雷帕霉素对体外培养人翼状胬肉成纤维细胞增生、移行和纤维化的抑制作用

摘要

目的 观察哺乳动物雷帕霉素靶点(mTOR)通路抑制剂雷帕霉素对人翼状胬肉成纤维细胞增生和移行的抑制作用.方法 收集2015年5—7月沈阳市第四人民医院初发翼状胬肉患者术中切除的胬肉组织,利用组织块贴壁法原代培养翼状胬肉组织成纤维细胞(PFBs),波形蛋白细胞免疫荧光法进行细胞鉴定,取第3～5代细胞进行后续实验.利用甲基偶氮四唑(MTT)法检测不同摩尔浓度雷帕霉素处理条件下的细胞活力.将细胞分为正常对照组和雷帕霉素组,划痕试验观察各组细胞的迁移情况,实时定量PCR法检测各组细胞培养24 h MKI67、α.平滑肌肌动蛋白(α.SMA)、fibronectin、caspase3、mTOR及LC3B mRNA表达情况.结果 体外培养的PFBs为长梭形,波形蛋白表达阳性.MTT检测显示,雷帕霉素处理的PFBs细胞活力呈剂量依赖性.30μmol/L雷帕霉素组细胞活力为0μmol/L雷帕霉素组的(76.67±8.84)％,差异有统计学意义(P<0.001),30μmol/L被选择为后续实验工作浓度.划痕后48 h,正常对照组细胞相对划痕宽度为(2.45±0.76)％,明显低于雷帕霉素组的(35.40±11.62)％,差异有统计学意义(P<0.05).实时荧光定量PCR结果显示,雷帕霉素组细胞增生标志物MKI67、细胞成肌纤维化标志物 α.SMA和细胞骨架蛋白fibronection、mTOR mRNA相对表达量均明显低于正常对照组,自噬标志物LC3B mRNA相对表达量明显高于正常对照组,差异均有统计学意义(均P<0.05).2个组凋亡相关基因caspase3 mRNA的相对表达量比较,差异无统计学意义(P=0.861).结论 雷帕霉素可能通过阻断mTOR通路抑制翼状胬肉成纤维细胞的增生、移行和纤维化,其具体作用机制仍有待进一步研究.雷帕霉素可能成为预防翼状胬肉进展及术后复发的有效药物.%Objective To investigate the inhibitory effect of rapamycin,an mammalian target of rapamycin (mTOR) pathway inhibitor,on the proliferation,migration and fibrosis of human pterygium fibroblasts (PFBs). Methods Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People's Hospital from May to July 2015. The tissues were cultured in vitro and the PFBs were identified by anti.human vimentin immunofluorescence assay. The 3 to 5 generation cells were used for the experiments. The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium ( MTT) . The cells were divided into normal control group and rapamycin group, and the scratch wound healing test was used to evaluate migration of the PFBs. The expressions of MKI67,α.smooth muscle actin (α.SMA), fibronectin,caspase3, mammalian target of rapamycin ( mTOR ) and LC3B mRNA were detected by real.time quantitative PCR. Results The cultured cells showed morphology of long spindle and were vimentin immunopositive. The cell viability in rapamycin treated PFBs demonstrated a dose.dependent decrease. At 24 hours after culture,The cell viability in 30μmol/L rapamycin group was (76. 67±8. 84)％ of that in 0μmol/L rapamycin group ( P<0. 001 ) . The relative residual scratch width in 30μmol/L rapamycin group was ( 35. 40 ± 11. 62 )％ 48 hours after scratch,which was significantly greater than (2. 45±0. 76)％ in the normal control group (P<0. 05). Real.time quantitative PCR showed that the mRNA expressions of MKI67,α.SMA,fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P<0. 05). The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P<0. 05). The mRNA expression of caspase3 was not significantly different between the two groups (P=0. 861). Conclusions Rapamycin can effectively inhibit the proliferation, migration and fibrosis of PFBs without affecting the cell survival. Detailed mechanism remains to be further studied. Rapamycin may serve as an anti.fibrosis agent to prevent the progression and recurrence of pterygium in the future.