K115对TGF-β1诱导的人Tenon囊成纤维细胞增殖和迁移的影响及其机制

Effects of Rho protein kinase inhibitor K115 on proliferation and migration of human Tenon’s fibroblasts induced by TGF-β1 and its mechanisms

目的探讨K115对转化生长因子-β1(TGF-β1)诱导的人Tenon囊成纤维细胞增殖和迁移的影响及其机制。方法人Tenon囊成纤维细胞取自于河北省人民医院眼科行青光眼手术的患者。采用CCK-8实验检测K115对人Tenon囊成纤维细胞增殖的影响,筛选出TGF-β1对细胞增殖最佳作用浓度为5.0μg·L^(-1),最佳作用时间为24 h;K115对细胞增殖起始作用浓度为5.0μmol·L^(-1),最佳作用浓度为10.0μmol·L^(-1)。依据这两个浓度进行分组,分别是对照组(不加TGF-β1与K115),TGF-β1组(加5.0μg·L^(-1) TGF-β1),TGF-β1+5.0μmol·L^(-1) K115组(加5.0μg·L^(-1)TGF-β1和5.0μmol·L^(-1)的K115),TGF-β1+10.0μmol·L^(-1) K115组(加5.0μg·L^(-1) TGF-β1和10.0μmol·L^(-1)的K115)。采用Transwell实验检测K115对人Tenon囊成纤维细胞迁移的影响;免疫荧光法测定人Tenon囊成纤维细胞中α-SMA蛋白的表达;流式细胞仪检测细胞凋亡情况。对实验所得各指标进行统计学分析。结果本研究细胞培养状态良好,6~8周可见人Tenon囊成纤维细胞铺满培养瓶瓶底。用CCK-8试剂盒检测各组细胞增殖情况结果显示,对照组、TGF-β1组、TGF-β1+5.0μmol·L^(-1) K115组、TGF-β1+10.0μmol·L^(-1) K115组光密度值分别为0.977±0.042、1.306±0.059、0.862±0.035、0.558±0.042,TGF-β1+5.0μmol·L^(-1) K115组、TGF-β1+10.0μmol·L^(-1) K115组光密度值均小于TGF-β1组(均为P<0.05),并且呈现浓度依赖性减小,K115浓度越大细胞增殖减少幅度越大。Transwell实验结果显示,对照组、TGF-β1组、TGF-β1+5.0μmol·L^(-1) K115组、TGF-β1+10.0μmol·L^(-1) K115组迁移细胞数分别为(38.67±3.06)个、(81.00±4.00)个、(44.33±3.21)个、(23.67±2.52)个;TGF-β1+5.0μmol·L^(-1) K115组与TGF-β1组比较,迁移细胞数降低,差异有统计学意义(P<0.05);TGF-β1+10.0μmol·L^(-1) K115组与对照组及TGF-β1组比较,迁移细胞数均明显降低,差异均有统计学意义(均为P<0.05)。各组细胞内α-SMA蛋白免疫荧光染色结果显示,对照组人Tenon囊成纤维细胞胞浆内可见散在分布的绿色荧光物质,TGF-β1处理后24 h(TGF-β1组),胞浆内绿色丝状荧光物质明显增加,荧光强度差异有统计学意义(P<0.05);与TGF-β1组比较,TGF-β1+5.0μmol·L^(-1) K115组和TGF-β1+10.0μmol·L^(-1) K115组细胞胞浆内的荧光强度呈浓度依赖性减弱,K115浓度越高荧光强度减弱越明显(均为P<0.05)。流式细胞仪检测结果显示,对照组细胞凋亡率为(2.400±0.346)%,TGF-β1组为(0.900±0.173)%,TGF-β1+5.0μmol·L^(-1) K115组为(1.333±0.252)%,TGF-β1+10.0μmol·L^(-1) K115组为(1.067±0.058)%;TGF-β1组的细胞凋亡率较对照组降低(P<0.05);TGF-β1+5.0μmol·L^(-1) K115组和TGF-β1+10.0μmol·L^(-1) K115组的细胞凋亡率均较对照组降低(均为P<0.05),而与TGF-β1组比较,差异均无统计学意义(均为P>0.05)。结论K115对TGF-β1诱导的人Tenon囊成纤维细胞增殖和迁移有明确的抑制作用,其作用机制可能与K115抑制了TGF-β1诱导的细胞内α-SMA的表达进而调控了细胞活化有关。

