摘要
目的探讨成人脂肪干细胞体外培养老化模型的建立及相关机制。方法成人脂肪干细胞来源于临床脂肪抽吸的患者，采用低速离心、Ⅱ型胶原酶消化、滤网过滤等方法分离出干细胞，在干细胞培养液和恒温恒湿培养箱中连续传代培养。定期观察细胞形态变化，并分别进行细胞衰老相关的β-半乳糖苷酶染色。结果脂肪干细胞数目累积倍增值（PD）〉35时细胞周期停滞，进入老化期。老化脂肪干细胞出现细胞体积膨大、核增大扁平、胞质内容物增多等现象；衰老相关的β-半乳糖苷酶染色显示表达在自我更新和老化脂肪干细胞之间差异有统计学意义（P〈0．01）。实时定量聚合酶链反应结果显示基因Nanog和基因Oct4的表达在自我更新和老化脂肪干细胞之间有显著差异。结论从多层次，多水平证实了成人脂肪干细胞体外传代培养，可复制其在体内的自然老化过程，建立脂肪干细胞的老化模型，为抗衰老药物提供新的模型。
Objective To explore the aging mechanisms of human adult adipose -derived stem ceils （hADSCs） in vitro. Methods The hADSCs come from the production of normal patients with liposuction. The mix production of liposuction was then centrifuged, digested with collagenase 11 and filleted to get single cells mix. The hADSCs were cultured in the 37℃ incubator and passed when the cells almost cover the bottom of the bottle. The senescence - associated β - galactosidase（ β - gal） staining （ SA - β - gal） and histone H2AX phosphorylation （ser139） （γH2AX） staining were done in both ＇young＇ and ＇old＇ hADSCs. Results The aging cells become big and have big flat nuclei and much more lososome in the plasma. The SA - β- gal staining and 5,H2AX staining were mostly positive in the aging hADSCs [ population doublings （PD） 〉35] comparing the self- renewing hADSCs （PD 〈20）. The real -time quantitative polymerase chain reaction （ Real - time PCR） results show that the transcribe levels of Nanog and Oct4 gene are very low in aging hADSCs compare with the selfrenewing ones. Conclusion The hADSCs will be senescent （SEN） when cultured and passed in vitro for long time. The aging hADSCs lost the Self-Renewing （SR）, differentiation functions and stay in the G0/G1 of cell cycle. The study of the senescence of hADSCs will be very useful in the regeneration medicine, tissue engineering and stem cell therapy.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第2期347-350,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（30801131）
教育部高等学校博士点专项科研基金（200804861045）
关键词
脂肪干细胞
衰老
体外培养
老化机制
Adiposederived stem cells
Senescence
Cell culture
Aging mechanism
