成骨分化的人脐带血间充质干细胞免疫原性研究  

IMMUNOGENICITY OF HUMAN UMBILICAL CORD BLOOD DERIVED MESENCHYMAL STEM CELLS AFTER OSTEOGENIC INDUCTION

作　　者：杨大威[1] 林和敏[2] 刘广鹏[3] 

机构地区：[1]哈尔滨医科大学附属第四医院骨科,哈尔滨150001 [2]温州医科大学附属眼视光医院整形科 [3]同济大学附属上海第十人民医院整形外科

出　　处：《中国修复重建外科杂志》2014年第6期752-757,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31271027;81171475);哈尔滨科技局技术研究与开发项目(2013RFXYJ041);National Natural Science Foundation of China(31271027;81171475);Research and Development Item of Harbin Science and Technology Bureau(2013RFXYJ041)

摘　　要：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�Objective To study the immunological properties of osteogenically differentiated umbilical cord blood derived mesenchymal stem cells （UCB-MSCs）. Methods UCB-MSCs were isolated from the umbilical cord vein, and were expanded; the cells at passage 3 were osteogenically induced for 2 weeks in vitro. The expressions of human leukocyte antigen I （HLA-I） and HLA-Ⅱ molecules were observed by flow cytometry analysis before and after osteogenic induction. Peripheral blood T lymphoc）rtes were isolated and cultured with osteoblastic induced or non-osteoblastic induced UCB- MSCs in different cell concentrations of 1 x10^2, 1 x 10^3, 1 x 10^4, and 1 x10^5 cells/well. The intake value of 3H-thymidine was calculated with luminescence counter. Then T lymphocytes were pretreated with PHA, and co-cultured with osteoblastic induced and non-osteoblastic induced UCB-MSCs as described above. IL-2 was further added to test the reversed effect of T lymphocytes proliferation stimulated by UCB-MSCs. Finally, to investigate whether the immunomodulatory effects on T lymphocytes proliferation depend on direct or indirect cell contact, the Transwell chamber culture system of UCB-MSCs and T lymphocytes was established. Results Flow cytometry analysis showed that non-osteoblastic induced UCB-MSCs expressed HLA-I but did not express HLA-Ⅱ; the expression of HLA-Ⅱ increased in osteoblastic induced UCB-MSCs. No T lymphocyte response was stimulated by non-osteoblastic induced UCB-MSCs, but osteoblastic induced UCB-MSCs could stimulate the proliferation of allogeneic T lymphocytes, especially after IFN-y treatment. Non-osteoblastic induced UCB-MSCs of 1x10^4 and1 x 10^5 cells/well could suppress the proliferation of T lymphocytes evoked by PHA, and this suppression could be reversed by the addition of IL-2. While osteoblastic induced UCB-MSCs did not have such suppressive effect. The results of the Transwell culture system also showed that non-osteoblastic induced UCB-MSCs could obviously inhibit the proliferation ofT lymphocytes, bu

关 键 词：脐带血间充质干细胞 成骨分化 免疫原性 淋巴细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]