中国组织工程研究

中国组织工程研究 ›› 0, Vol. ›› Issue (0): 56-60.doi: 10.3969/j.issn.1673-8225.2012.01.012

• 骨髓干细胞 • 上一篇    下一篇

转白血病抑制因子基因人胚肺成纤维细胞对脐血CD34+细胞体外扩增的影响

夏 卫1，郁 心1，姜 健1，田丽娜2，陈 瑶2，缪竞诚2   

  1. 1 无锡市红十字中心血站输血重点实验室，江苏省无锡市 214021；
    2 苏州大学医学部细胞与分子生物学教研室，江苏省苏州市 215123
  • 收稿日期:2011-05-13 修回日期:2011-07-15
  • 通讯作者: 郁心，硕士，副主任技师，无锡市红十字中心血站输血重点实验室，江苏省无锡市 214021 yu_heart@sina.com
  • 作者简介:夏卫，男，1964年生，江苏省无锡市人，汉族，1984年镇江医学院(江苏大学)毕业，副主任技师，主要从事血液免疫方面的研究。

Effects of human embryonic lung fibroblasts feeder layer transfected with leukemia inhibitoryfactor gene on the proliferation of umbilical cord blood CD34+ cells in vitro

Xia Wei1, Yu Xin1, Jiang Jian1, Tian Li-na2, Chen Yao2, Miao Jing-cheng2   

  1. 1Key Laboratory ofBlood Transfusion,Wuxi Red CrossBlood Centre, Wuxi214021, JiangsuProvince, China;
    2Department of Celland MolecularBiology, MedicineSchool of SoochowUniversity, Suzhou215123, JiangsuProvince, China
  • Received:2011-05-13 Revised:2011-07-15
  • Contact: Yu Xin, Master,Associate chieftechnician, KeyLaboratory of BloodTransfusion, WuxiRed Cross BloodCentre, Wuxi214021, JiangsuProvince, Chinayu_heart@sina.com
  • About author:cian, KeyLaboratory of BloodTransfusion, WuxiRed Cross BloodCentre, Wuxi214021, JiangsuProvince, China

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342

被引次数

2

摘要：

背景：单份脐血的造血细胞数量有限，难以满足成人的需要，如何有效地扩增脐血造血干/祖细胞是目前研究的热点。
目的：构建人白血病抑制因子基因修饰的人胚肺成纤维细胞，观察转基因细胞对脐血CD34+造血干/祖细胞体外扩增的影响。
方法：建立转人白血病抑制因子基因的饲养层细胞，用RT-PCR法和ELISA法鉴定目的基因的表达；采用免疫磁珠法分离脐血CD34+造血干/祖细胞，流式细胞术检测纯度；将CD34+造血干/祖细胞与饲养层细胞共培养，流式细胞术检测各组增殖效果；扩增后的造血干/祖细胞用跨膜迁移实验检测自发迁移率和基质细胞衍生因子1诱导迁移试验以鉴定体外扩增的造血干/祖细胞的归巢能力。
结果与结论：成功建立转基因饲养层细胞，RT-PCR法和ELISA法证实有目的基因表达，与人白血病抑制因子转基因饲养层细胞共培养7 d后CD34+造血干/祖细胞可大量扩增，同时表面黏附分子的表达量仍较高。体外迁移实验显示与转基因饲养层细胞共培养的造血细胞的诱导迁移率明显高于对照组，可以较好地保持其归巢能力。因此转人白血病抑制因子基因的饲养层细胞可有效扩增脐血CD34+造血干/祖细胞，延缓其分化，并且体外扩增后仍保持较高的归巢能力。

关键词: 人白血病抑制因子, 饲养层, CD34+细胞, 体外扩增, 造血干/祖细胞

Abstract:

BACKGROUND: The number of hematopoietic cells in single copy cord blood is limited, and it is difficult to meet the needs of adults. How to effectively amplify cord blood hematopoietic stem/progenitor cells (HSPCs) is a hot research issue currently.
OBJECTIVE: To construct gene-modified human embryonic lung fibroblast cells using human leukemia inhibitory factor (hLIF) and to observe the effect of transgenic cells on the amplification of HSPCs in vitro.
METHODS: The feeder layer trancfected with recombinat adenouirus Ad-hLIF was established and objective gene was detected by using RT-PCR and ELISA. Umbilical cord blood CD34+ HSPC were separated using magnetic-activated cell sorting and the purity was detected using flow cytometry. Homing ability of amplified HSPC in vitro was detected by transmembrane migration assay.
RESULTS AND CONCLUSION: Transgenic feeder cells were constructed successfully, and the expression of objective gene was verified by RT-PCR and ELISA. After culturing with feeder layer cells for seven days, the number of CD34+ HSPCs in the group containing hLIF was 15.73 much more than that in the group without hLIF. The expression rate of surface adhesion molecules was also still great. In vitro migration assay showed that the induced migration rate of hematopoietic cells in co-culturing with transgenic feeder layer was significantly higher than that of the control group, with a better homing ability. The feeder layer cells of adenovirus vector transfected with hLIF gene amplify umbilical cord blood CD34+ HSPC effectively in vitro and delay differentiation; what’s more, the CD34+ cells retains a great homing ability after amplifying in vitro.

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