小鼠卵黄囊间质干细胞向成骨细胞定向分化的实验研究

Osteogenic Differentiation of Murine Yolk Sac Mesenchymal Stem Cells in vitro

目的研究卵黄囊间质干细胞的成骨潜能。方法用０．１％的Ⅰ型胶原酶消化获得妊娠第８．５天的小鼠卵黄囊细胞；取贴壁细胞培养，并于接近融合时进行传代培养。对细胞进行形态学观察、碱性磷酸酶（ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ，ＡＫＰ）及Ⅰ、Ⅲ型胶原检测，传代４次后进行成骨诱导，每天观察细胞形态，并于培养７ｄ后测定ＡＫＰ、Ⅰ、Ⅲ型胶原及骨形态发生蛋白－２（ｂｏｎｅ ｍｏｒｐｈｏｇｅｎｅｔｉｃ ｐｒｏｔｅｉｎ－２，ＢＭＰ －２）的表达，培养８周后进行矿化检测。结果体外可获得纯化的卵黄囊间质干细胞，细胞大小形态均一，呈梭形，ＡＫＰ染色弱阳性，Ⅰ、Ⅲ型胶原阳性。在体外可诱导卵黄囊间质干细胞定向分化为多形性成骨样细胞，细胞由梭形向星形转化，并形成胞浆突起，相互连接成网状，细胞ＡＫＰ染色阳性，Ⅰ型胶原阳性，Ⅲ型胶原阴性，ＢＭＰ －２强阳性，８周后矿化区经Ｖｏｎ Ｋｏｓｓａ ＇ｓ染色呈阳性反应，符合成骨细胞的生物学特征。结论经体外纯化的卵黄囊间质干细胞可诱导向成骨细胞定向分化。

Objective To investigate the potential of yolk sac mesenchymal stem cells in osteogenic differentiation.Methods Murine yolk sacs were harvested on day8.5postcoitus,yolk sac cells were obtained after the yolk sacs were digested by0.1%typeⅠcollagenase for1hour,the non-adherent cells were removed after being cultured for1hour.The adherent cells were cultured in DMEM conta-ining of5ng/ml bFGF and15%FBS,and passaged when they became subconfluent.The morpholo-gic characteristics?and AKP?BMP -2?as well as typeⅠ?Ⅲcollagen of the yolk sac adherent cells were observed and tested.The attached cells were treated with10-8 mol/L dexamethasone?10mmol/Lβ-glycerophosphate?and50μg/ml vitamin C at passage4.Alternations of morphological characteristic?AKP activity?collagen of typeⅠ?Ⅲand mineralization were detected.Results Pure mesenchymal stem cells which were of spindle shape,uniform in size,positive in typeⅠ?Ⅲcollagen staining and weak positive in AKP activity could be induced to pleomorphism osteoblast-like cells in vitro.The cells were transformed from spindle shape to polygonal cells which were positive in typeⅠcollagen,negative in typeⅢcollagen,strong positive in BMP -2,and positive in Von Kossa' s stain at week8.The polygonal cells could form nodular structure and their AKP activity was increased.All these were coincidence with the characters of osteoblast.Conclusion Yolk sac mesenchymal stem cell can be purified and induced to osteoblast in vitro.