MG132对人晶状体上皮细胞增生、移行和上皮-间充质转化的抑制作用

Inhibitory effect of MG132 on proliferation, migration and epithelial-mesenchymal transition of human lens epithelial cells

背景 晶状体后囊膜混浊（PCO）发生的主要病理机制是白内障术后后囊膜残留的晶状体上皮细胞（LECs）的增生、移行和分化,研究发现蛋白酶体抑制剂MG132可抑制牛LECs的增生,但对人LECs的生物学影响尚不明确. 目的 研究蛋白酶体抑制剂MG132对体外培养的人LECs增生、移行和上皮-间充质转化（EMT）的抑制作用.方法 收集白内障超声乳化摘出术中连续环形撕囊的囊膜片用组织块培养法进行原代培养并传代,取第2代或第3代LECs进行常规消化,调整细胞密度至5×105个/ml,以200 μl/孔接种至96孔板常规培养24 h,分别加入成纤维细胞生长因子-2（FGF-2）、MG132或MG132＋FGF-2,以基础培养液培养的细胞作为对照组,培养24 h后用MTT法测定吸光度（A490）值,计算LECs的增生率.在移行能力测定中,应用传代的人LECs分别加入FGF-2、MG132或MG132＋FGF-2,以基础培养液培养的细胞作为对照组,用无菌棉棒在细胞层中划出细胞裸露区,继续培养24 h,计数移行至裸露区的细胞,评估细胞移行率.在传代的人LECs培养液中分别加入转化生长因子-β2（TGF-β2）、MG132或MG132＋TGF-β2,以基础培养液培养的细胞作为对照组,培养24 h后用免疫组织化学法检测细胞质中纤维连接蛋白（FN）的表达. 结果 对照组、FGF-2组、MG132组和MG132＋FGF-2组的LECs增生值（A490）分别为0.582±0.020、0.723 ±0.010、0.434±0.011和0.465±0.008,总体差异有统计学意义（F=110.482,P＜0.01）,FGF-2组LECs增生值明显高于对照组,MG132组和MG132＋FGF-2组的LECs增生值明显低于对照组,差异均有统计学意义（P＜0.05）.对照组、FGF-2组、MG132组和MG132＋FGF-2组LECs移行数目分别为8.67±1.08、11.58±1.59、2.67±0.09和2.75±0.09,差异有统计学意义（F=34.301,P＜0.01）,其中FGF-2组LECs的移行数明显多于对照组,而MG132组和MG132＋FGF-2组LECs移行数明显少于对照组,差异均有统计学意义（P＜0.05）.与对照组比较,MG132组LECs的增生率和移行率分别降低25.4％和75.0％,FGF-2组LECs增生率和移行率分别增加24.2％和33.6％,MG132＋FGF-2组LECs的增生率和移行率分别下降20.1％和68.3％.对照组、TGF-β2组、MG132组和MG132＋TGF-β2组LECs中FN表达量（A值）分别为1.242±0.023、2.329±0.113、1.043±0.021和1.163±0.018,差异有统计学意义（F=113.752,P＜0.01）,TGF-β2组LECs中FN表达量明显高于对照组,而MG132组和MG132＋TGF-β2组明显低于对照组,差异均有统计学意义（P＜0.05）.TGF-β2可以诱导人LECs转分化,FN表达增强;MG132与TGF-β2同时存在时可以减弱人LECs转分化,FN表达下降. 结论 MG132抑制体外培养的人LECs的增生与移行,并可阻止TGF-β2诱导的人LECs的EMT.

Background The primary pathologic mechanism of posterior capsular opacification （PCO） is proliferation,migration and epithelial-mesenchymal transition （EMT） of residuary lens epithelial cells （LECs） after cataract extract surgery.Researches showed that MG132,a proteasome inhibitor,can attenuate the proliferation of bovine LECs,but its effect on human LECs remains unclear.Objective This study was to investigate the inhibitory effect of MG132 on proliferation,migration and differentiation of human LECs in vitro.Methods Human lens capsule were collected during the surgery.Human LECs were primarily cultured by explant method and passaged.The second or third generation of cells were incubated to 96-well plates at the density of 5×105/ml （200 μl/well） for 24 hours.Fibroblast growth factor-2 （FGF-2,10 mg/L）,MG132 （10 μmol/L） or MG132＋FGF-2 was added into the culture medium for 24 hours separately,and regular cultured cells served as the control group.The proliferation value （absorbance,A490） of the cells was assayed by MTT colorimetric method.A bare area was made by a sterile cotton swab in the cell layer,and migrated cell number in the blank zone was counted to evaluate the migration ability of the cells after 24 hours.Transforming growth factor-32（TGF-β2）,MG132 or MG132＋TGF-β2 was added into the culture medium for 24 hours separately,and the expression of fibronectin （FN） in the cells was detected using immunochemistry.Results The proliferation values （A490） of the cells were 0.582±0.020,0.723±0.010,0.434± 0.011 and 0.465±0.008 in the control group,FGF-2 group,MG132 group and MG132 ＋ FGF-2 group,respectively,showing a significant difference among the groups （F =110.482,P〈0.01）.The A value was significantly higher in the FGF-2 group and lower in the MG132 group and MG132＋FGF-2 group than that of the control group （all at P〈 0.05）.The migrated cell number was 8.67 ± 1.08,11.58 ± 1.59,2.67 ± 0.09 and 2.75 ± 0.09 in the control group,FGF-2 group,MG132 group and MG132＋FGF-2 group,respectively,with a significant difference among the groups （F=34.301,P〈0.01）,and more cells in the blank zone were seen in the FGF-2 group and less cells were in the MG132 group and MG132＋FGF-2 group in comparison with the control group （all at P〈0.05）.Compared with the control group,the proliferative rate and migrating rate of the cells declined by 25.4% and 75.0% in the MG132 group as well as 20.1% and 68.3% in the MG132＋FGF-2 group,but in the FGF-2 group,they increased by 24.2% and 33.6%.The expressing levels （A value） of FN in the LECs were 1.242±0.023,2.329±0.113,1.043 ±0.021 and 1.163±0.018 in the control group,TGF-β2 group,MG132 group and MG132 ＋TGF-β2 group,respectively,with a significant difference among the groups （F =113.752,P〈0.01）,a considerably increased expressing value was seen in the TGF-β2 group and decreased value was in the MG132 group and MG132＋TGF-β2 group when compared with the control group （all at P〈0.05）.Conclusions MG132 can effectively inhibit the proliferation,migration and differentiation of human LECs in vitro.