体外培养乙型肝炎患者骨髓间充质干细胞的生物安全性及其质量控制

Safety evaluation and quality control of MSCs from hepatitis B patient in vitro

目的评价乙型肝炎患者骨髓问充质干细胞（MSCs）长期体外培养的安全性，为MSCs临床应用的质量控制提供基础。方法采用全骨髓接种法，用无血清培养基联合成纤维生长因子体外培养乙型肝炎患者的MSCs，观察细胞形态；采用流式细胞仪检测细胞表型；对细胞悬液行细菌、病毒、内毒素及血清残留检测；T淋巴细胞抑制试验观察MSCs对T淋巴细胞增殖的抑制率；软琼脂实验观察MSCs的致瘤性；染色体核型分析MSCs遗传稳定性。组间吸光度值比较用析因设计方差分析，细胞悬液和细胞粉碎液中牛血清白蛋白含量比较用t检验。结果乙型肝炎患者的MSCs经过体外培养传代至第10代，染色体核型及表面分子表达稳定，未见细菌、病毒污染，T淋巴细胞抑制率为45％，无致瘤性。结论乙型肝炎患者的MSCs在无血清培养基中亦具有较高的增殖潜能，体外传代可以大量扩增，满足移植细胞数量需求，长期传代核型及细胞表型稳定，无致瘤性，且可抑制T淋巴细胞增殖。

Objective To get mesenchymal stem cells （MSCs） from hepatitis B patient and to valuate the safety and quality after long term culture in vitro. Methods The cells obtained directly from bone marrow and cultured in Mesen Pro medium supplemented with FGF, and the morphology of MSCs was observed. Surface antigens of the MSCs were analyzed by flow-cytometry. The becteria, virus, endotoxin and residual serum of cell suspension were detected. The MSCs and perpheral blood T lymphocytes were cocultured in 48 well plates for 72 h and the T lymphocyte proliferation was measured by using MTT reduction method and the effect of MSCs on T lymphocyte transformation stimulated by PHA was also observed. The oncogenicity of MSCs was verified by the tumorigenesis test in sofo agar. The genetic stability of MSCs was examined by karyotype analysis. Result The MSCs from hepatitis B patient could bepassaged to many generations and had strong abilities of proliferation.They expressed stem cell-surface antigens and maintained normal karyotype, prevented the pollution of bacteria and viruses, inhibited the immune response of allogenic T lymphocytes and no oncogenicity found. Conclusion The MSCs have proliferative potentials, can be passaged in long-term cultures in Mesen Pro medium without oncogenicity, can maintain normal karyotype, can inhibit the immune response of T lymphocytes and can alleviate the grafe-versus diseases. The MSCs can be served as a new type of cells in cell and gene therapy.