血小板生成素糖基化与其体内生物学活性关系的研究.doc

???? 血小板生成素糖基化与其体内生物学活性关系的研究 ???? ????摘　要　为探讨血小板生成素（TPO）糖基化与其体内生物学活性的关系，给血小板缺乏症小鼠模型腹腔分别注射相同活性单位酵母系统表达的人TPO成熟肽或大肠杆菌表达的人TPO N-端结构域蛋白，连续给药5d，每天一次，末次给药后3d，比较两种TPO组动物外周血血小板数量、骨髓巨核细胞培养单位（CFU-Meg）、骨髓有核细胞DNA含量。结果高、中、低剂量的TPO成熟肽组动物的血小板数、巨核细胞集落数和骨髓DNA含量均明显高于TPO N-端结构域蛋白的相应组（P＜0．01）。显示酵母系统表达的TPO成熟肽比大肠杆菌表达的TPO N-端结构域有明显的生物学活性，这可能与它们的糖基化程度及半衰期有关。　　关键词 血小板生成素　糖基化　生物学活性　　中号 Q78 STUDIES ON RELATIONS OF THROMBOPOIETIN GLYCOSYLATION AND ITS BIOLOGICAL EFFECTS IN VIVO Liu Li，Wang Ying，Tian Shengli，Lu Xingwu，Meng Yan，Liu Lizhong，Xie Baoshu，Leng Aijun（Research Center of Molecular Biology of Air Force General Hospital，Beijing 100036） 　　Abstract To investigate the relation of thrombopoietin（TPO）glycosylation and its biological effects in vivo，human TPO mature peptide expressed by Piochia Pastoris or TPO N-terminal domain protein expressed by E．coli were injected into thrombocytopenia mice with the same activity unit in 5 days by intraperitoneal injection once for a day．On fourth day after last injection，peripheral platelet number，CFU-Meg and DNA content in bone marrow cells were compared in two kinds of TPO．The results showed that platelet number，CFU-Meg and DAN content in TPO mature peptide were remarkably higher than that of APO N-terminal domain protein in different dosage groups．TPO mature peptide expressed by Pichia Pastoris has significantly higher biological activity than that of TPO N-terminal domain protein expressed by E．coli．It may be related to their glycosylation degree and half-life．　　Key words Thrombopoietin，Glycosylation，Biological activity 　　血小板生成素（Thrombopoietin，TPO），又称巨核细胞发育形成因子（Megakaryocyte growth and development factor，MGDF）或mpl配基（Mpl Li-gand，ML），是1958年从血小板缺乏症动物的血浆中发现的一种特异地作用于巨核细胞增殖、分化及血小板生成的细胞因子。由于多种因素使TPO研究工作在近半个世纪里进展不大。90年代发现c-mpl原癌基因（c-myeloproliferative leukemia）是强有力的促巨核细胞生成调节物的受体，以c-mpl cDNA为依据，利用亲合层析法，成功地纯化出有活性的mpl配体蛋白，并根据其末端氨基酸序列合成寡核苷酸引物，利用PCR方法从人胎肝cDNA文库中筛选出ML cDNA，即人们一直寻找的TPO cDNA，至此TPO的研究工作才有了突破性进展。　　已有研究表明，TPO的糖基化不影响它的体外生物学活性［1～3］，但与体内生物学活性及半衰期的关