羊膜上皮细胞培养液抑制角膜基质细胞凋亡的实验研究

Suppression of rabbit corneal keratocytes apoptosis by culture medium of amniotic membrane epithelial cells

目的 对羊膜上皮细胞培养液抑制由肿瘤坏死因子(TNF-α)诱发的角膜基质细胞凋亡进行研究。方法 体外培养兔角膜基质细胞(RCK),30ng/ml TNF-α诱发角膜基质细胞凋亡。实验分为对照组、羊膜上皮细胞培养液组(羊膜组)和阴性对照组。应用FITC-dUTP-TUNEL和DAPI染色检测RCK细胞凋亡发生率,分别测定经24h培养后各组细胞凋亡特异性DNA梯形降解。Western blot检测细胞凋亡保护性因子Bcl-2、促进因子Bax在经羊膜上皮细胞培养液处理后于细胞中表达强度及Bcl-2/Bax比值变化。结果 羊膜上皮细胞培养液明显抑制由TNF-α诱发的兔角膜基质细胞凋亡,DAPI染色显示24h对照组,羊膜培养液组和阴性对照组细胞凋亡发生率分别为:42.4％±4.3％,2.2％±0.3％和2.7％±0.4％。DNA降解实验显示羊膜上皮细胞培养液明显抑制TNF-α诱导的DNA片段降解。Western blot显示羊膜上皮细胞培养液明显刺激RCK细胞Bcl-2蛋白的表达和分泌。结论 羊膜培养液中可能释放的多种细胞因子对TNF-α诱发的角膜基质细胞调亡具有明显的抑制作用,其作用机制之一是刺激Bcl-2在RCK细胞的表达,并使Bcl-2/Bax比值上调。

Objective To investigate the effects of culture medium of human amniotic membrane epithelial cells (AMEC) on corneal keratocyte cell apoptosis induced by tumor necrosis factor alpha (TNF-a). Methods AMEC were isolated and cultured in EGM basic medium for 5 days,and culture medium of AMEC was collected. Rabbit corneal keratocyte (RCK) cells were cultured and passage 2 or 3 was used. RCK cell apoptosis was induced by cultivating RCK with TNF-a (30ng/ml) for 24 hrs,and assayed by DAPI and TUNEL ( TdT-mediated dUTP-biotin nick-end labeling) staining and DNA laddering. The experiments were divided into control (EGM only) , AMEC medium (AMEC) and negative control (N-control; EGM only without TNF-ainducer) groups. Western blot was applied for checking Bcl-2 and Bax proteins in RCK cells. Results Apoptosis was successfully induced by TNF-a in RCK cells and was significantly suppressed by culture medium of AMEC. The apoptotic ratio of RCK cells was (42. 4±4. 3 ) % , (2. 2 ± 0. 3 ) % and ( 2. 7 ± 0. 4 ) % in control, AMEC medium and negative control group respectively. Both TUNEL staining and DIVA laddering showed that AMEC medium significantly inhibited the apoptosis of RCK cells. Culture medium of AMEC strongly up-regulated Bcl-2 protein, but no effect on Bax. Conclusion Anti-apoptotic factors from culture medium of AMEC can protect RCK cells from apoptosis induced by TNF-a through up-regulation of Bcl-2 protein and rising Bcl-2/Bax in RCK cells in part.