【作者】 王海峰；

【导师】 赵勇刚；

【作者基本信息】 河南科技大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:研究成人骨髓间充质干细胞(hMSC)的生物特性和发生,发育的规律,探讨其在临床应用,体外扩增的过程中的安全性,以及在临床应用的过程中是否会发生恶性转化。方法:我们研究了体外培养的hMSC,在不同的培养时间点,发生恶性转化的敏感性。穿刺抽取10名健康志愿者的骨髓,分离提取得到hMSC,体外培养至衰老或需要传代(P) 25,8周至12周(hMSC的衰老期),干预终止之前,密切观察监视hMSC的变化。通过阵列比较基因组杂交,常规核型分析和端粒荧光原位杂交,研究分析长期培养之前和之后干细胞的遗传特性。检测分析不同传代次数和阶段,hMSC端粒酶的表达活性,端粒酶逆转录酶(hTERT)的转录物,和端粒延长的具体机制。结果:不同的志愿者之间,hMSC的增殖能力,和寿命有着巨大的变化。其中八个志愿者,hMSC增殖能力在衰老之前逐渐减弱。余下的两个志愿者,为了进行数据分析在P25阶段,hMSC的培养被打断。阵列比较基因组杂交阵列全息图和细胞遗传学分析显示,干细胞在体外培养扩增没有出现染色体异常;端粒酶活性未变,hTERT没有转录表达,端粒在培养阶段被缩短。端粒的延长机制没有发现和证实。结论:骨髓来源的干细胞可以在体外安全地扩增,不易发生恶性转化,从而使hMSC的更加适合于干细胞疗法的临床应用。更多还原

【Abstract】 OBJECTIVE: Significant improvement in the understanding of mesenchymal stem cell (HMSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that HMSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)–derived HMSCs at different in vitro culture time points.METHODS: HMSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. HMSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of HMSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping,and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. HMSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages.Results: A huge variability in terms of proliferative capacity and HMSCs life span was noted between donors. In eight of 10 donors, HMSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two HMSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that HMSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the HMSCs tested.Conclusion: BM-derived HMSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.更多还原