孕中期羊水来源胎儿间充质干细胞体外诱导为平滑肌细胞的研究  

Fetal mesenchymal stem cells derived from second-trimester anmiotic fluid differentiate into smooth muscle cells in vitro

作　　者：朱哲[1] 潘骏[2] 李现铎[3] 耿红全[4] 陈方[1] 

机构地区：[1]上海交通大学医学院附属新华医院泌尿外科,200092 [2]上海市儿科医学研究所 [3]山东省千佛山医院泌尿外科 [4]上海交通大学医学院附属新华医院小儿泌尿外科,200092

出　　处：《中华小儿外科杂志》2006年第12期617-621,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(编号:30271351)

摘　　要：目的探讨孕中期羊水来源的胎儿间充质干细胞(MSC)体外诱导为平滑肌细胞的可能性。方法通过超声引导下羊膜腔穿刺获取人孕18～22周羊水,分离细胞,体外培养传代,机械方法挑取MSC克隆,体外扩增,流式细胞仪检测MSC表面抗原表达,以0．01 ng/ml、0．1 ng/ml、1 ng/ml和10ng/ml浓度的转录生长因子β(TGF-β)分别诱导第3代MSC 28 d,免疫组化染色检测各浓度TCF-β诱导后a-Actin在细胞内表达情况,透射电镜观察1 ng/mlTGF-β诱导28 d后细胞内肌丝含量,激光共聚焦显微镜测定该浓度组细胞在乙酰胆碱刺激下细胞内液钙离子浓度的变化。结果羊水来源的胎儿MSC在体外培养体系中增殖迅速,流式细胞仪检测结果显示CD44表达阳性,CD34、CD45和HLA-DR阴性。经1 ng/ml TGF-β诱导28 d后,细胞形态呈长梭形平滑肌样,融合后细胞分布形成特征性的“峰”和“谷”状,同时表达平滑肌细胞特异性蛋白标志a-Actin,细胞内出现黑色棒状肌丝,激光共聚焦显微镜显示该浓度TGF-β诱导后细胞内液钙离子基础值明显高于未诱导的细胞。在乙酰胆碱刺激下,细胞内液钙离子呈波峰样释放。结论羊水来源的胎儿MSC在体外可以定向分化为平滑肌细胞。Objective To investigate the potential of human fetal mesenchymal stem cells derived from second-trimester amniotic fluid to form smooth muscle cells. Methods Amniotic fluid was obtained by ultrasound-guided amniocentesis performed on pregnant women with a gestational age ranging from 18^th-22^nd weeks. The fetal MSC clones were isolated from the 3^rd amniotic fluid cells and am plified in vitro. The flow cytometer was used to analyze the expression of the MSC; surface antigens. After 4 weeks of induction with 0. 01 ng/ml,0. 1 ng/ml, 1 ng/ml and 10 ng/ml TGF-β, the expression of α-Actin and myonemes in the cells was studied with immunohistochemistry and TEM. The Achstimulated intracellular calciumion concentration was assessed with confocal microscopy. Results The MSC expanded rapidly in the culture medium. The isolated cultured MSCs consisted of a single phenotypic population and were uniformly positive for CD44 and negative for CD34 ,CD45 and HLA-DR. After 4 weeks of induction with 1 ng/ml TGF-β, MSCs assumed a spindle shape and a ＂hill-and-valley＂ growth pattern,expressing characteristic α-Actin. Myonemes were also expressed in the induced cells. Ach stimulation lead to efflux of the intracellular calcium. Conclusions The fetal MSC derived from second-trimester amniotic fluid can be induced to form smooth muscle cells in vitro.

关 键 词：羊水 干细胞 平滑肌细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]