脐血造血细胞体外扩增过程中DNA损伤的研究  

DNA Damage during Umbilical Cord Blood Expansion Ex Vivo

作　　者：王彩霞[1] 毛平[1] 张玉平[1] 段华新[1] 杜庆华[1] 

机构地区：[1]广州医学院附属市一人民医院血液科,广东广州510180

出　　处：《中国实验血液学杂志》2010年第2期450-453,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30471644);广东省自然科学基金重点项目(编号06112013);广州市科技局项目(编号2008A1-E4011-8)

摘　　要：本研究检测脐血(CB)单个核细胞(MNC)体外培养DNA损伤情况,以探讨体外培养的最佳收获时机。脐血MNC分别接种于含细胞因子无血清培养体系并进行集落接种,分别在0、7、14及21天收集细胞,用流式细胞仪检测CD34+及CD133+细胞数,采用单细胞凝胶电泳试验检测培养后不同时间点脐血造血细胞及集落细胞的DNA链断裂损伤情况。结果表明,体外短期培养(14天内)时,脐血CD34+及CD133+细胞数最多,脐血细胞DNA损伤率均低于5.0%,21天时CD34+及CD133+细胞数降到低于0天的比例,细胞DNA损伤率为28.2%,DNA损伤率比0天时高(p=0.000),损伤细胞彗星尾长度明显大于0天(p=0.000);而其余各培养时间点(7天和14天)损伤细胞彗星尾长度与培养0天组损伤细胞的彗星尾长度比较,差异无统计学意义(p=0.863及p=0.253);集落培养细胞DNA损伤率均低于5.0%。结论:利用本方法进行脐血造血细胞短期(14天)培养DNA损伤率均低于5.0%,但超过14天时DNA损伤明显增加,体外培养的最佳收获时机应在14天内。The aim of this study was to detect DNA damage during expansion ex vivo of umbilical cord blood （UCB） hematopoietic cells and explore the optimal harvest time for culture of CB hematopoietic cells. Mononuclear cells（MNCs） separated from UCB were cultured in a serum-free system supplemented with cytokines and colony forming units were assessed by semisolid culture at the same time. On day 0, 7, 14 and 21 cells were collected for single cell gel electrophoresis （SCGE） analysis and CFUs were also assayed by SCGE, CD34^＋ cells and CD133^＋ cells were quantitated by fluorescence-activated cell sorting （ FACS ）. The results showed that the percentage of CD34^＋ and CD133^＋ cells was found to be highst after short-term culture （ 〈 14 days） and the cord blood DNA damage rate was observed to be less than 5.0% at earlier time points, but at day 21 the DNA damage rate was 28.2%, which was higher than that at day 0 （p = 0. 000 ）, the tail length of the DNA comet was longer than that at day 0 （p = 0. 000 ）. The tail lengths of DNA damage on other time points were not significantly different from that at day 0. It is concluded that the DNA damage rate is less than 5.0% after short-term （ 〈 14 days） culture of UCB cells ex vivo by using this method. After 14 days DNA damage rate increases significantly. The optima/harvest time of cord blood cells after culture ex vivo would be within 14 days.

关 键 词：脐血 造血细胞 DNA损伤 体外扩增 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]