摘要
背景:DNA损伤及损伤后的应答异常会导致基因组不稳定,基因组不稳定是肿瘤形成的重要因素之一。在细胞衰老过程中,衰老细胞基因组稳定性下降,对于端粒、端粒酶的研究以及对于原癌基因和抑癌基因的研究,表明细胞衰老和肿瘤有密切的联系。目的:以氧化损伤为模型,探索DNA损伤应答异常是衰老细胞基因组不稳定性的直接原因。方法:培养骨髓间充质干细胞,流式细胞术进行检测鉴定。衰老相关β-半乳糖苷酶检测细胞衰老情况,BrdU掺入实验检测细胞增殖情况。建立体外培养人间充质干细胞衰老模型,通过CCK-8法检测细胞存活情况,DCFH-DA荧光探针检测细胞内活性氧,单细胞凝胶电泳观察细胞DNA损伤情况。结果与结论:人间充质干细胞经长期培养发生衰老,培养40代以后,衰老相关β-半乳糖苷酶染色阳性细胞明显增多,BrdU掺入能力显著下降,提示间充质干细胞长期培养后发生衰老。生存曲线结果显示H2O2作用下,年轻间充质干细胞存活率明显高于衰老间充质干细胞;活性氧及单细胞凝胶电泳结果显示,H2O2处理后衰老间充质干细胞DNA损伤更严重,修复时间更长,说明衰老间充质干细胞比年轻间充质干细胞对H2O2损伤更为敏感。
BACKGROUND： The abnormal DNA damage response may result in genomic instability, which is one of the pivotal factors of tumorigenesis. The genomic stability is decreased in senescent cells. Recent studies about telomere, telomerase, oncogenes and cancer suppressor genes indicate that cell aging is closely linked with cancer. OBJECTIVE： Using an oxidative damage model to determine whether the abnormal DNA damage response is the direct cause of genomic instability in senescent cells. METHODS： Bone marrow mesenchymal stem cells were cultured and identified by flow cytometry. Cellaging was detected using senescence-associated β-galactosidase. Cell proliferation was determined using BrdU incorporation test. Human mesenchymal stem cell aging model was established in vitro. Cell Counting Kit-8 was used to observe cell survival, and DCFH-DA fluorescent probe was used to detect intracellular reactive oxygen species. Then the cell damage repair ability was detected by single cell gel electrophoresis assay. RESULTS AND CONCLUSION： Human mesenchymal stem cells were found to be senescent after long term culture, implied by increased number of senescence-associated β-galactosidase-positive cells and decreased BrdU incorporation. The growth curve showed that the survival rate of senescent mesenchymal stem cells was lower than that of young mesenchymal stem cells after hydrogen peroxide treatment. Furthermore, supporting evidence from the reactive oxygen species detection and single cell gel electrophoresis assay demonstrates that senescent mesenchymal stem cells underwent more serious DNA damage and required longer repair time. All these results imply that senescent mesenchymal stem cells are more sensitive to hydrogen peroxide injury than their young counterparts.
出处
《中国组织工程研究》
CAS
CSCD
2013年第10期1801-1808,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81272822)
项目名称:Nanog调节膀胱癌干细胞DNA损伤应答介导顺铂耐药的作用与机制研究~~
关键词
干细胞
干细胞培养与分化
间充质干细胞
衰老
DNA损伤修复
氧化损伤
衰老相关β-半乳糖苷酶
活性氧
单细胞凝胶电泳
国家自然科学基金
干细胞图片文章
stem cells
stem cell culture and differentiation
mesenchymal stem cells
aging
DNA injury repair
oxidative injury
senescence-associated β-galactosidase
reactive oxygen species
single cell gel electrophoresis assay
National Natural Science Foundation of China
stem cell photographs-containing paper
