人羊水源性间充质干细胞的分离培养及鉴定

目的 体外分离培养人羊水间充质干细胞(HAFMSCs),观察生物学特性,并鉴定其多向分化能力.方法 采用改良两步法分离培养HAFMSCs,用MTT法观察细胞增殖能力.FACS检测表面抗原CD29,CD34,CD44,CD45,CD73,CD90,HLA-ABC,HLA-DR等的表达.RT-PCR法分析细胞中OCT-4,Nanog mRNA水平的表达.用成脂、成骨、内皮细胞培养基对HAFMSCs进行诱导,并分别采用油红O,Yon Kossa染色及细胞免疫荧光染色检测其分化能力.结果 羊水细胞于2-3d开始贴壁,10-15d形成细胞集落.传至第3代HAFMSCs呈旋涡状排列,生长曲线呈S形.FACS检测显示CD29,CD44,CD73,CD90,HLA-ABC表达阳性,CD34,CD45,HLA-DR表达阴性,RT-PCR法检测显示HAFMSCs中OCT-4、Nanog mRNA的表达阳性.成脂、成骨诱导21d后油红O、Von Kossa染色阳性,成内皮细胞诱导4周后细胞免疫荧光染色检测vWF抗体表达阳性.结论 成功分离培养出HAFMSCs,细胞具有干细胞特征,具有多向分化潜能.

Isolation, cultivation and identification of mesenchymal stem cells derived from human amniotic fluid

Objective To isolate and culture human amniotic fluid mesenchymal stem cells(HAFMSC),observe their biological characteristics,and identify their multi-differentiation ability.Methods HAFMSC were isolated from amniotic fluid of the second trimester of gestation with the modified two-step method.Their proliferating ability was observed with the MTT method.Expressions of surface antigens including CD29,CD34,CD44,CD45,CD73,CD90,HLA-ABC and HLA-DR were detected by FACS.Expression of OCT-4 in HAFMSC at Nanog mRNA level was detected by RT-PCR.HAFMSC were induced with media of adipogenic,osteogenic and endothelial cells.Their differentiation ability was observed with oil red O,von Kossa and cellular immunofluorescence staining.Results The HAFMSC were adhered 2-3 days after cultured,and cell colonies were observed 10-15 days after cultured and arranged in a whirl-like form with a "S" shape of growth curve after passage 3.FACS showed that the CD29,CD44,CD73,CD90 and HLA-ABC were positively expressed while the CD34,CD45 and HLA-DR were negatively expressed.RT-PCR demonstrated that the OCT-4 and Nanog mRNA were positively expressed in HAFMSC.Twenty-one days after differentiation was induced with adipogenic and osteogenic cells,the HAFMSC were positively stained with oil red O and cellular immunofluorescence staining displayed that the vWF antibody was positively expressed 4 weeks after differentiation was induced with osteogenic cells.Conclusion HAFMSC can be isolated from amniotic fluid and are characterized by mesenchymal stem cells with a multi-differentiation potential.