含重组人血小板生成素基因的小鼠成纤维细胞的克隆筛选与鉴定  

Screening and identifying of the cell clones containing recombinant human thrombopoietin gene

作　　者：韩安平 陆爱丽 王申五 侯纬敏 王德炳 

机构地区：[1]北京医科大学人民医院血液病研究所 [2]北京医科大学医院血液病研究所中心实验室 [3]北京医科大学医院生物化学与分子生物学系

出　　处：《北京医科大学学报》1999年第2期110-112,123,共4页Journal of Peking University(Health Sciences)

基　　金：“九五”国家科技攻关项目!（９６－９０６－０１－１９）;高等学校博士学科点专项科研基金

摘　　要：获取稳定表达重组人血小板生成素的ＮＩＨ３Ｔ３成纤维细胞克隆，为开展成纤维细胞介导的ＴＰＯ基因治疗研究提供细胞学基础。方法：以重组人ＴＰＯｃＤＮＡ为目的基因，构建真核细胞表达重组体ｐｃＤＮＡ３／ＴＰＯ，脂质体将其转染于ＮＩＨ３Ｔ３细胞，经Ｇ４１８筛选，获得稳定表达重组人ＴＰＯ的细胞克隆，应用ＰＣＲ，ＲＴ－ＰＣＲ及Ｗｅｓｔｅｒｎ印迹等技术鉴定目的基因的导入及表达。To obtain the engineered NIH3T3 fibroblast clones which stably express the recombinant human thrombopoietin (TPO) so as to explore the feasibility of gene therapy on animal with fibroblasts-mediated TPO cDNA delivery. Methods: Constructing the eukaryotic expression vector pcDNA3/TPO with human recombinant TPO cDNA as the therapeutic gene; transfecting NIH3T3 fibroblasts with pcDNA3/TPO by means of lipofection; obtaining the TPO-secreting NIH3T3 cell clone (3T3/TPO+ ) through G418 screening; identifying the DNA, mRNA and protein with methods of PCR, RT-PCR and Western blotting, respectively. Results: NIH3T3 cells could be transfected with pcDNA3/TPO by lipofection, and these cells could stably express and secret TPO.Conclusion:The needed cell clones containing human TPO cDNA have been obtained, and they can be used for the investigation of gene therapy with human TPO cDNA on experimental mice.

关 键 词：血小板生成素 成纤维细胞 基因表达 克隆 筛选 

分 类 号：Q343.11[生物学—遗传学] R394.8[医药卫生—医学遗传学]