体外培养人的骨髓间充质干细胞的生物安全性分析

发布时间：2018-05-02 00:43

本文选题：乙型肝炎 + 骨髓间充质干细胞　；参考：《广西医科大学》2011年硕士论文

【摘要】：目的：通过体外培养人的骨髓问充质干细胞(MSCs),评价其生物学特性及长期体外培养的安全性,为行MSCs移植治疗终末期肝病的临床应用的质量控制提供深入基础研究对象及方法：选择中山大学附属第三医院感染科2009年10月——2010年4月行自体干细胞移植治疗的乙肝肝硬化和肝衰竭患者的骨髓液作为培养对象,采用全骨髓贴壁培养的方法,用无血清培养基联合成纤维生长因子(FGF)行患者的MSCs的体外培养,观察细胞形态,采用流式细胞检测仪行细胞表面分子鉴定,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其对T淋巴细胞增殖抑制率,软琼脂克隆实验观察其致瘤性,染色体核型分析其遗传稳定性,并用培养过MSCs的条件培养基培养肿瘤细胞,观察其对肿瘤细胞增殖的影响。结果：患者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,计算T淋巴细胞抑制率约为45%(P=0.00),无致瘤性,但在肿瘤细胞存在情况下,体外培养时可能通过直接作用或其分泌可溶性因子的间接作用促进肝癌细胞的生长。结论：患者的MSCs在无血清培养基中亦具有较高的增殖潜能,经过体外传代后可以大量扩增,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,可以抑制T淋巴细胞,具有降低移植物抗宿主反应的可能性,是作为细胞治疗和基因治疗的理想种子。但在体外与肝癌细胞共同培养时可能通过其分泌细胞因子的的营养作用促进肝癌细胞的增殖,是否能将其作为移植物治疗肝脏肿瘤引起的肝衰竭有待于进一步深入的探讨。
[Abstract]:Objective: to evaluate the biological characteristics and long-term safety of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro so as to provide a foundation for quality control of clinical application of MSCs transplantation in the treatment of end-stage liver diseases. Subjects and methods: bone marrow fluid of patients with hepatitis B cirrhosis and liver failure treated by autologous stem cell transplantation from October 2009 to April 2010 were selected as culturing objects in the third Hospital infection Department affiliated to Sun Yat-sen University. Whole bone marrow adherent culture was used to culture patients with MSCs in vitro using serum-free medium combined with synthetic fiber growth factor (FGFs). Cell morphology was observed, cell surface molecular identification was performed by flow cytometry, and bacteria were isolated from cell suspension. Virus, endotoxin, serum residues, T lymphocyte inhibition test, soft Agar clone assay were used to detect the tumorigenicity of T lymphocytes, and chromosome karyotype was used to analyze its genetic stability. Tumor cells were cultured on MSCs conditioned medium and their effects on tumor cell proliferation were observed. Results: the chromosome karyotype and surface molecular expression of MSCs were stable, no bacteria and virus contamination were observed. The inhibition rate of T lymphocytes was calculated to be 45P0. 00G, no tumorigenicity, but in the presence of tumor cells. In vitro culture may promote the growth of hepatoma cells by direct action or indirect effect of soluble factor secretion. Conclusion: the MSCs of the patients also has high proliferative potential in serum-free medium. After passage in vitro, it can be expanded in large quantities to meet the needs of the number of transplanted cells. The long-term subculture karyotype and cell phenotype are stable and non-tumorigenic. It can inhibit T lymphocytes, which has the possibility of reducing graft versus host reaction and is an ideal seed for cell therapy and gene therapy. However, in vitro co-culture with hepatoma cells may promote the proliferation of hepatoma cells through the nutrient effect of cytokines. Whether it can be used as graft in the treatment of liver failure caused by liver tumors needs further study.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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