水蛭提取液对p38丝裂原活化蛋白激酶介导的人视网膜色素上皮细胞信号转导途径的影响

Influence of leech extract on signal transduction of human retinal pigment epithelial cell mediated by p38 mitogen activated protein kinase

目的研究水蛭提取液对p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)介导的人视网膜色素上皮(human retinal pigment epithelial,hRPE)细胞跨膜信号转导途径的影响。方法选3-5代生长良好的hRPE细胞,细胞同步化后分为:正常对照组、凝血酶组、水蛭组、合药组(凝血酶+水蛭组),根据前期实验数据,用64g.L-1的水蛭提取液与500NIHU.L-1的凝血酶分别(或共同)孵育hRPE细胞30min后,采用流式细胞术及免疫细胞化学方法检测孵育hRPE细胞内p38MAPK的表达。结果根据各组P-p38MAPK免疫细胞化学平均光密度值发现,凝血酶组(1.1950±0.0226)与正常对照组(1.0550±0.0187)相比,OD值上升,差异有显著统计学意义(P<0.01);水蛭提取液组(0.7567±0.0710)与正常对照组相比,OD值下降,差异有显著统计学意义(P<0.01);合药组(0.5550±0.0650)与凝血酶组相比,OD值下降,差异有显著统计学意义(P<0.01)。根据各组P-p38MAPK流式细胞术检测的荧光量比较(OD值)发现,凝血酶组(6.4417±0.0720)与正常对照组(5.4567±0.1825)相比,荧光量升高,差异有显著统计学意义(P<0.01);水蛭组(4.4483±0.2015)与正常对照组相比,荧光量下降,差异有显著统计学意义(P<0.01);合药组(4.1433±0.0680)与凝血酶组相比,荧光量下降,差异有显著统计学意义(P<0.01)。结论水蛭提取液能抑制hRPE细胞的增生,其机制与p38 MAPK介导的信号转导通路有关。

Objective To investigate the influence of leech extract on signal transduction of human retinal pigment epithelial（hRPE） cells mediated by p38 mitogen activated protein kinase （MAPK）. Methods Well growth hRPE cells at the 3rd to the 5th generation were chosen, and divided into normal control group, thrornbin group, leech extract group and combined leech extract and thrombin group after cell synchronization. Based on the previous experiments, 54 g ·L^-1 leech extract and 500 NIHU·L^-1 thrombin were used to incubate hRPE cells for 30 minutes, respectively. The expression of p38 MAPK was measured by flow cytometry and immtmocytochemistry. Results The optical density（OD） value of P-p38 MAPK in thrombin group was 1. 195 0 ± 0. 022 6 higher than that in normal control group 1.055 0 ±0. 018 7（P〈0.01） ,0. 756 7 ±0.071 0 in leech extract group lower than that in normal control group （ P 〈 0.01 ）, and 0. 555 0 ± 0. 055 0 in combined leech extract and thrombin group lower than that in thrombin group （P 〈0. 01 ）. The fluorescence quantity of P-p38 MAPK in thrombin group was 6. 441 7 ± 0.072 0 higher than that in normal control group 5. 456 7 ± 0. 182 5 （P 〈 0.01 ） ,4. 448 3 ± 0. 201 5 in leech extract group lower than that in normal control group （P 〈 0. 01 ）, and 4. 143 3 ± 0.058 0 in combined leech extract and thrombin group lower than that in thrombin group （P 〈 0.01 ）. Conclusion Leech extract can inhibit the proliferation of hRPE cells,which may have relation with the signal transduction mediated by p38 MAPK.