背景:孕中期羊水干细胞的分离培养及生物学性状研究已取得了一定进展,但孕足月羊水是否也可作为干细胞来源目前报道不多。目的:探讨从孕足月羊水中分离培养羊水干细胞的可行性。设计、时间及地点:观察性实验,于2007-10/2008-08在解放军总医院妇产科及解放军军事医学科学院干细胞及再生医学研究室完成。材料:孕足月羊水来自在解放军总医院行剖宫产、排除胎儿畸形及母体全身性疾病的孕妇,告知后同意留取羊水标本10例;鼠龄2个月雄性BALB/C裸鼠,体质量15～20g,用于致瘤实验的观察。方法:从10例孕晚期(孕38～40周)羊水中分离扩增羊水干细胞进行传代培养,取第5,10代对数生长期的细胞在酶标仪450nm波长处检测吸光度(A)值,绘制羊水干细胞生长曲线;待细胞70%汇合时换为脂肪诱导培养基用于检测体外分化能力;分别于第4代,第11代行染色体核型分析;取第4～6代羊水干细胞1×107个注射到BALB/C裸鼠皮下,观察8周。主要观察指标:①羊水干细胞的形态学特点及生长曲线。②流式细胞仪及免疫荧光法检测羊水干细胞表面标志的表达。③体外向脂肪细胞分化的能力。④染色体核型及致瘤实验结果。结果:10例孕晚期羊水中有4例分离到梭形可持续传代的细胞群,细胞增殖旺盛;羊水干细胞原代生长较慢,传代后生长迅速,第5代,第10代细胞的生长曲线形态相似;流式细胞仪检测证实孕足月羊水干细胞表达CD44,CD29,CD105等间质来源标志,免疫荧光检测显示表达胚胎来源标志SSEA-4及oct-4;换为脂肪诱导培养基后6d,细胞内出现透明、浑圆的小液滴,表明向脂肪样细胞分化;所有标本的染色体核型均为46XX或46XY,第11代染色体核型保持正常;羊水干细胞注射到裸鼠体内后无肿瘤形成。结论:孕晚期羊水也可分离培养到干细胞,可以作为干细胞的新来源。 BACKGROUND: The progress of the isolation, culture and biological characterization of the second trimester amniotic fluid stem cells has been made. However, whether full term amniotic fluid can be used as the source of stem cells is not reported yet. Objective: To investigate the feasibility of isolation and culture of amniotic fluid stem cells from pregnant term amniotic fluid. DESIGN, TIME AND SETTING: Observational experiments were performed at the Department of Obstetrics and Gynecology, PLA General Hospital, and the Stem Cell and Regenerative Medicine Laboratory, Academy of Military Medical Sciences, People’s Liberation Army from October 2007 to August 2008. MATERIALS: Pregnant full-term amniotic fluid was obtained from pregnant women who underwent cesarean section at the General Hospital of the Chinese People’s Liberation Army and to exclude fetal malformations and maternal systemic diseases. Ten months after consent, samples of amniotic fluid samples were obtained. Male BALB / C nude mice Quality 15 ~ 20g, for the observation of tumorigenic experiments. Methods: The amniotic fluid stem cells were isolated and cultured in 10 third trimester pregnancies (38-40 weeks pregnant), and the absorbance (A) value was measured at 450 nm wavelength on the 5th and 10th generation of logarithmic growth phase , To draw the amniotic fluid stem cell growth curve; when the cell confluence of 70% to adipogenic induction medium for the detection of differentiation in vitro; respectively, 4th and 11th generation line chromosome karyotype analysis; take 4th to 6th generation of amniotic fluid stem cells 1 × 107 were injected subcutaneously into BALB / C nude mice for 8 weeks. MAIN OUTCOME MEASURES: ① Morphological characteristics and growth curve of amniotic fluid stem cells. ② Flow cytometry and immunofluorescence method to detect the expression of amniotic fluid stem cell surface marker. ③ in vitro differentiation of adipocytes ability. ④ chromosome karyotype and tumorigenicity test results. RESULTS: Four of the ten cases of amniotic fluid during the third trimester of pregnancy were isolated from the spindle-shaped cell population with sustained proliferation. The primary growth of amniotic fluid stem cells was slow, and the growth of the amniotic fluid stem cells was rapid. The growth curves of the 5th and 10th passage cells FACS analysis showed that all the mesenchymal markers such as CD44, CD29 and CD105 were expressed on the full-term amniotic fluid stem cells by flow cytometry. The expression of embryonic-derived markers SSEA-4 and oct-4 were detected by immunofluorescence and changed to adipogenic induction medium on day 6 , The cells appeared transparent, rounded small droplets, indicating differentiation of adipose-like cells; all specimens of chromosome karyotype are 46XX or 46XY, the 11th generation chromosome karyotype remains normal; amniotic fluid stem cells injected into nude mice without tumor form. Conclusion: Amniotic fluid can also be isolated and cultured into stem cells in the third trimester of pregnancy, which can be used as a new source of stem cells.