Ad—hLIF转基因饲养层细胞对CD34^＋造血干／祖细胞增殖和分化的影响  

Effect of feeder cells transduced with Ad-hLIF on the expansion of CD34^＋ cells in indirect co-culture

作　　者：井莹莹[1] 杨吉成[1] 盛伟华[1] 胡志清[1] 郁心 单云波[1] 刘铁连[1] 韩亚丽[1] 包婉蓉[1] 张日[3] 朱南康[4] 缪竞诚[1] 

机构地区：[1]苏州大学细胞与分子生物学教研室,215123 [2]无锡市红十字中心血站 [3]苏州大学附属第一医院血液科 [4]苏州大学放射医学研究所

出　　处：《中华微生物学和免疫学杂志》2009年第3期234-238,共5页Chinese Journal of Microbiology and Immunology

基　　金：国防科工委基础科研计划资助项目（K0102061501）

摘　　要：目的用腺病毒载体介导的人白血病抑制因子基因（Ad—hLIF）感染WT-38人胚肺成纤维细胞制备饲养层细胞，体外观察转基因细胞对CD34^＋造血干／祖细胞增殖和分化的影响，体内研究对辐射损伤SCID小鼠造血功能恢复的效果。方法用RT—PCR和ELISA法鉴定Ad—hLIF转基因饲养层细胞目的基因的表达后，将经免疫磁珠法分离和流式细胞术检测后的CD34^＋细胞在饲养层和／或细胞因子培养体系中扩增28d，检测不同时问点的单个核细胞（MNC）数量及CD34^＋细胞阳性率；扩增后的MNC经CFDASE荧光标记后移植入辐射损伤SCID小鼠体内，RT—PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的归巢情况。结果感染50MOI（multiplicityofinfection）Ad·hLIF的饲养层细胞均有绿色荧光，RT—PCR和ELISA法结果显示hLIF日的基因能在WI-38饲养层细胞中表达，免疫磁珠法分离的CD34^＋造血干／祖细胞经流式细胞术检测其纯度可达95．60％±2．58％，MNC在Ad-hLIF转基因饲养层培养体系持续扩增，最高可达356．95±0．87倍，其中CD34^＋细胞仅在0～14d能维持较高水平，最高可扩增52．11±1．13倍，以后逐渐降低。将其移植辐射损伤SCID小鼠后，可明显提高小鼠存活率，4周内小鼠骨髓中不仪可观察到CFDASE荧光标记的细胞，而凡经RT．PCR法鉴定后，还可检测到表达Alu人源基因的人脐血造血归巢细胞。结论成功建立的Ad—hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34^＋造血干／祖细胞，并延缓其分化。扩增的CD34^＋细胞对辐射损伤SCID小鼠具有造血功能恢复的功能。Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34^＋ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell （HSPC） transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34^＋ HSPC separated by magnetic-activated cell sorting（MACS） was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell （MNC） and CD34^＋ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Resuits The green fluorescence was observed in the transgenic cells infected with 50MOI（ multiplicity of infection） Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34^＋ HSPC separated by MACS could reach 95.60% ± 2.58% , Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95 ± 0.87 fold, and maximal expansion of CD34^＋ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34^＋ HSPC in vitro and delay it differentiate, what＇s more, it has high transplant efficacy and haematogenesis activity.

关 键 词：hLIF 腺病毒载体 造血干/祖细胞 SCID小鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]