CLC-2氯离子通道靶向阻滞对人结膜成纤维细胞纤维化的抑制作用及其机制  

Inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts

作　　者：吴沙仁高娃 崔仁哲[1] 卢迪[1] 丁浩轩 孙丽霞 Wu Sharengaowa;Cui Renzhe;Lu Di;Ding Haoxuan;Sun Lixia(Department of Ophthalmology,Yanbian University Affiliated Hospital,Yanji 133000,China)

机构地区：[1]延边大学附属医院眼科,延吉133000

出　　处：《中华实验眼科杂志》2022年第4期294-302,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81760171);吉林省教育厅"十三五"科学技术项目(JJKH20191117KJ)。

摘　　要：目的探讨CLC-2氯离子通道靶向阻滞对人结膜成纤维细胞(HConF)纤维化过程的抑制作用。方法将HConF分为空白对照组、脂质体2000(Lipo2000)组、无义小干扰RNA(siRNA)组和CLC-2 siRNA转染组,其中空白对照组不做任何处理,其他各组分别采用含有相应转染试剂的培养基培养。采用实时荧光定量PCR法检测各组细胞中CLC-2 mRNA的表达水平;采用CCK-8试剂盒检测各组HConF的增生能力,即吸光度(A)值;采用流式细胞仪检测各组HConF凋亡比例;采用细胞划痕实验和Transwell迁移实验检测各组HConF迁移能力;采用胶原收缩实验检测各组胶原面积收缩率;采用Western blot法检测各组细胞胶原蛋白(Collagen)Ⅰ、CollagenⅢ、PI3K、Akt、p-PI3K和p-Akt的蛋白表达水平。结果空白对照组、Lipo2000组、无义siRNA组和CLC-2 siRNA转染组CLC-2 mRNA相对表达量和细胞A值总体比较差异均有统计学意义(F=90.110、198.680,均P<0.001),其中CLC-2 siRNA转染组CLC-2 mRNA相对表达量和细胞A值明显低于无义siRNA组,差异均有统计学意义(均P<0.001)。空白对照组、Lipo2000组、无义siRNA组和CLC-2 siRNA转染组细胞凋亡率分别为(4.78±1.10)%、(4.54±1.51)%、(4.82±0.88)%和(28.90±0.91)%,总体比较差异有统计学意义(F=363.260,P<0.001),其中CLC-2 siRNA转染组细胞凋亡率明显高于无义siRNA组,差异有统计学意义(P<0.001)。各组细胞迁移率和细胞迁移数量总体比较差异均有统计学意义(F=74.493、1625.431,均P<0.001),其中CLC-2 siRNA转染组细胞迁移率明显低于无义siRNA组,细胞迁移数量明显少于无义siRNA组,差异均有统计学意义(均P<0.01)。各组细胞胶原面积收缩率总体比较差异有统计学意义(F=104.692,P<0.001),其中CLC-2 siRNA转染组细胞胶原面积收缩率明显低于无义siRNA组,差异有统计学意义(P<0.001)。各组CollagenⅠ和CollagenⅢ蛋白相对表达量及p-PI3K/PI3K比值和p-Akt/Akt比值总体比较差异均有统计学意义(F=112Objective To investigate the inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts(HConF).Methods HConF were divided into blank control group,lipofectamine 2000(Lipo2000)group,nonsense small interfering RNA(siRNA)group,and CLC-2 siRNA transfected group.The HConF were cultured in medium containing the corresponding transfection reagents according to grouping.No intervention was given to blank control group.The expression level of CLC-2 mRNA of HConF was detected by real-time fluorescence quantitative PCR;absorbance(A)value indicating the proliferative ability of HConF was determined by CCK-8 kit;the apoptosis ratio of HConF was tested by flow cytometry;the migration ability of HConF was identified by cell scratch test and Transwell migration assay;the contraction rate of HConF was assayed by collagen contraction test;the expression levels of collagenⅠ,collagenⅢ,PI3K,Akt,p-PI3K and p-Akt proteins were measured by Western blot.Results Significant differences were found in relative expression levels of CLC-2 mRNA and A value among four groups(F=90.110,198.680;both at P<0.001).The relative expression level of CLC-2 mRNA and A value were significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group,showing statistically significant differences(both at P<0.001).The proportion of apoptotic HConF in blank control group,Lipo2000 group,nonsense siRNA group,and CLC-2 siRNA transfected group was(4.78±1.10)%,(4.54±1.51)%,(4.82±0.88)%and(28.90±0.91)%,respectively,and a statistically significant difference was found(F=363.260,P<0.001).The proportion of apoptotic HConF was significantly higher in CLC-2 siRNA transfected group than nonsense siRNA group,with a statistically significant difference(P<0.001).Statistically significant differences were found in cell migration rate and the number of migrating cells among four groups(F=74.493,1625.431;both at P<0.01).The cell migration rate of HConF in CLC-2 siRNA transfected group was significantly lower and t

关 键 词：青光眼 滤过性手术 氯离子通道 滤过泡 瘢痕化 细胞凋亡 细胞迁移 胶原收缩 

分 类 号：R775[医药卫生—眼科]