人血源性间充质干细胞培养与体外成骨研究  

STUDY ON CULTURE AND IN VITRO OSTEOGENESIS OF BLOOD-DERIVED HUMAN MESENCHYMAL STEM CELLS

作　　者：曹聪[1] 董英海[1] 董宇启[1] 路丽明[2] 崔磊[2] 刘伟[2] 曹谊林[2] 

机构地区：[1]上海第二医科大学附属仁济医院骨科,上海200127 [2]上海组织工程研究与开发中心

出　　处：《中国修复重建外科杂志》2005年第8期642-647,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海市科委重点攻关基金资助项目(ODJ14001-7);上海第二医科大学自然科学研究基金资助项目(2001校14);上海第二医科大学附属仁济医院重点科研资助项目~~

摘　　要：目的建立分离、培养成人外周血来源的间充质干细胞(mesenchymalstemcells,MSCs)的方法,观察成人血源性MSCs体外成骨潜能。方法抽取成年志愿者外周静脉血30份,每份15ml。用淋巴细胞分离液(密度为1.077g/ml)梯度离心,取单个核细胞在含有20%胎牛血清的α-MEM培养液中培养扩增MSCs,流式细胞仪分析MSCs表型。第2次传代培养时在培养液中添加成骨诱导因子(地塞米松、β-磷酸甘油、维生素C和1,25二羟维生素D3)。第5代传代细胞检测碱性磷酸酶、型胶原(collagentype,Col)、骨钙素(osteocalcin,OC)和骨粘连蛋白(osteonectin,ON)表达,连续培养1个月后检测钙结节形成情况。结果成人外周血中存在MSCs,集落形成率为0.27±0.22/106单个核细胞,表达CD44、CD54、CD105和CD166,而不表达与造血系相关的CD14、CD34、CD45及内皮细胞特异的CD31。地塞米松、β-磷酸甘油、维生素C和1,25二羟维生素D3可作用于传代培养的血源性MSCs,表现为碱性磷酸酶染色阳性细胞增多,逆转录-聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)产物电泳条带显示ColI、OC和ON表达,盐酸四环素荧光标记证实有钙结节形成。结论建立的成人MSCs分离、培养条件可分选出外周血贴壁生长的细胞中一组独特的细胞群。成人血源性MSCs具有较强的体外成骨潜能,可望成为一种新的骨组织工程种子细胞。Objective To establish a method of isolating and culturing adult human blood derived mesenchymal stem cells（MSCs） and to investigate their osteogenic potential in vitro. Methods Thirty peripheral blood samples were collected from 30 adult volunteers （15 ml per person）. Aduh human MSCs derived from peripheral blood were isolated from the lymphocyte separation fluid fraction of mononuclear cells, cultured in α-Modified Eagle＇s Medium with low glucose containing 20% fetal bovine serum, and proliferated through a process of subcuhuring. The phenotype of MSCs was analyzed with flow eytometry. For in vitro osteogenic differentiation, MSCs from the second passage grew in the presence of osteogenic supplements （100 nmol/L dexamethasone, 10 mmol/L β glycerophosphate, 50 μmol/L vitamin C, and 10 nmol/L 1,25-2-hydroxide vitamin D3）. In the fifth passage cells, the activity of alkaline phosphatase, the expression level of collagen type 1, osteocalcin and osteonectin were determined. And the calcium tubercle formation would be examined after the continual one-month culture of the fifth passage. Results MSCs exsited in the pheripheral blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0. 27±0. 22/10^6 mononuclear cells. The MSCs expressed CD44,CD54,CD105,and CD166,but did not CD14, CD34, CD45,and CD31. Under the function of osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression levels of collagen type Ⅰ , osteocalcin and osteonectin. And the calcium tubercle formation was examined through tetracycline fluorescence labeling method. Conclusion The isolation and culture conditions established for adult human MSCs may select a distinct population of peripheral blood derived adherent cells. Adult human blood derived MSCs possess osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.

关 键 词：血细胞间允质干细胞骨生成 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]