Turner综合征羊水干细胞的分离及特征

Isolation and characterization of amniotic fluid-derived stem cells in Turner’s syndrome

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 cells. However, few studies are reported on amniotic fluid-derived mesenchymal stem cells in 45, X/46, XX （Turner＇s syndrome）. OBJECTIVE： To develop a simple culture protocol to isolate amniotic fluid-derived mesenchymal stem cells and investigate the biological characteristics of amniotic fluid-derived mesenchymal stem cells.METHODS： We developed a gradient dilution culture protocol to isolate a population of 45, X/46, XX （Turner＇s syndrome） arnniotic fluid-derived mesenchyrnal stern cells from second-trimester amniocentesis. The morphology of amniotic fluid-derived mesenchymal stem cells was observed by light microscope. The karyotype was analyzed Specific cell surface antigens and cell cycle of the clonal amniotic fluid-derived mesenchymal stem cells at passage 4 were characterized by flow cytometry. Osteogenic differentiation of amniotic fluid-derived mesenchymal stem cells was confirmed by alkaline phosphatase staining and alizarin red staining. RESULTS AND CONCLUSION： The cultured human amniotic fluid-derived m esenchymal stem cells proliferated rapidly after passage. Karyotype mapping showed abnormal female chromosome type with 45, X/46, XX observed. The amniotic fluid-derived mesenchymal stem cells had an immunophenotype similar to that of common mesenchymal stem cells and were positive for CD29, CD44, CD90 and CD105, but negative for CD34 and CD45. The cell cycle measurement showed that amniotic fluid-derived mesenchymal stern cells cultured in vitro could maintain strong proliferation ability. Alkaline phosphatase staining and alizarin red staining results confirmed that amniotic fluid-derived mesenchymal stem cells could be successfully induced to differentiate into osteocytes under specific culture media. These results demonstrated that arnniotic fluid-derived mesenchymal stem cells in 45, X/46, XX （Turner＇s syndrome） were successfully isolated and the cells had a great potential of proliferation and showed the characteristics of mesenchymal stem cells.