羊水多潜能干细胞的体外培养及其生物学特性

In vitro culture and biological characteristics of amniotic fluid-derived multipotent stem cells

背景:近来发现羊水中含有具有多向分化潜能的干细胞,具有向内胚层、中胚层和外胚层分化的能力。目的:建立羊水多潜能干细胞的体外培养方法,并探讨其生物学特性。设计、时间及地点:单一样本实验,于2007-04/2008-01在解放军军事医学科学院生物工程研究所完成。材料:5份羊水标本来自孕16～22周流产孕妇自愿捐献。方法:从人孕中期羊水中,采用贴壁筛选法分离羊水多潜能干细胞。主要观察指标:采用免疫荧光、流式细胞术和反转录聚合酶链反应等方法分析细胞的生物学特性和鉴定细胞表型。结果:体外培养的羊水多潜能干细胞呈梭形或类上皮细胞样,细胞增殖迅速,细胞倍增时间约为24h;细胞周期中G1、S、G2期的细胞所占百分比分别为72.88%、5.77%、21.35%;细胞表达Oct4、Nanog、SSEA-4、CD44和CD105,不表达CD34和CD45。结论:采用贴壁筛选法获得的羊水多潜能干细胞增殖能力强,同时表达胚胎和成体干细胞的部分标志,可能为介于胚胎干细胞与成体干细胞之间的一种过渡阶段的干细胞。

BACKGROUND： Recently, some reports mentioned that amniotic fluid contained multipotent stem cells, which could differentiate into endoderm, mesoderm and ectoderm. OBJECTIVE： To establish in vitro culture procedure of amniotic fluid-derived stem cells （AFMSCs）, and to study its biological characteristics. DESIGN, TIME AND SETTING： A single sample experiment was conducted at the Institute of Bioengineering, Academy of Military Medical Sciences of Chinese PLA from April 2007 to January 2008. MATERIALS： Five samples of amniotic fluid were collected from 5 aborted pregnant women of pregnancy for 16-22 weeks. METHODS： AFMSCs were isolated from second-trimester amniotic fluid by adherence. MAIN OUTCOME MEASURES： The biological characteristics and phenotypes of AFMSCs were detected by immunofluorescence, flow cytometry and reverse Ixanscription-polymerase chain reaction （RT-PCR）, RESULTS： AFMSCs were characterized by spindle and epithelioid appearance. The cells proliferated rapidly, and the doubling time of cells was about 24 hours. The percentages of Gb S, and （32 of cell cycle were respectively 72.88%, 5.77%, and 21.35%. The cells were positive for Oct4, Nanog, SSEA-4, CD44 and CD45, and negative for CD34 and CD45. CONCLUSION： AFMSCs have a great potential of proliferation, which express both embryonic and adult stem cell markers. They may be an intermediate stage between embryonic stem cells and adult stem cells.