人血小板生成素cDNA转染COS-7细胞及表达产物对小鼠骨髓巨核细胞的作用

THROMBOPOIETIN cDNA TRANSFECTED IN COS7 CELLS AND ITS EFFECT ON MEGAKARYOCYTE IN MICE

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摘要将克隆全长１．１ｋｂ血小板生成素（ＴＰＯ）ｃＤＮＡ构建重组ＰＣＩｎｅｏＴＰＯ表达载体，利用ＬｉｐｏｆｅｃｔｉｎＴＭ介导转染ＣＯＳ－７细胞中，对阳性克隆ＣＯＳ－７细胞的ＤＮＡ、ｍＲＮＡ和培养上清分别进行ＰＣＲ扩增Ｓｏｕｔｈｅｒｎｂｌｏｔ、斑点杂交和ＴＰＯ夹心ＥＬＩＳＡ检测，观察表达产物对小鼠骨髓巨核细胞的作用。结果表明：全长１．１ｋｂＴＰＯｃＤＮＡ转染ＣＯＳ－７细胞获得表达，最高表达量可达４８．２８Ｕ／ｍｌ，其产物使骨髓巨核细胞集落形成单位（ＣＦＵ－ＭＫ）集落数增加４倍，巨核细胞增大至４２．１０±６．７０μｍ（Ｐ＜０．０１）；动态观察骨髓ＣＦＵ－ＭＫ集落数于实验第８天达最高峰，约持续２ｄ，第１０天后开始下降。 he fulllength 1.1 kb thrombopoietin(TPO) cDNA from fetal liver was cloned into the expressing vector PCIneo to construct the recombinant plasmid PCIneoTPO. COS7 cells were grown to log phase and transfected using LipofectinTM reagent. TPO cDNA of COS7 cells transfected was analyzed with PCR amplificationSouthern blot, mRNA expressed with Dot blot and supernatant with TPO ELISA. The expressing TPO stimulating megakaryocytes in mice in vitro was observed. The results showed that TPO secreted from COS7 cells transfected could increase the significantly fourfold number of megakaryocyte colony forming unit(CFUMK) and enhance the size of MK to 4210±6.70 μm(P<001). CFUMK reached peaking level after day 8, then continued for 2 days, and decreased after day 10. These data demonstrate that COS7 cells tranfected with the construct PCIneoTPO by mediated LipofectinTM may successfully express TPO in the supernant liquid which promotes megakaryocytopoiesis.