摘要
目的 探讨骨髓间充质干细胞 (mesenchymalstemcells,MSCs)无支架条件下在体外向软骨组织定向分化的条件。方法 取引产胎儿骨髓悬液 ,经Percoll密度梯度离心 ,获取单个核细胞 ,细胞贴壁、扩增后 ,再以高糖DMEM(TGF β15ng/ml)进行成软骨细胞预诱导 ,4～ 7d后获得成纤维样单层贴壁的MSCs。以此MSCs为种子细胞 ,2× 10 6个细胞离心成团 ,在TGF β1与IGF Ⅰ及其它辅助成分的协同作用下 ,体外培养 3wk后形成组织团块。经固定、包埋、切片后行甲苯氨蓝染色 ,免疫组化方法测定特异性Ⅱ型胶原。通过图象分析软件检测组织块蛋白多糖和Ⅱ型胶原表达强度 ,并与胎儿正常软骨比较。结果 胎儿骨髓单个核细胞经过预诱导 ,细胞表达特异性Ⅱ型胶原 ,甲苯氨蓝染色蓝染明显增强 ;再经离心成团、体外诱导培养后形成有光泽软骨样组织块 ,其甲苯氨蓝染色呈蓝染 ,特异性Ⅱ型胶原为阳性表达 ,图象分析表明其蛋白多糖及Ⅱ型胶原表达强度与胎儿正常软骨相似。结论 胎儿骨髓间充质干细胞经高糖DMEM预诱导后可向软骨细胞分化 ;离心成团后 ,在TGF β1与IGF Ⅰ及其它辅助成分的协同作用下具有形成类软骨组织的能力 ,本实验建立的定向诱导培养系统在无支架条件下可使MSCs向软骨样组织分化。
Objective To explore the culture conditions that chondrocytic differentiation of mesencchymal stem cells in virtro. Methods The MSCs were isolated from the nucleated cells fraction of human embryonic marrow washing fluid by density gradient centrifuge.And then the MSCs were induced into chondrocyte by TGF-β 1,dexamethasone,10% FBS in high glu DMEM after initial culture of 10 days in low glu DMEM.Initial MSCs were trypsinized,counted and centrifuged to a small pellet in a 15ml polypropylene tube.The pellet were induced into chondrocytic tissue by limited culture conditions in virtro. Results The histological observation of the cartilage-like tissue showed positive staining of toluidine blue in 3 weeks.The expression of the type Ⅱ collagen was positive too.These were similar to natural human embryonic articular cartilage. Conclusions MSCs can be used as functional cells to construct tissue engineered cartilage.The culture system could be used to induce MSCs to cartilage-like tissue without scaffold material.
出处
《合肥医学院学报》
2003年第3期221-225,共5页
Journal of Zunyi Medical University
