【作者】 葛剑云；

【导师】 蔡海波；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 硕士

【摘要】 体外扩增技术是解决新鲜脐血中造血干/祖细胞数量稀少问题的有效策略,而扩增后细胞的质量和安全性是影响其临床应用的重要因素。本文以脐血CD34+细胞为对象,分别采用有或无血清培养体系,对体外培养细胞的生理状态、遗传稳定性、染色体核型和克隆形成能力进行了系统的分析,获得下列结果：(1)无论细胞培养基有无血清,细胞的比生长速率、S/G2/M期细胞比例、以及细胞内端粒酶活性均有相似的变化趋势,存在一定的相关性；(2)与CD34-细胞相比,在培养过程中CD34+细胞的凋亡率始终维持在较低水平上,低于新鲜分离的CD34+细胞中的凋亡率,表明CD34+细胞具有很强的抗凋亡特性；(3)在细胞培养过程中,与遗传稳定性相关基因Bmil、hTERT和Survivin mRNA的表达水平随培养时间延长均有不同程度的上调,但与K562细胞中的稳定高表达不同,提示细胞培养没有产生细胞恶化的信号;(4)6个脐血样本用含血清培养体系扩增,染色体核型异常比例较高,达2/3。但是这些异常的发生不具有定向性和稳定遗传性,且未表现出异常增殖的特征,提示有血清培养体系扩增的脐血造血干/祖细胞可能有临床安全性风险,但没有足够证据显示培养细胞出现了恶性转化。这些研究对确保培养细胞移植的安全性与有效性具有重要意义。更多还原

【Abstract】 It has been demonstrated that ex vivo expansion is a feasible strategy which could overcome the cell dose limitation in fresh cord blood (CB). However the quality and safety of expanded cells are critical for their clinical application. In this research, physiological parameters and genetic stability of CB CD34+ cells cultured with media at present of or absence of serum were examined extensively, producing following results:(1) Cell specific growth rate, S/G2/M phase cell proportion and telomerase activity had a similar variation tendency during culture with or without serum, suggesting the parameters were correlated each other to some extent. (2) The apoptosis of expanded CD34+ cells was much lower than cultured CD34- cells or freshly isolated CD34+ cells, sugggesting that cultured CD34+ cells are of anti-apoptosis. (3) The expression of Bmil, hTERT and Survivin genes related to cell genetic stability were upregulated differently in cultured CD34+ cells than those in freshly isolated CD34+ cells, although the expressions were not as efficient as those in K562 cells. Thus, the expresson pattern of these genes could not indicate neoplastic transformation occurs during the culturing. (4) Although high proportion of chromosomal alterations exsisted in cultured CD34+ cells cells in serum originated from 6 CB samples, the expanded cells with altered chromosomes could not proliferate neoplastically. Therefore, malignant transformation seems unlikely when expanded CD34+cells in serum were applied clinically, although potential risks were not excluded completely. These efforts were of importance for clinical transplantation efficiently and safely with expanded cells.更多还原