Ad-LIF-OSM双基因转染饲养细胞对脐血造血细胞体外增殖和分化的影响  

The effect of adenovirus mediated LIF and OSM co-expressing on proliferation and differentiation ofumbilical cord blood CIY34~ hernatopoietic stem/progenitor cell

作　　者：包婉蓉[1] 郁心[1] 盛伟华[1] 张凤娟[1] 王家融[1] 杨吉成[1] 缪竞诚[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院细胞与分子生物学教研室,215123

出　　处：《中华微生物学和免疫学杂志》2011年第10期865-869,共5页Chinese Journal of Microbiology and Immunology

基　　金：江苏省卫生厅科研基金资助项目（H200955）;国防科工委基础科研计划资助项目（A3820060130）

摘　　要：目的构建表达人白血病抑制因子（LIF）和抑瘤素M（OSM）双基因的WI-38人胚肺成纤维细胞，并以此细胞作为饲养层细胞观察其对CD34造血干／祖细胞体外增殖和分化的影响。方法以双启动子转移载体pAdTrack-CMV-LIF-polyA＋promoter。为基础，将OSM基因片段酶切后插入，构建出转移质粒pAdTrack-CMV-LIF-polyA＋promoter。OSM。将构建正确的转移质粒与腺病毒骨架质粒共转化，获得重组腺病毒载体pAdEasy-1-pAdTrack-CMV-LIF-polyA＋promoter。OSM，通过包装，收获重组腺病毒（Ad-LIF-OSM）。将重组腺病毒感染饲养层细胞，经RT．PCR、ELISA法检测外源基因在细胞中的表达；体外与CD34造血干／祖细胞共培养后，通过Transwell法和细胞计数检测，比较各实验组干／祖细胞体外扩增与分化情况。结果测序结果显示重组载体中的LIF和OSM基因序列正确；转基因饲养层细胞中能检测到外源LIF和OSM基因的转录和表达；外源LIF和OSM基因在造血干／祖细胞体外培养中能够发挥作用。结论成功构建携带人LIF和OSM的双基因重组腺病毒载体（Ad-LIF-OSM），Ad-LIF．OSM在造血干／祖细胞体外培养的过程中能够有效地扩增CD34造血干／祖细胞，并延缓其分化。Objective To establish Ad-LIF-OSM transgenic feeder cells for the expansion of CD34 hematopoietic stem/progenitor cell and tentatively study its effect in expansion and differentiation of cord blood hematopnietic stem cell（HSC） in vitro. Methods In the foundation of pAdTrack-CMV-LIF-pelyA＋promotera, the OSM gene was inserted to the vector plasmid. Then we structure the transfer plasmid pAdTraek-CMV-LIF- polyA＋promoterA-OSM. The transfer vector and backbone vector were further cotransfected for homologous re- combination. The result pAdEasy-l-pAdTrack-CMV-LIF-polyA ＋ promoter-OSM homologous recombination plasmid were transfected into the human embryonic kidney 293 （QBI-293A） cells, leading to formation of the recombinant adenviruses Ad-LIF-OSM which co-expressing LIF and OSM. Infect the feeder layer cells with groups of Adenovirus, detection the expressing of LIF and OSM in WI-38 cells by RT-PCR and ELISA. Compares the stem cells differentiation and proliferation of the different experimental groups in vitro by transwell and cell counting. Results The sequencing results show that the OSM genes were anastomotic in Ad-LIF-OSM. LIF and OSM gone could be detected in feeder layer cells which infected by Ad-LIF-OSM. Exogenetic LIF and OSM have special effect in culturing HSC in vitro. Conclusion The adenoviral vector co-expressing LIF and OSM （Ad-LIF-OSM） were successfully constructed. Ad-LIF-OSM transgenic feeder cells can effectively proliferate umbilical cord blood CD34 HSPC in vitro and delay it differentiate.

关 键 词：腺病毒 白血病抑制因子 抑瘤素M 共表达载体 造血干/祖细胞 

分 类 号：R733[医药卫生—肿瘤] R3[医药卫生—临床医学]