Turner综合征羊水干细胞的分离及特征*★◆

摘要

背景：羊水干细胞的分离培养及生物学特性相关研究取得了明显进展，但未见45，X/46，XX(Turner综合征)羊水细胞建系的报道。目的：建立体外培养人羊水来源干细胞的方法，初步探讨羊水干细胞的生物学特性。方法：孕中期羊膜腔穿刺获得1例染色体核型异常(45，X/46，XX)羊水标本，采用梯度稀释后低密度种植的方法获得羊水干细胞，体外传代培养，倒置显微镜观察细胞的贴壁生长情况，细胞染色体检查确认核型，流式细胞仪检测羊水干细胞特异性表面标志和细胞周期，进行成骨诱导分化及碱性磷酸酶染色和茜素红染色鉴定。结果与结论：羊水干细胞在体外培养体系中增殖迅速，核型为45，X/46，XX，流式细胞仪检测羊水干细胞表达CD29、CD44、CD90和CD105间充质干细胞表面标志，不表达造血干细胞标志CD45和CD34；大部分细胞处于 G1期，增殖能力强。羊水干细胞经成骨诱导分化后，碱性磷酸酶染色和茜素红染色阳性。结果可见该实验成功分离获得45，X/46，XX (Turner 综合征)羊水干细胞，其增殖能力强，表现间充质干细胞的特性。%BACKGROUND: Great progress has been achieved on studies on isolation, culture and biological characteristics of amniotic fluid-derived mesenchymal stem cel s. However, few studies are reported on amniotic fluid-derived mesenchymal stem cel s in 45, X/46, XX (Turner’s syndrome). OBJECTIVE: To develop a simple culture protocol to isolate amniotic fluid-derived mesenchymal stem cel s and investigate the biological characteristics of amniotic fluid-derived mesenchymal stem cel s. METHODS: We developed a gradient dilution culture protocol to isolate a population of 45, X/46, XX (Turner’s syndrome) amniotic fluid-derived mesenchymal stem cel s from second-trimester amniocentesis. The morphology of amniotic fluid-derived mesenchymal stem cel s was observed by light microscope. The karyotype was analyzed. Specific cel surface antigens and cel cycle of the clonal amniotic fluid-derived mesenchymal stem cel s at passage 4 were characterized by flow cytometry. Osteogenic differentiation of amniotic fluid-derived mesenchymal stem cel s was confirmed by alkaline phosphatase staining and alizarin red staining. RESULTS AND CONCLUSION: The cultured human amniotic fluid-derived mesenchymal stem cel s proliferated rapidly after passage. Karyotype mapping showed abnormal female chromosome type with 45, X/46, XX observed. The amniotic fluid-derived mesenchymal stem cel s had an immunophenotype similar to that of common mesenchymal stem cel s and were positive for CD29, CD44, CD90 and CD105, but negative for CD34 and CD45. The cel cycle measurement showed that amniotic fluid-derived mesenchymal stem cel s cultured in vitro could maintain strong proliferation ability. Alkaline phosphatase staining and alizarin red staining results confirmed that amniotic fluid-derived mesenchymal stem cel s could be successful y induced to differentiate into osteocytes under specific culture media. These results demonstrated that amniotic fluid-derived mesenchymal stem cel s in 45, X/46, XX (Turner’s syndrome) were successful y isolated and the cel s had a great potential of proliferation and showed the characteristics of mesenchymal stem cel s.