人脐血基质细胞生物学特性的实验研究

The study on biological characteristics of stromal cells from human cord blood

目的:探讨人脐血基质细胞的生物学特性,为骨组织工程选择理想的种子细胞来源。方法:采用加入10%FBS、20ng/mlbFGF的L-DMEM培养基培养人脐静脉血基质细胞,以人骨髓基质细胞(BMSCs)做为对照。应用倒置显微镜观察细胞形态学特征,描绘生长曲线,流式细胞仪检测细胞周期。同时原代细胞进行成骨诱导分化培养。碱性磷酸酶染色和Von-Kossa染色法检测成骨能力。结果:①人脐血基质细胞原代培养形态呈长梭形,需21～30d长满瓶底,传代后细胞增殖缓慢并逐渐死亡。②在成骨诱导培养基里,脐血基质细胞增殖速度减慢,诱导前后形态无明显改变。碱性磷酸酶染色:脐血基质细胞阳性率为46.0%。Von-Kossa染色提示脐血基质细胞成骨诱导后形成钙化结节。结论:脐血基质细胞增殖能力有限,较骨髓基质细胞难于培养,成骨诱导证实可以向成骨细胞分化。

Objective：To study the biological characteristics of stromal cells from human cord blood, and select seed cells for bone tissue engineering. Methods：The stromal cells of fetal cord blood were cultured in L-DMEM containing 10%FBS, 20ng/ml bFGF, while bone marrow stromal cells （BMSCs） of human was taken as the control. The cells were observed under the invert microscope, and then the growth curves were drawn and the cell cycles were tested by flowing cytometry. The primary cells were incubated in the differentiation medium containing induction agents to explore their ability of osteogenesis. The degree of osteogenic differentiation was tested by ALP and Von-Kossa staining. Results： ①The cord blood stromal stem cells presented spindle shape, and confluenced after 21-30 days. The proliferation ability of cells was lower than that of BMSCs. ②In osteogenic medium, the cord blood stromal cells grew slowly, and there was no obvious change in morphology. Alkaline phosphatase staining showed that positive cells was about 46%. After 21 days, hydroxyapatite nodules appeared through Von-Kossa staining in cord blood stromal cells. Conclusions：The cord blood stromal cells are more difficult to culture than human BMSCs, and the prolific ability of cord blood stromal cells was limited. Nevertheless, it can be differentiated into osteoblast in differentiation condition.