小鼠卵黄囊间质干细胞的分离纯化及多向分化潜能的研究

MULTILINEAGE POTENTIAL OF PURIFIED MURINE YOLK SAC MESENCHYMAL STEM CELLS IN VITRO

目的 :分离、纯化小鼠卵黄囊间质干细胞 ,并观察其定向分化成骨、成软骨及成脂的潜能。方法 :分离、纯化孕 8.5d的小鼠卵黄囊间质干细胞 ,传代 4次后进行成骨、成软骨及成脂诱导。观察细胞形态 ,检测AKP ,Ⅰ ,Ⅱ ,Ⅲ型胶原及BMP - 1和BMP - 2的表达 ,并进行组化染色 ,检测成骨、成软骨及成脂情况。结果 :体外可获得纯化的卵黄囊间质干细胞 ,大小形态均一 ,呈梭形。成骨诱导后细胞向星形转化 ,AKP、Ⅰ型胶原、BMP - 2及VonKossa’s染色阳性 ,Ⅲ型胶原阴性。成软骨诱导后 ,细胞向多角形和圆形转化 ,AKP、Ⅱ型胶原、BMP - 1及Alcianblue染色阳性 ,Ⅰ型胶原阴性。成脂诱导后细胞肥大 ,油红O染色阳性。结论 :体外纯化的卵黄囊间质干细胞可诱导成骨、成软骨及成脂 。

Objective:To investigate the multilineage potential of purified murine yolk sac mesenchymal stem cells in vitro. Methods:Murine yolk sacs were harvested on day 8.5 postcoitus; yolk sac cells were obtained after the yolk sacs were digested by 0.1% typeⅠcollagenase for 1 hour; the non-adherent cells were removed after being cultured for 2 hours. The adherent cells were cultured in DMEM containing of 5ng/ml bFGF and 15% FBS, and passaged when they became subconfluent. At passage 4th, to promote osteogenic, chondrogenic and adipogenic differentiation, the attached cells were treated with 10 -8 mol/L dexamethasone,10 mmol/L β-glycerophosphate,50μg/ml vitamin C ; TGF-β; 10μg/ml Insulin,10 -5 mol/L indomethacin,10 -6 mol/L dexamethasone, respectively. Alternations of morphological characteristic,AKP activity,collagen of typeⅠ,Ⅱ,Ⅲ and BMP1,2 were detected. Results:Purified mesenchymal stem cells which were of spindle shape , uniform in size could be induced to pleomorphism osteoblast-like cells which were positive in typeⅠcollagen,BMP-2 and Von Kossa's stain in vitro. The cells could also be induced to differentiate to chondrocytic lineage which were positive in typeⅡcollagen,BMP-1 and Alcian blue stain. And the cells could be induced to adipocytes, which were positive in Oil red O stains. Conclusion:Purified yolk sac mesenchymal stem cells have the multilineage potential to differentiate to bone, cartilage and fat in vitro.