人羊膜上皮细胞培养液抑制角膜新生血管的实验研究

Suppression of corneal neovascularization by culture supernatant of human amniotic epithelial cells in vitro

背景角膜新生血管（CNV）是一种常见的眼部病变，研究其发病机制及其抑制剂是眼科研究的热点和难点。目的研究人羊膜上皮细胞（AECs）培养液对CNV的抑制作用及机制。方法消化法培养及鉴定人AECs，并收集培养液，酶联免疫吸附试验（ELISA）法检测色素上皮衍生因子（PEDF）和白细胞介素1受体拮抗剂（IL-1Ra）在培养液中的质量浓度。兔角膜上皮细胞分离后分别用无血清DMEM培养基、人AECs培养液、混合培养液（DMEM＋人AECs培养液）培养48h，采用实时定量聚合酶链反应（QRT—PCR）法检测不同培养液培养的角膜上皮细胞中血管内皮生长因子（VEGF）及碱性成纤维细胞生长因子（bFGF）的表达。用含质量分数10％胎牛血清的DMEM培养基培养人脐静脉血管内皮细胞（UVECs），并分别加入无血清DMEM、混合培养液和人AECs培养液，划痕法和细胞计数试剂盒8（CCK8）法检测各组培养液对人UVECs迁移的影响。分别在上述3种培养基中加入终质量浓度为50txg／L的bFGF，CCK8法检测各培养液中人UVECs的增生情况。在原子力显微镜（AFM）下观察人AECs培养液对人UVECs超微结构的影响。结果人羊膜培养和传代细胞角蛋白免疫组织化学染色阳性证实为人AECs。与无血清DMEM组相比，人AECs培养液组的兔角膜上皮细胞VEGFmRNA（1．00＋0．22VS．2．98_＋0．46）及bFGFmRNA（1．00＋0．36VS．2．55-＋0．48）的表达均明显下降，差异均有统计学意义（p_-0．001、0．002）；培养后不同时间人AECs培养液组人UVECs的增生吸光度（A）值明显下降，差异均有统计学意义（P〈0．05），人UVECs的迁移率下降，差异有统计学意义（P〈0．05）。AFM观察见人AECs培养液组的血管内皮细胞膜粗糙、表面颗粒紊乱，细胞间连接及伪足减少。ELISA法检测人AECs培养液中PEDF的质量浓度为（70．41±0．68）Ixg／L，IL-1Ra的质量浓度为（153．56±0．36）-s／L，无血清DMEM组中未检出。结论人AECs培养液可抑制角膜上皮VEGF及bFGF的表达，抑制血管内皮细胞的增生和迁移，细胞结构和功能改变，这可能是其抑制CNV的机制之一。

Background Corneal neovascularization （CNV） is a common eye disease. The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology. Objective This study was to investigate the effects of culture supernatant from human amniotic epithelial ceils （AECs） on CNV in vitro and its mechanism. Methods Human AECs were obtained from a placenta and cultured in serum-free medium for 48 hours, and the supernatant was collected. The levels of interlenkin-1 receptor antagonist （IL-1Ra） and pigment epithelium-derived factor （PEDF） in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Rabbit corneal epithelial cells （CECs） were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours, serum-free medium was used as the control group. The expression of vascular endothelial growth factor （VEGF） mRNA and basic fibroblast growth factor （bFGF） mRNA in rabbit CECs were measured by quantitative real-time PCR （QRT-PCR）. Human umbilical cord vein endothelial ceils （UVECs） were cultured in the three mediums above,and the proliferation of human UVECs （absorbanee value,A value） was tested by the cell counting kit 8 （CCK8）. Migration assay was performed by the wound healing method for the human UVECs. The membrane ultra-structure of human UVECs was examined under the atomic force microscope （ AFM）.Results Cultured and passaged human AECs showed a positive response for keratin. The expression of VEGF mRNA （1.00±0.22） and bFGF mRNA （1.00 ±0.36） in rabbit CECs was suppressed by the human AECs culture supernatant, with a significant reduction in comparison with the serum-free DMEM group （2.98 ±0.46,2.55±0.48 ） （P=0. 001,0. 002）. The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group （ 1. 941 ± 0. 036 versus 2. 144 ± 0. 059 ） （ P = 0. 000 ） , and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM, The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps, and decreased intercellular connection and cellular pseudopodia were found on the AFM image. The concentration of IL-1Ra was （153.56±0.36） ng/L and that of PEDF was （70.41 ±0. 68 ） p,g/L in the human AECs culture supernatant. Nothing was deteched in serum-free DMEM group. Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells. The effect may be associated with IL-1Ra and PEDF secreted by human AECs. These results suggest that human AECs may be a potential therapy for the inhibition of CNV.