Ad-hOSM转基因饲养层细胞对脐血CD34^+造血干/祖细胞增殖及归巢能力的影响  

The effect of transgenic cell strains expressing recombinant adenovirus vector of hOSM gene on the proliferation and homing ability of umbilical cord blood CD34^+ hematopoietic stem/progenitor cells

作　　者：田丽娜[1] 郁心 包婉蓉[1] 陈瑶[1] 韩亚丽[1] 张凤娟[1] 缪竞诚[1] 

机构地区：[1]苏州大学医学部细胞与分子生物学教研室,江苏苏州215123 [2]无锡市中心血站,江苏无锡214021

出　　处：《细胞与分子免疫学杂志》2011年第3期270-273,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国防科工委基础科研计划资助项目(K0102061501)

摘　　要：目的:建立转人抑瘤素M(hOSM)基因腺病毒载体的饲养层细胞,观察转基因细胞对脐血CD34+造血干/祖细胞扩增的影响,比较扩增前、后造血干/祖细胞体外迁移能力的变化。方法:建立转hOSM基因腺病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术(FCM)检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,FCM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(Transwell实验)检测自发迁移率和SDF-1诱导迁移率以鉴定体外扩增的造血干/祖细胞的归巢能力。结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实有目的基因表达,免疫磁珠法分离的脐血CD34+造血干/祖细胞纯度可达(96.8±2.28)%,与Ad-hOSM转基因饲养层细胞共培养7 d后CD34+造血干/祖细胞可扩增15.73倍,表面黏附分子CXCR4和CD54表达量仍较高,培养后的细胞用Tran-swell板做体外迁移实验,与转基因饲养层细胞共培养的干细胞,其诱导迁移率为(40.68±1.35)%,明显高于对照组,可以较好的保持其归巢能力。结论:转hOSM基因腺病毒载体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。AIM： To establish the transgenic cell strains expressing recombinant adenovirus vector of human Oncostain M（hOSM）gene which is supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34＋ hematopoietic stem/progenitor cell（HSPC） and compare its migration capacity before and after amplification in vitro.METHODS： Establish the transgenic cell strains expressing recombinant adenovirus vector of hOSM gene,and the objective gene 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 FCM.After culturing with feeder layer cells,detect the rate of proliferation by flow cytometry（FCM）.To compare the homing ability of HSPC after amplification in vitro,detect the spontaneous migration rate and migration rate induced by SDF-1 using transmembrane migration assay（Transwell experiment）.RESULTS： The green fluorescence was observed by fluorescence microscope in the transgenic cell strains,and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34＋ HSPC separated by MACS could reach（96.8±2.28）%.After culturing with feeder layer cells for 7 days,the CD34＋ cells were 15.73 times in group containing hOSM more than in group without hOSM.The expression rate of adhension molecules on the surface of CD34＋cells were also higher in the group containing hOSM than without hOSM.After using Transwell assys to detect the homing ability of culturing cells,the induction migration rate of stem cells clturing on transgenic cell strains was（40.68±1.35）%,significantly higher than the controll,which reveals a better homing ability.CONCLUSION： Recombinant adenovirus vector of hOSM gene as feeder layer cells can effectively proliferate umbilical cord blood CD34＋ HSPC in vitro and delay it differentiate,what＇s more,the stem cells retain a high homing ability after culturing on transgenic cell strains in vitro.

关 键 词：hOSM 造血干/祖细胞 腺病毒载体 迁移率 

分 类 号：R392.12[医药卫生—免疫学]