摘要
背景:随着传代次数的增加,体外培养的人骨髓间充质干细胞被发现逐渐呈现衰老细胞的特征,但是,年龄是否影响人骨髓间充质干细胞的衰老进程则报道罕见。目的:观察年龄对体外培养的人骨髓间充质干细胞数量、增殖和衰老以及氧化应激的影响。设计、时间及地点:单因素设计,细胞学体外对比观察,于2007-07/12在中南大学湘雅二医院完成。材料:骨髓来源于中南大学湘雅二医院门诊28名健康自愿者,其中健康老年人14名,男10名,女4名;年龄60～73岁,平均65岁。健康年轻人14名,男9名,女5名;年龄18～28岁,平均25岁。方法:无菌条件下穿刺健康自愿者髂后上嵴,抽取骨髓2.0～3.0mL,注入含肝素的无菌抗凝管中,加等体积的无菌PBS稀释,离心后以1×105/cm2骨髓来源的有核细胞接种于6孔板培养,于接种4d后换液,去除未贴壁细胞,以后每3d换液。主要观察指标:倒置显微镜下观察细胞形态及生长情况,流式细胞仪检测表面抗原,比较两组原代细胞集落形成数量、首次传代和第2代骨髓间充质干细胞传代时间、早代龄骨髓间充质干细胞经衰老相关β-半乳糖苷酶染色阳性的衰老细胞计数及其上清液丙二醛水平。结果:两组体外培养的原代和第2代骨髓间充质干细胞在形态上无差异,早期呈集落样生长。流式细胞仪检测显示,两组骨髓间充质干细胞的免疫表型一致,CD44、CD105阳性表达,不表达CD34。老年人骨髓间充质干细胞首次传代与次代传代时间显著长于年轻人,集落形成单位显著少于年轻人(P<0.05)。第5代老年人骨髓间充质干细胞开始出现较多宽大扁平的衰老细胞,经β-半乳糖苷酶染色胞浆呈蓝绿色。第5代年轻人骨髓间充质干细胞大多保持梭形细胞外观,β-半乳糖苷酶染色阴性。老年人骨髓间充质干细胞上清液中丙二醛水平随代数增加而逐渐升高。骨髓间充质干细胞的β-半乳糖苷酶染色阳性细胞百分比和上清液丙二醛水平显著高于同代年轻人(P<0.05)。结论:人骨髓间充质干细胞的数量和增殖随年龄增长而降低;体外培养的早代龄老年人骨髓间充质干细胞出现衰老征象,伴随氧化应激而增高。
BACKGROUND： It was demonstrated that the cultivated human bone mesenchymal stem cells （BMSCs） presented senescent cell morphology with the passage number increased. However, it is poorly understood whether age would affect senescence process of BMSCs. OBJECTIVE： To explore the effects of age on number, proliferation, cell senescence and oxidative stress of cultivated BMSCs. DESIGN, TIME AND SETTING： The single factor design, in vitro cytology contrast experiment was performed at the Second Xiangya Hospital, Central South University between July and December 2007. MATERIALS： The bone marrow were harvested from 28 healthy volunteers, including 14 aged people, 10 male and 4 female, aged 60 73 years, with mean age of 65 years old. Fourteen young people comprise 9 male and 5 female, aged 18 28 years, with mean age of 25 years old. METHODS： Totally 2.0-3.0 mL bone marrow were extracted from volunteers from posterior superior iliac spine under aseptic condition, and then injected into anticoagulative tube containing heparin, diluted with isovolumic PBS, followed by centrifugation, next, 1 ×10^5/cm^2, cells were cultured in 6-well culture plate. After 4 days of incubation, the culture solution was renewed and adherent cells were removed. The culture solution was renewed every 3 days. MAIN OUTCOME MEASURES： Cell morphology and growth status were observed by a inverted microscope, expression of surface antigen was detected by flow cytometry, the fibroblast colony-forming unit（CFU-F）, senescence-associated β-galactosidase（SA-β-Gal） staining and supernatant malondialdehyde （MDA） content were compared between two groups. RESULTS： BMSCs expressed antigens of CD44 and CD105, but not CD34. There was no difference in morphology of the primary and the second passage BMSCs between two groups, however, the elder BMSCs needed more time to reach the first and second confluence than the young BMSCs （P 〈0.05）. CFU-F of the primary BMSCs was higher in young group （P 〈0.05）. Many senescent BMSCs, which were large and flat in shape and showed aquamarine in cytoplasma after SA-β-Gal staining, appeared among the fifth passage BMSCs from the elders. Most of the fifth passage BMSCs from the youths were spindle-shaped and did not show aquamarine in cytoplasma after SA-β-Gal staining. The supernatant MDA content increased in BMSCs from the elders with increase in number of passages （P〈0.05）. The percentage of SA-β-Gal positive cell and supernatant MDA content in the fifth passage BMSCs were significantly higher in elder group than young group （P 〈0.05）. CONCLUSION： Human BMSCs number and proliferation drop with age. Furthermore, early passage BMSCs from elders present senescent cell morphology with increased oxidative stress.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第32期6243-6248,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30500209)
教育部新世纪优秀人才计划(NCET-06-0684)~~