Objective To study the regulation of Rho protein kinase inhibitor K115 on the proliferation and migration of human Tenon’s fibroblasts induced by TGF-β1 and its related mechanisms.Methods Human Tenon’s fibroblasts were collected from patients undergoing glaucoma surgery in the Ophthalmology Department of Hebei Provincial People’s Hospital.CCK-8 assay was used to detect the effect of K115 on the proliferation of human Tenon’s fibroblasts.The optimal concentration of TGF-β1 on cell proliferation was 5.0μg·L^(-1),and the optimal time was 24 hours.The initial concentration of K115 on cell proliferation was 5.0μmol·L^(-1),and the optimal concentration was 10.0μmol·L^(-1).According to the screening results of TGF-β1 and K115 concentration,the subsequent experiments were set as control group(without TGF-β1 and K115),TGF-β1 group(5.0μg·L^(-1) TGF-β1 was added),TGF-β1+5.0μmol·L^(-1) K115 group(5.0μg·L^(-1)TGF-β1 and 5.0μmol·L^(-1) K115 were added),and TGF-β1+10.0μmol·L^(-1) K115 group(5.0μg·L^(-1)TGF-β1 and 10.0μmol·L^(-1) K115 were added).Transwell assay was used to detect the effect of K115 on the migration of human Tenon’s fibroblasts.The expression ofα-SMA protein in human Tenon’s fibroblasts was determined by immunofluorescence assay.Cell apoptosis was detected by flow cytometry.Statistical analysis was carried out on data obtained from the experiment.Results The cultured cells were in good condition,and human Tenon’s fibroblasts could be seen on the bottom of the culture bottle at 6-8 weeks.The results of CCK-8 kit showed that the optical density(OD)values of control group,TGF-β1 group,TGF-β1+5.0μmol·L^(-1) K115 group,TGF-β1+10.0μmol·L^(-1) K115 group were 0.977±0.042,1.306±0.059,0.862±0.035,0.558±0.042,respectively.The OD values of TGF-β1+5.0μmol·L^(-1) K115 group and TGF-β1+10.0μmol·L^(-1) K115 group were lower than those of TGF-β1 group(both P<0.05),showing a concentration-dependent decrease.The decrease of cell proliferation was greater with the increase of K115 concentration.Transwell assay results showed that the migratory cell counts of control group,TGF-β1 group,TGF-β1+5.0μmol·L^(-1) K115 group and TGF-β1+10.0μmol·L^(-1) K115 group were 38.67±3.06,81.00±4.00,44.33±3.21 and 23.67±2.52,respectively.The migratory cell count in TGF-β1+5.0μmol·L^(-1) K115 group was lower than that in TGF-β1 group,and the difference was statistically significant(P<0.05).Compared with the control group and TGF-β1 group,the migratory cell count in TGF-β1+10.0μmol·L^(-1) K115 group was significantly decreased,and the differences were statistically significant(both P<0.05).There were scattered green fluorescent substances in the cytoplasm of human Tenon’s fibroblasts in the control group.After treatment with TGF-β1 for 24 h(TGF-β1 group),the amount of green filamentous fluorescent substances in the cytoplasm was significantly increased,and the difference of fluorescence intensity was statistically significant(P<0.05).Compared with TGF-β1 group,the intensity of green filamentous fluorescence in the cytoplasm of TGF-β1+5.0μmol·L^(-1) K115 group and TGF-β1+10.0μmol·L^(-1) K115 group decreased in a concentration-dependent manner.The higher the concentration of K115 was,the more obviously the fluorescence intensity decreased(both P<0.05).The results of flow cytometry showed that the apoptosis rate in control group was(2.400±0.346)%,TGF-β1 group was(0.900±0.173)%,TGF-β1+5.0μmol·L^(-1) K115 group was(1.333±0.252)%,and TGF-β1+10.0μmol·L^(-1) K115 group was(1.067±0.058)%.The apoptosis rate in the TGF-β1 group was lower than that in the control group(P<0.05).The apoptosis rates in TGF-β1+5.0μmol·L^(-1) K115 group and TGF-β1+10.0μmol·L^(-1) K115 group were lower than that in control group(both P<0.05),but there was no significant difference compared with TGF-β1 group(both P>0.05).Conclusion K115 has a definite inhibitory effect on TGF-β1 induced human Tenon’s fibroblasts proliferation and migration.The mechanism might be that K115 inhibitions the expression ofα-SMA induced by TGF-β1 and then regulates the activation of cells.