孕足月羊水干细胞的分离培养  

Isolation and culture of stem cells from term amniotic fluids

作　　者：刘慧[1] 刘大庆[2] 闫志风[1] 李宝伟[2] 管利东[2] 岳文[2] 吕阳[2] 裴雪涛[2] 李亚里[1] 

机构地区：[1]解放军总医院妇产科,北京市100853 [2]解放军军事医学科学院野战输血研究所干细胞与再生医学研究室,北京市100850

出　　处：《中国组织工程研究与临床康复》2009年第14期2733-2737,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军医药卫生科研基金资助项目(06G101)~~

摘　　要：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： Studies on the isolation, culture and biological character of second trimester amniotic fluid stem cells have obtained some progresses. However, studies addressing whether term amniotic fluids can serve as a novel source of stem cells are few. OBJECTIVE： To investigate the possibility of isolating stem cells from term amniotic fluids. DESIGN, TIME AND SETTING： The observational study was performed at the Department of Gynaecology and Obstetrics, General Hospital of Chinese PLA and Research Room of Stem cells and Regeneration Medicine, Academy of Military Medical Sciences of Chinese PLA from October 2007 to August 2008. MATERIALS： Term amniotic fluids were obtained following Caesarean delivery at the General Hospital of Chinese PLA. Fetal anomaly and systemic disease pregnant women were excluded. Ten samples of amniotic fluid were collected. 2-month male BALB/C nude mice, weighing 15-20 g, were used. METHODS： Term amniotic fluids （38 -40 pregnancy weeks） were cultured and stem cells were isolated from the ten samples. The fifth and tenth passage cells in the logarithmic phase were harvested. Absorbance values were measured at 450 nm with a microplate reader. Growth curve of amniotic fluid stem cells were drawn. When 70% of cells were confluent, adipogenic induction medium was employed to examine differentiation potency in vitro. Chromosome karyotype of amniotic fluid stem cells was analyzed at the fourth and 11th passages. At the fourth to sixth passage, amniotic fluid stem cells （1 ×10^7） were subcutaneously infused into the 2-month BALB/C nude mice for 8 weeks. MAIN OUTCOME MEASURES： Morphology characteristics, growth curve, and in vitro differentiation capacity of amniotic fluid stem cells were measured. Flow cytometry and immunofluorescence were applied to detect the expression of surface markers of amniotic fluid stem cells. RESULTS： Stem cells from 4 of 10 term amniotic fluids samples were successfully isolated. The amniotic fluid stem cells were spindle shaped and grew rap

关 键 词：羊水 干细胞 孕晚期 细胞培养 

分 类 号：R394.2[医药卫生—医学遗传学]