人血小板生成素cDNA转染及表达产物对实验性血小板减少症的治疗作用

Transfection and expression of human thrombopoietin cDNA for treatment of thrombocytopenia in mice

目的探讨将人血小板生成素（ｈＴＰＯ）ｃＤＮＡ转染ＣＯＳ－７细胞中及表达产物对实验性小鼠血小板减少症（卡铂诱导）的治疗作用。方法以哺乳动物表达载体ＰＣＩｎｅｏ为运载体，构建受控于ＳＶ４０早期启动子ｈＴＰＯｃＤＮＡ的表达载体，利用脂质体介导转染技术，将重组ＰＣＩｎｅｏ－ｈＴＰＯ表达载体转染ＣＯＳ－７细胞中，经Ｇ４１８作用后，对挑选阳性克隆ＣＯＳ－７细胞中ｈＴＰＯｃＤＮＡ和ｍＲＮＡ分别进行ＰＣＲ扩增Ｓｏｕｔｈｅｒｎｂｌｏｔ检测和斑点杂交鉴定；对转染ＣＯＳ－７细胞培养上清进行ｈＴＰＯＥＬＩＳＡ夹心法检测；应用ｈＴＰＯｃＤＮＡ转染的ＣＯＳ－７细胞表达产物对实验性小鼠血小板减少症进行腹腔注射。结果ｈＴＰＯｃＤＮＡ不仅可在ＣＯＳ－７细胞中获得表达，且分泌到细胞培养上清中，最高表达量为４８．２８ｎｇ／ｍｌ。实验组小鼠第１０天，骨髓巨核细胞集落形成单位（ＣＦＵ－ＭＫ）集落数增加近３倍（Ｐ＜０．０１）。骨髓ＣＦＵ－ＭＫ以小集落数为主，巨核细胞体积增大（Ｐ＜０．０１）；外周血中血小板数保持在（８０±２６）×１０９／Ｌ，Ｐ＜０．０１。结论ｈＴＰＯｃＤＮＡ转染ＣＯＳ－７细胞中获得有效地表达，且表达的ｈＴＰＯ对实验性小鼠血小板减少症具?

bjectives To explore that COS7 cells efficiently transfected with human thrombopoietin (hTPO) cDNA from fetal liver and hTPO potentially treated carboplatininduced thrombocytopenia in mice. Methods Using molecular cloning technique, expression vectors PCIneo under the control of SV40 early. Applying liprofectin mediating method, recombinant plasmids PCIneohTPO were transfected into COS7 cells in addition to effect of G418. After hTPO cDNA and mRNA in colony COS7 cells were respectively identified by PCRSouthern and dot blot, hTPO was analysed with sandwich ELISA and administrated intraperitoneally to mice with thrombocytopenia. Results COS7 cells transfected with constructed PCIneohTPO expressed and secreted hTPO up to 48.28 ng/ml in the supernatant. We kinetically observed that the number of megakaryocytecolonyforming unit (CFUMK) in bone marrow enhanced to threefold (P<0.01), especially the number of small CFUMK, accompanied by an increased mean megakaryocyte volume (P<0.01) and circulating platelets returned to a normal level (80±26, ×109/L, P<0.01) in peripheral blood. Conclusion Recombinant PCIneohTPO can efficiently be transfected into COS7 cells and hTPO is an effective cytokine for treating carboplatininduced thrombocytopenia in mice.