小鼠卵黄囊间充质干细胞的培养及鉴别

A STUDY OF INCUBATION AND INITIAL IDENTIFICATION OF MURINE YOLK SAC MESENCHYMAL STEM CELLS

目的研究小鼠卵黄囊间充质干细胞(YS-MSCs)的分离、培养和鉴定。方法显微分离妊娠10d的小鼠胚胎卵黄囊,经0.1%的Ⅰ型胶原酶消化1h得到卵黄囊细胞,取贴壁细胞培养,并于接近汇合时进行传代培养,透射电镜下观察YS-MSCs的超微结构;流式细胞仪检测YS-MSCs表面标志;钙钴法测定YS-MSCs碱性磷酸酶(AKP)活性;细胞组织化学检测YS-MSCs化学变化;地塞米松、胰岛素定向诱导YS-MSCs分化为脂肪细胞,油红O检测中性脂肪。结果体外可获得YS-MSCs,细胞大多数呈梭形;透射电镜下YS-MSCs表面有微绒毛,胞浆中有丰富的线粒体、粗面内质网、高尔基复合体;核大,形态不规则;流式细胞术分析YS-MSCs免疫表型显示,原代和第1代YS-MSCs均为CD44、CD105阳性,CD34也有少量表达;细胞化学YS-MSCs PAS-过碘酸雪夫染色阳性,苏丹黑-B(SB)及碱性磷酸酶(AKP)染色阴性;YS-MSCs经成脂诱导后,胞浆中有脂滴形成,经油红O染色脂滴呈鲜红色。结论小鼠YS-MSCs的生物学特性与成体间充质干细胞相似,且更为原始,提示其可作为组织工程的种子细胞来源。

Objective To investigate the isolation, incubation and identification of murine yolk sac mesenchymal stem cells（YS-MSCs）. Methods Murine yolk sacs were harvested on the lOth day of postcoitus microisolation. Yolk sac cells were obtained after the yolk sacs were digested by type I collagenase of 0.1% for 1 hour. Adherent cells were cultured in DMEM containing 10% FBS and 1% SP,and passaged when they became subconfluent.The ultrastructure of YS-MSCs was observed under transmission electron microscope; the surface markers of YS-MSCs were detected by flow cytometry; the alkaline pbesphatase （AKP） expression of YS-MSCs was tested; and cytochemical changes of YS-MSCs were observed with cytochemical detection. Adipogenic differentiation of YS-MSCs was induced by 1μmol/L dexamethasone and 10mg/L insulin, and oil red O was used for fat staining. Results YS-MSCs were obtained in vitro, and most of them were of spindle shape; under transmission electron microscope, there were microvilli on the surface of YS-MSCs, there were abundant mitochondria, rough endoplasmic reticulum and golgi complex in the endochylema. The cell nuclei of YS-MSCs were big and the shape of cell nucleus was irregular; the analysis of flow cytometry suggested that the primary and first generation of YS-MSCs were positive for CD44, CD105, and the expression of CD34 was small; Cytochemistry showed that YS-MSCs were positive for glycogen, and negative for SB and AKP; Adipogenic differentiation of YS-MSCs was induced by 1μmol/L dexamethasone and 10mg/L insulin; accumulation of lipid-rich vacuoles appeared in the cells, positive for oil red O staining. Conclusion The biological characteristics of murine yolk sac mesenchymal stem cells are similar to, and more primitive than, those of adult mesenchymal stem cells. It suggests that YS-MSCs may be used as an ideal resource of seed cells of tissue engineer.