人胚胎视网膜色素上皮细胞在微孔聚酰胺纳米纤维膜上的培养

Culture of human fetal retinal pigment epithelial cells on electrospun polyamide nanofibers

目的研究人胚胎视网膜色素上皮(RPE)细胞在微孔聚酰胺纳米纤维膜(EPN)上的生长和分化情况,观察EPN能否作为一种理想的膜性支架而支持RPE细胞的生长和分化,为后续Bruch膜替代物的研究奠定基础。方法将培养的人胚胎RPE细胞分别以5×105/cm2的密度种植在EPN和常规塑料(对照组)的Transwell植入物的24孔培养板中,低钙培养基(Ca2+浓度0.1mmol/L)中培养,待细胞贴壁生长后,改用正常钙培养基(Ca2+浓度1.8mmol/L)培养4周。用相差显微镜每天观察2组细胞的形态及生长情况;分别以免疫荧光染色及Westernblot法鉴定RPE细胞中CK-18、细胞连接蛋白ZO-1及RPE65的表达,观察培养的RPE细胞遗传表型的维持情况。结果培养在EPN上的RPE细胞与对照组一样,能够较好地贴壁生长,形成单细胞层。EPN较常规塑料更适于RPE细胞贴附生长。EPN的纤维排列类似Bruch膜内胶原层纤维排列。EPN上培养的人胚胎RPE细胞CK-18表达阳性,与对照组相比,ZO-1和RPE65均呈较强表达,较好地保持了原代RPE细胞的表型。结论生长在EPN上的人胚胎RPE细胞能够较好地生长、分化并形成单细胞层,有效地维持了原代RPE细胞的表型。为后续Bruch膜替代物组织工程学研究提供了新的思路。

Background Retinal pigment epithelial（RPE）cellular transplantation alone is thought to have a unideal outcome in the treatment of age-related macular degeneration（AMD）,because Bruch＇s membrane occur alteration with aging.Therefore,prosthetic replacement of Bruch＇s membrane may be an important adjunct to successful RPE transplantation.ObjectivePresent study was to investigate the attachment,growth and differentiation of human fetal RPE cells seeded onto electrospun polyamide nanofibers（EPN）.MethodsHuman RPE cells was isolated from the fetal eyes derived from therapeutic abortuses at 20-21 weeks gestation.Primary RPE cells plastic culture was established in DMEM containing 0.1mmol/L Ca2＋ and 10% fetal bovine serum.Upon subconfluence,the cells were subcultured at the density of 5×105 cells/cm2 in the above-mentioned culture medium onto the exposed surface of EPN in 24-plastic culture wall.In control,RPE cells were subcultured at the same cell density and in the same culture medium onto the plastic transwall in 24-plastic culture well.After attachment,the Ca2＋ concentration in the medium was elevated to 1.8mmol/L for 4 weeks.Growth and morphology of the cells were monitored under the phase contrast microscope,and the phenotype was identified by immunoinfluorescence staining with antibodies against cytokeratin 18,tight junction protein ZO-1.The expression of RPE65 protein was detected by Western blot.The protocal of this study was approved by the Ethic Committee of Second Xaingya Hospital,and written informed consent for the use of material was obtained from the pregnant woman prior to the medical procedure.ResultsRPE cells attached well and exhibited a complete monolayer within both EPN layers and plastic layers.EPN was preferable to plastic in the attachment of RPE cells.EPN resembled the inner collagenous layer of Bruch＇s membrane.Compared to adult in situ RPE cells,fetal RPE cells cultured on nanofibers surface could achieve a comparable phenotype.Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures within the EPN substrates.Results also showed that the RPE cells within the EPN substrates adopted a strong expression of ZO-1 and RPE65 in comparison to the plastic control.ConclusionEPN may be used as a basement membrane-containing matrix to maintain the phenotype of RPE cells in vitro and work as a potential Bruch＇s membrane substitutes and facilitate subsequent transplantation.