体外成骨诱导增加人骨髓间充质干细胞刺激PBMCs增殖

Osteogenic inducement in vitro increases the proliferation of human peripheral blood mononuclear cell stimulated by human bone marrow derived mesenchymal stem cells

目的初步观察人骨髓间充质干细胞(hum an bone m arrow derived m esenchym al stem cell,hMSCs)及其成骨分化后代的免疫原性,为异基因hMSCs作为骨组织工程种子细胞研究提供理论基础。方法应用Percoll密度梯度离心法从人骨髓中分离、培养细胞hMSCs;将第3代hMSCs及其体外诱导成骨3周的分化后代分别与人外周血单个核细胞(hu-m an peripheral b lood mononuc lear cell,PBMC)混合培养,观察上述细胞对PBMC的刺激指数。结果Percoll(1.073 g/m l)分离培养的原代hMSCs为均匀一致的梭形,呈漩涡样排列。其在体外成骨诱导3周时Von-Kossa染色见细胞间质有大量的钙盐沉积,免疫细胞化学检测见到大多数细胞骨钙素表达阳性。混合细胞培养结果显示:不同细胞数量的hMSCs对PBMCs的刺激指数(stimu lation index,SI)均小于2;而1×104、5×103、103的hMSCs-Osteo对PBMCs的刺激指数大于2。结论hMSCs的免疫原性较低,在体外不能刺激PBMCs增殖。但hMSCs的成骨分化后代的免疫原性增加,一定数量细胞在体外能够刺激PBMCs增殖,其在异基因宿主体内可能引起免疫排斥反应。本实验结果提示:异基因骨髓间充质干细胞要作为骨组织工程种子细胞,必须克服免疫方面的障碍。

Abstract： Objective To investigate immunogenicity of human bone marrow derived mesenchymal stem cells （hMCSs） and their offsprings after osteogenic induction in vitro. Methods hMSCs were isolated from bone marrow of the iliac crest of voluntary healthy adults by density gradient centrifugation of Percoll （ 1. 073 g/ ml） , and cultured and carried out osteogenic induction in vitro, hMSCs of third passage and the hMSCs off- springs after osteogenic induction （hMSCs-Osteo） for 3 weeks were cocuhured with human peripheral blood mononuclear cell （PBMC）. Then the stimulation index of hMSCs and hMSCs-Osteo were observed in this coculture system. Results The isolated cultured hMSCs were homogeneous spindle-like cells and had a circinate arrangement, hMSCs were induced into osteogenesis when they were cultured in osteogenic medium for three weeks demonstrated by calcium salts deposited in matrix, and osteocalcin were expressed in cytoplasm, hMSCs failed to elicit a proliferative response from allogeneic lymphocytes, but hMSCs-Osteo enhanced the proliferative response from allogeneic lymphocytes with the SI （ stimulation index） over 2. Conclusion hMSCs show a lower immunogenic feature in vitro, but osteogenic inducement increases immunogenic feature of hMSCs, which indicate that allogeneic hMSCs would arose immune rejection in recipients when used as seeding cells of bone tissue engineering.