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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(n 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、Collagen Ⅲ、PI3K、Akt、p-PI3K和p-Akt的蛋白表达水平。n 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n A值总体比较差异均有统计学意义(n F＝90.110、198.680，均n P<0.001)，其中CLC-2 siRNA转染组CLC-2 mRNA相对表达量和细胞n A值明显低于无义siRNA组，差异均有统计学意义(均n 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(n F＝363.260，n 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(n 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(n F＝74.493、1 625.431，均n 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n 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(n F＝104.692，n 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(n P<0.001)。各组Collagen Ⅰ和Collagen Ⅲ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(n F＝112.073、456.931、340.889、43.021，均n P<0.001)，其中CLC-2 siRNA转染组Collagen Ⅰ和Collagen Ⅲ蛋白相对表达量及p-PI3K/PI3K比值和p-Akt/Akt比值明显低于无义siRNA组，差异均有统计学意义(均n P<0.05)。n 结论:靶向抑制CLC-2氯离子通道基因表达可通过PI3K/Akt信号通路促进HConF凋亡，抑制细胞迁移、胶原合成及胶原收缩，抑制纤维化过程。“,”Objective: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 (n 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, collagen Ⅲ, PI3K, Akt, p-PI3K and p-Akt proteins were measured by Western blot.n Results:Significant differences were found in relative expression levels of CLC-2 mRNA and n A value among four groups (n F=90.110, 198.680; both at n P<0.001). The relative expression level of CLC-2 mRNA andn A value were significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, showing statistically significant differences (both at n 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 (n F=363.260, n 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 (n P<0.001). Statistically significant differences were found in cell migration rate and the number of migrating cells among four groups (n F=74.493, 1 625.431; both at n P<0.01). The cell migration rate of HConF in CLC-2 siRNA transfected group was significantly lower and the number of migrating cells was significantly smaller than those of nonsense siRNA group, with statistically significant differences (both atn P<0.001). A statistically significant difference in contraction rate was found among four groups (n F=104.692, n P<0.001). The contraction rate of HConF was significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, and the difference was statistically significant (n P<0.001). Statistically significant differences were found in relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio, and p-Akt/Akt ratio among four groups (n F=112.073, 456.931, 340.889, 43.021; all at n P<0.001). The relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio and p-Akt/Akt ratio in CLC-2 siRNA transfected group were significantly lower than those of nonsense siRNA group, showing statistically significant differences (all atn P<0.05).n Conclusions:Targeted blocking of CLC-2 chloride channel gene expression can inhibit fibrosis of HConF by promoting apoptosis of HConF through PI3K/Akt signaling pathway and inhibit fibrotic processes such as cell migration, collagen synthesis and collagen contraction.