氯通道阻断剂5-硝基-2-(3-苯丙胺)苯甲酸在内皮素-1对体外培养牛角膜内皮细胞增殖中的影响

目的观察氯通道阻断剂5-硝基-2-(3-苯丙胺)苯甲酸(NPPB)在内皮素-1(ET-1)对体外培养牛角膜内皮细胞增殖中的作用。方法实验设7组,对照组、1g·L-1二甲基亚砜(DMSO)组、200pmol·L-1ET-1组、200pmol·L-1ET-1+1g·L-1DMSO组和200pmol·L-1ET-1+50、100、200μmol·L-1NPPB3个实验组。采用倒置光学显微镜观察牛角膜内皮细胞形态。采用四甲基偶氮唑盐(MTT)比色分析法观察细胞增殖,计算细胞存活率。采用免疫细胞化学检测细胞增殖细胞核抗原(PCNA)表达,并采用计算机图像分析测定平均光吸收值。应用流式细胞仪检测细胞周期时相的变化,计算细胞增殖指数。结果 NPPB能够使细胞形态发生异常,细胞内分裂相减少。50、100μmol·L-1NPPB使培养牛角膜内皮细胞MTT光吸收值、细胞存活率和PCNA阳性细胞的平均光吸收值较200pmol·L-1ET-1组均显著降低,并具有浓度依赖性。而NPPB浓度达到200μmol·L-1时,细胞趋于死亡。经100μmol·L-1的NPPB处理后,与对照组和200pmol·L-1ET-1组相比,出现明显的凋亡峰,G1期细胞比例明显增高,S期及G2/M期细胞比例则明显降低,增殖指数明显下降。结论 NPPB浓度依赖性抑制ET-1对培养BCECs的增殖作用,使细胞周期停滞在G1期。

Effect of chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid on proliferation of cultured bovine corneal endothelium cells induced by endothelin-1

Objective To study the role of chloride channel blocker 5-nitro-2-( 3-phenylpropylamino) benzoic acid( NPPB) on proliferation of bovine corneal endothelium cells induced by endothelin-1( ET-1). Methods The experiment was set up seven groups,including control group,1 g·L -1 dimethyl sulfoxide ( DMSO) group,200 pmol·L -1 ET-1 group,200 pmol·L -1 ET-1 combined with 1 g· L -1 DMSO group,and three experimental groups with 200 pmol· L -1 ET-1 combined with 50,100 and 200 μmol·L -1 NPPB respectively. The morphous of bovine corneal endothelium cells were observed with inversion light micro-scope. Cell proliferation was observed by MMT assay and cell survival rate was calculated. The expression of proliferating cell nu-clear antigen ( PCNA) was detected by immunocytochemistry staining and the average light absorption value was analyzed by computer medical image analysis system. The phase change of cell cycle was detected by flow cytometry and cell proliferation in-dex was calculated. Results NPPB could make cell aberrant morphogenesis,induced to decrease intra-cellular cleavage phase. Compared with 200 pmol·L -1 ET-1 group,50,100 μmol·L -1 NPPB decreased significantly MTT light absorption value,cell survival rate and the average light absorption value of positive expression of PCNA of cultured bovine corneal endothelium cells,and showed a concentration dependent. At the concentration of 200 μmol · L -1 NPPB,cells tended to die. Compared with 200 pmol·L -1 ET-1 group and control group,after treated with 100 μmol·L -1 NPPB,the rate of G1 stage increased,the rate of S stage and G2 /M stage and cell proliferation index decreased significantly,and obvious apoptotic peak presented. Conclusion NPPB concentration dependent had the inhibition effect on proliferation on bovine corneal endothelium cells induced by ET-1,and resulted in the cell cycle arrest at G1 stage.