背景白内障囊外摘出术后晶状体囊残留的晶状体上皮细胞（LECs）的生物学行为改变是后发性白内障的主要病理改变。Rho激酶抑制剂盐酸法舒地尔具有抑制细胞骨架的重构、迁移、黏附及纤维化等作用，但对LECs的作用尚不清楚。
目的探讨盐酸法舒地尔对人LECs株HLEB-3细胞增生、迁移的作用及其对细胞外基质（ECM）合成的影响。
方法常规培养HLEB-3细胞，对照组细胞仍进行常规培养，药物组培养液中加入不同浓度的盐酸法舒地尔，使其终浓度分别为10、20、40和60 μmol/L，分别于培养后12、24和48 h采用细胞计数试剂盒8（CCK-8）法检测各组细胞的增生情况，计算不同剂量盐酸法舒地尔对细胞增生的抑制率；分别于处理后12、24、36和48h采用Transwell小室迁移实验检测各组的迁移细胞数；并于上述时间点采用ELISA法检测各组细胞上清中Ⅰ型胶原（COL-Ⅰ）、纤维连接蛋白（FN）的质量浓度及细胞内α-平滑肌肌动蛋白（α-SMA）质量浓度，对各组间的检测结果进行比较。
结果不同浓度盐酸法舒地尔组HLEB-3细胞增生抑制率均明显高于对照组，随着盐酸法舒地尔浓度的增加，HLEB-3细胞增生抑制率明显增加，组间总体差异有统计学意义（ F 浓度=966.727， P=0.000）；随着盐酸法舒地尔作用时间的延长，HLEB-3细胞增生抑制率明显增加，总体比较差异有统计学意义（ F 时间=280.428， P=0.000）。Transwell小室迁移实验显示，实验后48 h，对照组穿过聚碳酸酯膜的细胞数为（29.20±1.28）个，10、20、40和60 μmol/L盐酸法舒地尔组迁移的细胞数分别为（24.40±1.33）、（17.00±1.10）、（14.60±0.68）和（6.60±1.29）个，随着盐酸法舒地尔浓度的增加，HLEB-3细胞的迁移数逐渐减少，差异有统计学意义（ F=57.34， P<0.05）．随着盐酸法舒地尔浓度的增加和作用时间的延长，细胞上清液中COL-Ⅰ和FN质量浓度逐渐降低，差异均有统计学意义（COL-Ⅰ： F 浓度=143.992， P=0.000； F 时间=113.745， P=0.000.FN: F 浓度=81.761， P=0.000； F 时间=69.602， P=0.000）；细胞内α-SMA质量浓度逐渐下降，差异均有统计学意义（ F 浓度=78.156， P=0.000； F 时间=65.162， P=0.000）。
结论盐酸法舒地尔可抑制体外培养HLEB-3的增生、迁移及ECM的合成，其作用呈剂量和时间依赖性。
BackgroundBiological behavior changes of human lens epithelial cells (LECs) and extracellular matrix (ECM) synthesis are major events in the pathogenesis of posterior capsule opacification (PCO). Rho-kinase inhibitor fasudil can inhibit the cytoskeleton remodeling, migration and fibrosis, however, the mechanism of its effect in LECs is below understood.
ObjectiveThis study was to investigate the role of Rho-kinase inhibitor fasudil in proliferation, migration of human LECs and ECM synthesis in vitro.
MethodsHuman LECs line HLEB-3 cells were regularly cultured and assigned to the control group and the drug treatment groups. Different doses of fasudil were added into the medium to make the final concentrations 10, 20, 40 and 60 μmol/L, respectively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay in 12, 24 and 48 hours after addition of drug to calculate the growth inhibitory rate. Transwell test was employed to detect the migrating number of the cells in 12, 24, 36 and 48 hours after action of drug. The concentrations of collagen type Ⅰ (COL-Ⅰ), fibronectin (FN) in cell supernatant and α-smooth muscle actin (α-SMA) content in the cells were measured by ELISA.
ResultsThe cell growth inhibitory rates in different doses of fasudil groups were higher than that in the control group. The cell growth inhibitory rates were gradually elevated as the increases of fasudil concentrations and prolong of action time ( F concentration=966.727, P=0.000; F time=280.428, P=0.000). Transwell migration assay showed that the number of migration cells across polycarbonate membrane was 29.20±1.28 in the control group after 48 hours of incubation, which was much more than 24.40±1.33, 17.00±1.10, 14.60±0.68, 6.60±1.29 in the 10, 20, 40 and 60 μmol/L fasudil groups, respectively, with a significant difference among the five groups ( F=57.34, P<0.05). COL-Ⅰ and FN protein contents in supernatant were gradually declined with the increase of drug concentrations and lapse of time (COL-Ⅰ: F concentration=143.992, P=0.000; F time=113.745, P=0.000.FN: F concentration=81.761, P=0.000; F time=69.602, P=0.000), and the α-SMA levels in the cells were significantly reduced with the increase of drug concentrations and lapse of time ( F concentration=78.156, P=0.000; F time=65.162, P=0.000).
ConclusionsFasudil can inhibit the proliferation, migration and ECM synthesis of human LECs in vitro in a dose- and time-dependent manner.
任师杰,张凤妍,祁颖,等. Rho激酶抑制剂盐酸法舒地尔对人LECs增生、迁移及ECM合成的抑制作用[J]. 中华实验眼科杂志,2015,33(4)：311-315.
DOI:10.3760/cma.j.issn.2095-0160.2015.04.005版权归中华医学会所有。
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