论文部分内容阅读
目的研究经成骨诱导分化的人脐带血间充质干细胞(umbilical cord blood derived mesenchymal stem cells,UCB-MSCs)表面免疫分子的表达以及体外调节T淋巴细胞增殖反应的功能。方法取自愿捐赠的脐静脉血分离获取UCB-MSCs,体外扩增至第3代,行成骨诱导培养2周。流式细胞仪检测成骨诱导分化前后UCB-MSCs免疫分子人类白细胞抗原(human leukocyte antigen,HLA)Ⅰ类分子、HLA-Ⅱ类分子的表达。首先以成骨分化的UCB-MSCs刺激异体T淋巴细胞增殖,未诱导的UCB-MSCs设为对照。共设立UCB-MSCs 1×102、1×103、1×104和1×105个/孔4种不同细胞密度,分为IFN-γ预处理组和未处理组。然后以植物血凝素(phytohemagglutinin,PHA)刺激T淋巴细胞增殖,分别加入上述不同数量级的未诱导与成骨诱导分化的UCB-MSCs,观察对T淋巴细胞增殖的影响,并同时加入IL-2检测对UCB-MSCs抑制T淋巴细胞增殖作用的逆转。最后建立Transwell共培养体系,研究成骨诱导分化的UCB-MSCs对混合T淋巴细胞反应的抑制作用是否为接触抑制性。结果未诱导的UCB-MSCs高表达HLA-Ⅰ类分子,不表达HLA-Ⅱ类分子;经成骨诱导分化的UCB-MSCs HLA-Ⅱ类分子的阳性表达率升高。未诱导的UCB-MSCs对异体T淋巴细胞增殖有显著抑制效果,而成骨诱导分化的UCB-MSCs对T淋巴细胞增殖的抑制作用明显减弱(且无细胞数量依赖性),并且在IFN-γ作用后,有刺激T淋巴细胞增殖的效果。密度为1×104个/孔及1×105个/孔时未诱导的UCB-MSCs可抑制PHA刺激的T淋巴细胞增殖,但加入IL-2后该抑制作用被逆转;而成骨诱导分化的UCB-MSCs则不具有抑制T淋巴细胞增殖的作用。Transwell共培养结果表明,与直接接触培养相似,未诱导的UCB-MSCs可显著抑制T淋巴细胞增殖反应,而成骨诱导分化的UCB-MSCs抑制能力明显减弱。结论经成骨诱导分化的UCB-MSCs抑制混合T淋巴细胞增殖反应的效果明显低于未经成骨诱导分化的UCB-MSCs,不适合作为骨组织工程或细胞治疗的同种异体种子细胞来源。
Objective To study the expression of immune molecules on the surface of umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) induced by osteogenic differentiation and the function of regulating T lymphocyte proliferation in vitro. Methods UCB-MSCs were isolated from umbilical cord blood donated by voluntary donation and expanded to the third passage in vitro. The osteoblasts were cultured for 2 weeks. Flow cytometry was used to detect the expression of HLA class I and HLA class II UCB-MSCs before and after osteogenic differentiation. First, allogenic osteoblastic UCB-MSCs were used to stimulate the proliferation of allogeneic T lymphocytes. Uninduced UCB-MSCs were used as controls. UCB-MSCs were established at 1 × 102, 1 × 103, 1 × 104, and 1 × 105 cells / well, and were divided into IFN-γ pretreatment group and untreated group. Then, the proliferation of T lymphocytes was stimulated with phytohemagglutinin (PHA), and UCB-MSCs of different orders of magnitude without induction and osteogenic differentiation were respectively added to observe the effect on the proliferation of T lymphocytes. At the same time, IL-2 To detect the reversal effect of UCB-MSCs on the proliferation of T lymphocytes. Finally, a Transwell co-culture system was established to investigate whether the inhibitory effect of osteoblast-differentiated UCB-MSCs on mixed T lymphocyte responses is contact inhibition. Results The non-induced UCB-MSCs had high expression of HLA-Ⅰ molecules and no HLA-Ⅱ molecules. The osteoblast-differentiated UCB-MSCs showed increased expression of HLA-Ⅱ molecules. Uninduced UCB-MSCs had a significant inhibitory effect on the proliferation of allogeneic T lymphocytes, while osteoblast-differentiated UCB-MSCs showed a markedly diminished (and no cell-dependent) inhibitory effect on the proliferation of T lymphocytes, After the role of stimulation of T lymphocyte proliferation effect. UCB-MSCs that were not induced at densities of 1 × 104 cells / well and 1 × 105 cells / well inhibited the PHA-stimulated proliferation of T lymphocytes, but the inhibition was reversed after addition of IL-2, whereas osteogenic differentiation UCB-MSCs do not have the effect of inhibiting T lymphocyte proliferation. The results of Transwell co-culture showed that UCB-MSCs could significantly inhibit the proliferation of UCB-MSCs induced by osteoblasts, which was similar to that of UCB-MSCs. Conclusion The osteoblastic differentiation-induced UCB-MSCs have a significantly lower inhibitory effect on mixed T lymphocyte proliferation than UCB-MSCs without osteogenic differentiation and are not suitable as a source of allogeneic seed cells for bone tissue engineering or cell therapy.