小鼠胚胎卵黄囊与成体间充质干细胞的比较:生物学特性相似吗?

Biological characteristics of mouse yolk sac-derived mesenchymal stem cells versus adult mesenchymal stem cells

背景:与成体间充质干细胞相比,来源于早期胚胎的卵黄囊间充质干细胞有更强的自我更新能力和可塑性,且在胚胎发育过程中可分化为成血管细胞和造血干细胞,启动胚胎血管发生和造血组织发育。目的:探讨小鼠卵黄囊间充质干细胞的生物学特征。设计、时间及地点:重复测量设计,细胞学体外观察,于2006-03/2007-01在首都医科大学组织学与胚胎学实验室完成。材料:妊娠10d的ICR小鼠80只,由首都医科大学动物部提供。方法:显微分离ICR小鼠胚胎卵黄囊,经Ⅰ型胶原酶消化得到卵黄囊细胞,取贴壁细胞培养,于接近90%融合时按1∶2进行传代。取生长状态良好的第1代细胞,以地塞米松、胰岛素定向诱导卵黄囊间充质干细胞向脂肪细胞方向分化;以血管内皮生长因子和碱性成纤维细胞生长因子体外诱导其向血管内皮细胞方向分化。主要观察指标:相差显微镜下观察卵黄囊间充质干细胞形态;流式细胞仪检测卵黄囊间充质干细胞表面标志和细胞周期;钙钴法测定卵黄囊间充质干细胞碱性磷酸酶活性;细胞组织化学检测卵黄囊间充质干细胞化学变化;油红O检测诱导成脂能力;免疫荧光法鉴定诱导成血管内皮细胞能力。结果:体外培养获得的小鼠卵黄囊间充质干细胞大多数呈梭形;原代和第1代细胞均为CD44,CD105阳性,CD34也有少量表达;67.4%细胞处于G0/G1期,13.9%细胞处于S期;P1代细胞碱性磷酸酶染色及苏丹黑B反应呈阴性,PAS染色呈阳性;成脂诱导1周后,胞浆中有脂滴形成,经油红O染色脂滴为鲜红色;成血管内皮细胞诱导后,细胞由原来梭形或多角形渐变为圆形或椭圆形,且内皮细胞标志物FLK1染色呈阳性。结论:小鼠卵黄囊间充质干细胞的生物学特征与成体间充质干细胞相似。

BACKGROUND：Compared with adult mesenchymal stem cells（MSCs） ,yolk sac-derived MSCs（YS-MSCs） of early embryo have stronger self-renewal capacity and plasticity. Moreover,they could differentiate into angioblast and hemopoietic stem cells during embryonic development and trigger embryonic angiogenesis and hemopoietic tissue development. OBJECTIVE：To investigate the biological characteristics of mouse YS-MSCs. DESIGN,TIME AND SETTING：Repetitive measurements and in vitro cytological observation. The study was performed at the Department of Histology and Embryology,Capital Medical University between March 2006 and January 2007. MATERIALS：A total of 80 ICR mice of gestational 10 days were provided by Department of Animal,Capital Medical University. METHODS：The yolk sacs were harvested by microisolation,and yolk sac cells were obtained after the yolk sacs were digested by type Ⅰ collagenase. The adherent cells were cultured,and passaged according to 1：2 when they were 90% confluent. The first passage of YS-MSCs which grew in good condition was obtained,and adipogenic differentiation of YS-MSCs was induced by dexamethasone and insulin,and angioblast was induced by vascular endothelial growth factor and basic fibroblast growth factor. MAIN OUTCOME MEASURES：The shape of YS-MSCs were observed by contrast phase microscopy;the surface markers and cell cycle of YS-MSCs were detected by flow cytometry;the alkaline phosphatase（AKP） expression of YS-MSCs was tested by calcium-cobalt method;the cytochemical changes of YS-MSCs were observed with cytochemistry;Oil Red O was used for fat staining;the expression of endothelial marker of induced cells were identified by immunofluorescence staining method. RESULTS：YS-MSCs were obtained in vitro,and most of them were spindle shaped. The analysis of flow cytometry suggested that the primary and first passage of YS-MSCs were positive for CD44 and CD105,while the expression of CD34 was small. The analysis of cell cycle revealed that 67.4% of YS-MSCs were in the G0/G1 stage,and 13.9% of YS-MSCs were in the S stage. Cytochemistry showed that YS-MSCs were positive for glycogen,and negative for Sudan black and AKP. After 1 week of adipogenic differentiation,accumulation of lipid-rich vacuoles positive in oil red O staining within the cells appeared. After induction of angioblast,the induced cells was obviously changed from spindle or polygon to round or oval-shaped,and immunofluorescence staining analysis of the confluent cells in situ showed positive for endothelial-specific markers like FLK1. CONCLUSION：The biological characteristics of mouse YS-MSCs are similar to those of adult mesenchymal stem cells.