目的 探讨Hedgehog蛋白对血-视网膜内屏障中人视网膜微血管内皮细胞(HRMEC)功能的影响及其可能的信号通路.方法 取在正常培养基中培养的HRMEC,分为正常对照组、0.5μmol/L激动剂组和1.0μmol/L激动剂组,分别加入终浓度为0、0.5和1.0μmol/L的Hedgehog激动剂purmorphamine;取高糖培养基中培养的HRMEC,分为高糖对照组、1.5μmol/L抑制剂组和2.5μmol/L抑制剂组,分别加入终浓度为0、1.5和2.5μmol/L的Smoothed抑制剂Erismodegib.采用MTS细胞增生实验和Transwell细胞迁移试验检测各组HRMEC增生A490值和相对迁移率.采用Western blot方法检测各组细胞鸟苷酸结合蛋白偶联受体(GPCRs)通路下游PLCγ1、Akt和Erk蛋白磷酸化水平变化.结果 高糖对照组细胞Hedgehog蛋白相对表达量为6.24±0.11,明显高于正常对照组的1.00±0.00,差异有统计学意义(t=667.573,P<0.001).0.5μmol/L激动剂组和1.0μmol/L激动剂组A490值分别为1.349±0.050和1.422±0.053,相对迁移率分别为2.34±0.14和3.59±0.32,明显高于正常对照组的1.203±0.101和1.00±0.00,差异均有统计学意义(均P<0.01).1.5μmol/L抑制剂组和2.5μmol/L抑制剂组细胞A490值分别为0.849±0.010和0.737±0.030,相对迁移率分别为0.43±0.02和0.27±0.01,明显低于高糖对照组的1.000±0.040和1.00±0.00,差异均有统计学意义(均P<0.01).0.5μmol/L激动剂组和1.0μmol/L激动剂组PLCγ1磷酸化比值、Akt磷酸化比值和Erk磷酸化比值均明显高于正常对照组,1.5μmol/L抑制剂组和2.5μmol/L抑制剂组PLCγ1磷酸化比值、Akt磷酸化比值和Erk磷酸化比值均明显低于高糖对照组,差异均有统计学意义(均P<0.01).结论 高糖诱导HRMEC中Hedgehog蛋白表达,Hedgehog蛋白可能通过调节GPCRs通路中PLCγ1、Akt和Erk磷酸化水平来调节HRMEC的功能.%Objective To explore the impact of Hedgehog protein on human retinal microvascular endothelial cell(HRMEC) and its signaling pathway. Methods The cultured HRMECs were divided into normal control group,0. 5μmol/L agonist group and 1. 0μmol/L agonist group,and were cultured in medium with final concentration of 0,0. 5 and 1. 0μmol/L Hedgehog agonist,respectively;HRMECs cultured in high glucose medium were divided into high glucose control group,1. 5μmol/L inhibitor group and 2. 5μmol/L inhibitor group. Erismodegib,the Smoothed inhibitor with final concentration of 0,1. 5 and 2. 5 μmol/L was added into corresponding group,respectively. MTS method and Transwell cell migration method were used to detect the proliferation( A490 value) and relative mobility of HRMEC. The phosphorylation of PLCγ1, Akt and Erk proteins were detected by Western blot. Results The relative expression of Hedgehog protein in the high glucose control group was 6. 24±0. 11,which was significantly higher than 1. 00±0. 00 in the normal control group(t=667. 573,P<0. 001). The A490 value was 1. 349±0. 050 and 1. 422±0. 053,and the relative mobility rate was 2. 34±0. 14 and 3. 59±0. 32 in the 0. 5μmol/L agonist group and the 1. 0μmol/L agonist group, respectively, which were significantly higher than 1. 203 ± 0. 101 and 1. 00 ± 0. 00 in the normal control group(all at P<0. 01). The A490 value was 0. 849±0. 010 and 0. 737±0. 030,and the relative mobility rate was 0. 43 ± 0. 02 and 0. 27 ± 0. 01 in the 1. 5 μmol/L inhibitor group and the 2. 5 μmol/L inhibitor group, respectively,which were significantly lower than 1. 000±0. 040 and 1. 00±0. 00 in the high glucose control group(all at P<0. 01). The phosphorylation ratios of PLCγ1,Akt and Erk in the 0. 5μmol/L agonist group and the 1. 0μmol/L agonist group were significantly higher than those in the normal control group ( all at P<0. 01 ) . The phosphorylation ratios of PLCγ1,Akt and Erk in the 1. 5μmol/L inhibitor group and the 2. 5μmol/L inhibitor group were significantly lower than those in the high glucose control group ( all at P<0. 01 ) . Conclusions High glucose induces the expression of Hedgehog protein in HRMEC. Hedgehog protein may regulate the function of HRMEC by regulating the phosphorylation of PLCγ1,Akt and Erk in G Protein-coupled receptors pathway.
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