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建立体外稳定生长的人羊水干细胞(AFS)系并对其生物学特性作进一步鉴定。使用贴壁法分离培养羊水干细胞,通过扩增传代,建立体外稳定生长的人羊水干细胞系,采用RT-PCR检测干性基因的表达,流式细胞仪分析细胞表型,混合淋巴细胞培养检测对PBMC体外增殖的免疫调节作用并通过细胞计数研究其体外增殖潜能。成功建立三株体外稳定传代和生长的人羊水干细胞系,AFS表达干细胞特异性基因:Oct4、Sox2、Nanog、Klf-4、c-Mye和Rex-1;同时发现AFS高表达负性共刺激分子B7-H1、B7-H2、B7-H3、B7-H4、BTLA和不表达HLA-DR、CD40、CD80和CD86,AFS可抑制PBMC的体外增殖,不仅显示其具有低免疫原性,而且具有免疫调节作用。与此同时,AFS具有很高的体外增殖潜能。本研究建立的AFS系具有良好的干细胞生物学特性,可以作为一种来源充足的低免疫原性种子细胞用于组织工程的研究和应用。
The establishment of in vitro stable human amniotic fluid stem cells (AFS) line and its biological characteristics for further identification. The amniotic fluid stem cells were isolated and cultured by adherence method. Human amniotic fluid stem cell lines were established by passage of amplification. The expression of dry gene was detected by RT-PCR. The phenotypes were analyzed by flow cytometry. In vitro proliferation of PBMC immunoregulatory effect and by cell count study of its in vitro proliferation potential. Successfully established three human amniotic fluid stem cell lines stably passaged and grown in vitro. AFS expressed stem cell-specific genes Oct4, Sox2, Nanog, Klf-4, c-Mye and Rex-1. AFS inhibits the in vitro proliferation of PBMCs, B7-H1, B7-H2, B7-H3, B7-H4, BTLA and does not express HLA-DR, CD40, CD80 and CD86 not only showing low immunogenicity but also immunization Regulatory effect. At the same time, AFS has high potential for in vitro proliferation. The AFS system established in this study has good stem cell biological properties and can be used as a well-sourced, low-immunogenic seed cell for tissue engineering research and applications.