摘要
目的:探讨在体外特定培养条件下诱导绵羊骨髓间充质干细胞定向分化为软骨细胞表型的可行性。方法:实验于2005-09/2006-02在首都医科大学细胞与遗传实验室完成。选择1岁龄骨骼成熟的健康绵羊,雌雄不拘,体质量20～30kg。①采用Percoll分离液,经密度梯度离心法自成年绵羊髂骨骨髓分离得到间充质干细胞,体外培养至第3代时,将骨髓间充质干细胞分为2组,实验组细胞贴壁后更换培养液,以无血清H-DMEM特定培养液诱导(内含转化生长因子β310μg/L、地塞米松10-7mol/L、胰岛素样生长因子I10μg/L、维生素C50mg/L),对照组加含10%胎牛血清的L-DMEM培养液,3d换液一次。②在相差显微镜和透射电镜下观察细胞形态和超微结构,分别在诱导后的第7天、14天取出玻片,进行细胞形态学、组织化学和免疫组织化学分析,甲苯胺蓝染色和Ⅱ型胶原免疫组化鉴定间充质干细胞诱导后的软骨细胞表型。结果:①相差显微镜观察实验组诱导14d后,细胞形态由梭形逐渐向多角形、多边形转变,并出现聚集成堆现象,而对照组细胞仍保持均一的梭形,增殖能力非常旺盛。②透射电镜可见实验组诱导14d后的骨髓间充质干细胞周边绒毛增多,胞浆内富含粗面内质网、高尔基体和线粒体,表明细胞合成代谢旺盛。而对照组的骨髓间充质干细胞的细胞壁较光滑,胞核呈条状,核壁有许多皱襞和突起,细胞器也比较丰富。③免疫组织化学观察实验组诱导14d后甲苯胺蓝染色和Ⅱ型胶原染色阳性。而对照组诱导14d后甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化染色则为阴性。结论:骨髓间充质干细胞在特定的诱导条件下能够分化为软骨细胞表型,可为软骨组织工程提供较为理想的种子细胞来源。
AIM： To study the feasibility of adult sheep bone marrow-derived mesenchymal stem cells （MSCs） differentiate into chondrogenic phenotype in a defined, medium in vitro.
METHODS： The experiment was carried out in the Laboratory of Cell and Genetics Capital University of Medical Sciences from September 2005 to February 2006. The healthy sheep of skeletal maturation and either gender were adopted in this study, aging 20-30 kg.（1）MSCs were isolated by density gradient centrifugation with Pereoll solution from bone marrow of sheep iliac crest. The third passage MSCs were divided into 2 groups： experimental group and control group. The experimental ceils after adhesion were induced with H-DMEM serum-free medium containing 10 μg/L transforming growth factor （TGF）-β3, 10 μg/L insulin-like growth factor （IGF）-I, 10^-7 mol/L Dexamethasone and 50 mg/L vitamin （Vit） C. The control cells were cultured in L-DMEM containing 10% fetal bovine serum, which was changed every 3 days. （2）The shape and ultrastructure of cells were observed under phase contrast microscope （PCM） and transmission electron microscope （TEM）, additionally toluidine blue stain and type Ⅱ collagen immunohistochemistry were applied for chondrogenic phenotype identification after the analysis of cellular morphology, histochemistry and immunohistochemistry on the 7^th and 14^th days after induction respectively.
RESULTS： （1）PCM; After 14 days of induction, MSCs changed from a spindle-like appearance to a polygon by aggregations in the experimental group, and remained the homogeneous spindle shape but grew productively in the control group.（2）TEM： After 14 days of induction, a large amount of rough endoplasmic reticulum, Golgi complex and mitochondria were observed in plasm, indicating the cells had a high synthesis metabolism in the experimental group. While the control ceils presented smooth cell wall, strip nucleus, abundant cell organs, and many plica and prominence in the nucleus wall.（3）After 14
出处
《中国临床康复》
CSCD
北大核心
2006年第41期69-71,I0004,共4页
Chinese Journal of Clinical Rehabilitation
