CAJ | 학술논문

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+promoterΔ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+promoterΔ -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(AdLIF-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.
목적 구건표체인백혈병억제인자(LIF)화억류소M(OSM)쌍기인적WI-38인배폐성섬유세포,병이차세포작위사양층세포관찰기대CD34+.조혈간/조세포체외증식화분화적영향.방법 이쌍계동자전이재체pAdTrack-CMV-LIF-polyA+promoterΔ위기출,장OSM기인편단매절후삽입,구건출전이질립pAdTrack-CMV-LIF-polyA+promoterΔ -OSM.장구건정학적전이질립여선병독골가질립공전화,획득중조선병독재체pAdEasy-1-pAdTrack-CMV-LIF-polyA+promoter△-OSM,통과포장,수획중조선병독(AdLIF-OSM).장중조선병독감염사양층세포,경RT-PCR、ELISA법검측외원기인재세포중적표체；체외여CD34+조혈간/조세포공배양후,통과Transwell법화세포계수검측,비교각실험조간/조세포체외확증여분화정황.결과 측서결과현시중조재체중적LIF화OSM기인서렬정학；전기인사양층세포중능검측도외원LIF화OSM기인적전록화표체；외원LIF화OSM기인재조혈간/조세포체외배양중능구발휘작용.결론 성공구건휴대인LIF화OSM적쌍기인중조선병독재체(Ad-LIF-OSM),Ad-LIF-OSM재조혈간/조세포체외배양적과정중능구유효지확증CD34+조혈간/조세포,병연완기분화.
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 hematopoietic stem cell(HSC) in vitro.Methods In the foundation of pAdTrack-CMV-LIF-polyA+promoterΔ,the OSM gene was inserted to the vector plasmid.Then we structure the transfer plasmid pAdTrack-CMV-LIF-polyA+promoterΔ-OSM.The transfer vector and backbone vector were further cotransfected for homologous recombination. The result pAdEasy-1-pAdTrack-CMV-LIF-polyA + promoterΔ-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 gene 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.