体外扩增过程中造血细胞生理状态和遗传稳定性研究

发布时间：2018-02-01 05:42

本文关键词： 脐血造血干/祖细胞体外扩增细胞状态遗传稳定性　出处：《华东理工大学》2011年硕士论文　论文类型：学位论文

【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。
[Abstract]:In vitro amplification technique is an effective strategy to solve the problem of rare number of hematopoietic stem / progenitor cells in fresh umbilical cord blood. The quality and safety of expanded cells are the important factors affecting its clinical application. In this paper, umbilical cord blood CD34 cells were used as the object, with or without serum-free culture system, the physiological status of cultured cells in vitro. Genetic stability, chromosome karyotype and clone forming ability were systematically analyzed. The following results were obtained: 1: 1) the specific growth rate of cells in S- / G _ 2 / M phase, regardless of whether the cell culture medium had serum or not, was obtained. The telomerase activity in the cells showed a similar change trend, and there was a certain correlation between the telomerase activity and the telomerase activity. (2) compared with CD34- cells, the apoptotic rate of CD34 cells was maintained at a lower level during the culture process, which was lower than that in freshly isolated CD34 cells. The results showed that CD34 cells had strong anti-apoptotic properties. 3) during cell culture, the expression levels of Bmila hTERT and Survivin mRNA, which are related to genetic stability, were upregulated in varying degrees with the extension of culture time. However, different from the stable high expression in K562 cells, cell culture did not produce the signal of cell deterioration. 6 umbilical cord blood samples were amplified by serum-containing culture system, and the chromosome karyotype abnormality rate was 2 / 3. But the occurrence of these abnormalities was not directional and stable. There was no abnormal proliferation, which suggested that the umbilical cord blood hematopoietic stem / progenitor cells expanded with serum culture system might be at risk of clinical safety. However, there is not enough evidence to indicate the malignant transformation of cultured cells. These studies are important to ensure the safety and effectiveness of cultured cells transplantation.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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