluorescence staining, real-time quantitative RT-PCR, Western blotting, enzyme-linked immunosorbent assay P2Y1 receptor, GDNF localization, detection of GFAP, GDNF and signal molecules expression changes. Results Compared with the simple ischemia group, blocking the P2Y1 receptor with the selective antagonist MRS2179 could reduce the GFAP produced by in vivo and in vitro astrocytes and increase the GDNF production. In vitro hypoxia-naive and block P2Y1 receptor: phosphorylation of protein kinase B (Akt) and cAMP response element binding protein (CREB) increased phosphorylation of JAK2 and STAT3 (Ser727 ); JAK2 inhibitor AG490 decreased the expression of GFAP while phosphorylating STAT3 (Ser727); PI3-K inhibitor LY294002 decreased phosphorylation of Akt and CREB; MEK1 / 2 inhibitor U0126 decreased both phosphorylation JAK2, STAT3 (Ser727), Akt and CREB. Conclusions P2Y1 receptors are involved in the process of GFAP and GDNF production in astrocytes under transient ischemic condition. The related signaling pathways are JAK2 / STAT3 and PI3-K / AKT / CREB, respectively. There are crosstalk between the two pathways.