羊水作为间充质干细胞来源的可行性研究

Amniotic fluid:A novel source for mesenchymal stem cells

目的探讨孕中期羊水作为胎儿间充质干细胞（MSCs）来源的可行性。方法采用机械手段挑取孕中期羊水培养体系中类成纤维细胞的MSCs集落，体外培养扩增，观察细胞生长特性，流式细胞仪检测MSCs表面抗原表达及细胞周期，RT-PCR检测Oct-4、SCF、M-CSF、FL细胞因子的表达。结果用机械手段挑取出的细胞接种1～2d后贴壁，形态呈梭形，7～8d融合呈成纤维细胞状。体外扩增原代可获得（4～6）×10^6个细胞，10代可获得（1～5）×10^12个细胞，10代后细胞增殖能力下降。流式细胞仪检测结果显示：CD44表达阳性，CD34、CD45、HLA—DR不表达。RT-PCR检测结果显示：胎儿MSCs表达Oct-4、SCF、M-CSF、FL细胞因子。结论采用机械手段可从孕中期羊水培养体系中挑取出胎儿MSCs，与其他来源的MSCs特点相符。孕中期羊水可以作为一种新的胎儿间充质干细胞（MSCs）来源。

Objective To investigate the possibility of using second-trimester amniotic fluid as a novel source for mesenchymal stem cells （MSCs）. Methods Fetal MSCs which displayed fibroblastlike morphology were separated from the amniocyte culture and sub-cultured. The proliferation and growth characteristics were observed in primary and passage cultures. The cell-surface antigens and cell cycle of fetal MSCs were analyzed with flow cytometry. The expressions of Oct-4, SCF, M-CSF, FL were assayed with RT-PCR. Results Isolated cells were fetal MSCs. After culturing for 1 -2 days, MSCs were found to adhere to the culture bottle and were spindle-shaped. Seven to eight days later, the cells began to fuse like fibroblasts. After primary culture, approximately （4-6） × 10^6 cells were obtained. After passage 10, （1-5） × 10^12 cells were obtained. The proliferation ability decreased gradually after ten passages. These expanded MSCs were positive for CD44 and negative for CD34, CD45 and HLA-DR. RT-PCR analyses revealed that expanded MSCs express Oct-4, SCF, M-CSF, FL. Conclusions Fetal MSCs could be isolated mechanically from amniocyte culture systems. These cells were similar in cell morphology and phenotype to other tissues derived from MSCs. Amniotic fluid is a novel source for mesenchymal stem cells.