法舒地尔对转化生长因子-β_2诱导的人晶状体上皮细胞迁移及细胞外基质合成的影响

Effects of fasudil on human lens epithelial cell migration and extracellular matrix synthesis induced by transforming growth factor-β_2

目的探讨法舒地尔对转化生长因子-β2(transforming growth factor-β2,TGF-β2)诱导的人晶状体上皮细胞(lens epithelial cells,LECs)HLE-B3迁移及细胞外基质合成的影响。方法将培养的HLE-B3分为以下4组:A组:含体积分数10%胎牛血清的完全培养基;B组:含10μg·L-1TGF-β2的完全培养基;C组:含15μmol·L-1法舒地尔的完全培养基;D组:含10μg·L-1TGF-β2+15μmol·L-1法舒地尔的完全培养基。CCK-8法检测不同浓度的法舒地尔对细胞增殖的影响,Transwell小室迁移实验检测各组细胞迁移能力,ELISA法检测各组细胞上清I型胶原(collange-I,COL-I)、纤维连接蛋白(fibronectin,FN)的表达。结果随着法舒地尔干预浓度的增加,对HLE-B3细胞的增殖抑制作用逐渐增强,各浓度组间差异有统计学意义(P=0.000)。随着法舒地尔作用时间延长,对HLE-B3细胞增殖的抑制作用也逐渐增强,各组间差异有统计学意义(P=0.000)。Transwell小室迁移实验显示,48 h时A组、B组、C组、D组细胞穿过聚碳酸酯嵌套膜的个数分别为(29.17±1.17)个、(87.60±5.31)个、(15.60±1.07)个、(44.61±2.06)个,各组间差异有统计学意义(F=111.614,P=0.000);B组较A组、D组较C组迁移细胞明显增多(均为P=0.000);D组较B组、C组较A组迁移细胞明显减少(P=0.000、P=0.003),但D组较A组、B组较C组细胞迁移数量明显增多(P=0.003、P=0.000)。各组细胞上清液中FN、COL-I的含量于12 h开始升高,24 h、36 h时持续升高,48 h时增高明显,各时间点各组间以及组内各时间点间差异均有统计学意义(均为P<0.05)。与A组相比,各时间点B组FN和COL-I蛋白的表达明显增加(均为P<0.05);各时间点D组较B组显著下降(均为P<0.05);但C组与A组比较差异均无统计学意义(均为P>0.05);D组与A组、C组与B组、C组与D组比较,COL-I、FN差异均有统计学意义(均为P<0.05)。结论法舒地尔可抑制TGF-β2诱导的细胞迁移和细胞外基质的合成,可能在后发性白内障的防治过程中发挥作用。

[ Abstract] Objective To investigate the effects of fasudil on human lens epithe- lial cell （HLEC）-B3 migration and extracellular matrix synthesis induced by transfor- ming growth factor-β2 （TGF-β2）. Methods The cultured HLEC-B3 were divided into 4 groups：group A was the control group,the cells in group B were exposed in 10 μg · L -1 TGF-β2,group C in 15μmol·L-1fasudil and group D in 15 μmol· L-1 fasudil ＋10μg· L-1 TGF-β2. Cell proliferation was assessed by CCK-8 assay, and the mobility of HLE-B3 was evaluated by Transwell assay. Then ELISA method was used to check the protein content of the fibronectin （FN） and the collagen-I （COL-I） in the supernatant. Results After treatment with different concentrations of fasudil, the proliferation of HLE-B3 was significantly inhibited, there was statistical difference among each group （P = 0.000）. Fasudil inhibited HLE-B3 proliferation in a time dependent manner, there was statistical difference among different time points （ P = 0. 000 ）. Transwell migration assay showed that the number of migration cells across the polycarbonate membrane at 48 hours in group A,B,C,D were 29.17 ± 1.17,87.60 ±5.31,15.60 ±1.07,44.51 ±2.06, respectively,there was statistical difference among each group （F =111. 614,P = 0. 000）, group B was obvious more than group A, and group D was obvious more than group C （ all P = 0.000）, group D was obvious less than group B, group C was obvious less than group A （P = 0. 000 , 0. 003 ）, but group D was obvious more than group A, group B was more than group C （P = 0.003,0. 000 ）. The contents of FN and COL-1 in supernatant of each group increased from 12 hours, sustained at 24 hours and 36 hours, obvious increased at 48 hours, there were statistical differences among different groups at different time points and among different time points of different groups （ all P 〈 0. 05）. Compared with group A,the expression of FN and COL-l in group B at different time points obvious increased （ all P 〈 0.05 ）, group D were obvious less than group B at different time points （ all P 〈 0. 05 ）, but there was no statistical difference between group C and group A （ all P 〉 0. 05 ）, and there were statistical differences between group A and D, group C and B, group C and D （ all P 〈 0.05 ）. Conclusion Fasudilcan inhibit the HLEC migration and extracellular matrix synthesis induced by transforming growth factor-β2, which may pro- vide new options for prevention and treatment of posterior capsule opacification.