脐血间充质干细胞的培养鉴定及成骨诱导后的免疫原性研究

Study on in vitro culture of human umbilical cord blood mesenchymal stem cells and its osteogenic differentiation

目的通过观察体外培养人脐血间充质干细胞(Umbilical cord blood-mesenchymal stem cells,UCB-MSCs)并作诱导成骨分化,探讨其作为组织工程的骨种子细胞的可行性。方法体外分离培养UCB-MSCs,流式细胞仪检测细胞表面抗原的表达及其变化规律;体外成骨诱导UCB-MSCs2周,采用Alizarin Red染色和钙离子半定量测定的方法评价细胞成骨活性。取P2代UCB-MSCs细胞,流式细胞仪检测加入IFN-γ前后的HLA-Ι、HLA-Ⅱ、CD40、CD80等分子的表达情况;流式细胞仪检测UCB-MSCs在成骨诱导分化前后HLA-Ι类分子、HLA-Ⅱ类分子的表达情况以观察成骨诱导分化的MSCs免疫抗原性的变化。结果第1、3、5代细胞的CD29、CD105和CD166均呈阳性表达,而造血细胞系的表面标记CD31、CD34和CD45则呈低表达,且随传代次数增加含量逐渐下降。UCB-MSCs成骨诱导2周后,Alizarin Red染色为阳性。对照组则无明显钙盐沉积。钙离子半定量的检测结果则表明,UCB-MSCs成骨诱导2周后的钙离子含量远高于未诱导组。流式细胞检测结果显示,人UCB-MSCs高表达HLA-Ι类分子,不表达HLA-Ⅱ类分子和CD40,CD80等共刺激分子;经IFN-γ刺激诱导后,HLA-Ι类分子的表达未发生改变,HLA-Ⅱ类分子呈阳性表达,而CD40和CD80的表达仍为阴性。人UCB-MSCs在成骨诱导分化前后HLA-Ι类分子均为阳性表达;而HLA-Ⅱ类分子在成骨分化前为阴性,诱导后阳性表达率增高。结论①UCB-MSCs能诱导分化为成骨细胞,有望成为较理想的组织工程骨种子细胞;②成骨分化使UCB-MSCs的免疫原性发生相应变化。

Objective To culture human umbilical cord blood mesenchymal stem ceils （UCB-MSCs） in vitro and induce them into os- teoblasts, in order to study their feasibility as the seed cells of bone tissue engineering. Methods Human UCB-MSCs were isolated and expanded in vitro. The CD markers of UCB-MSCs were identified by flow cytometric analysis. After 2 weeks＂ osteogenic induction, their osteogenic differentia- tion was evaluated by Alizarin Red staining and measurement of calcium semi quantitative content.The expression of HLA- I ,HLA- 11 ,CD40 and CD80 was detected by flow cytometry before and after adding IFN-% and also the expression of HLA- I and HLA- 11 before and after the osteogenic differentiation. Results The expression of CD29, CDI05 and CD166 of UCB-MSCs was positive, while the hematopoietic cells surface markers of CD31, CD34 and CD45 appeared low expression and gradually decreased with cell passaging number. Alizarin Red staining was positive after 2 weeks＂ osteogenic induction while the control group had no obvious calcium deposition. Semi-quantitative determination results showed that the calci- um content of the osteogenic induced groups was much higher than that of non-induced group. Flow eytometry analysis showed high expression of HLA- I and no expression of HLA- Ⅱ, CD40 and CD80. The expression of HLA- I did not change after the stimulation of IFN-% while that of HLA- 1I showed positive changes, and the expression of CD40 and CD80 still maintained negative. HLA- I molecules were positive expressed on UCB-MSCs whether osteogenic differentiated or not. HLA- Ⅱ molecules were negative before osteogenie differentiation while expression rate increased after induction. Conclusion ①UCB-MSCs could be successfully osteogenically induced and served as seed cells for bone tissue engineering. ②Immunogenieity of UCB-MSCs could be changed after osteogenic differentiation.