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目的 克隆人垂体瘤转化基因 1(hPTTG1)cDNA并确定其在不同细胞内的分布和其在体外是否具有直接的肿瘤转化作用。方法 (1)用快速扩增 (RACE)克隆hPTTG1cDNA ;(2 )用Northern印迹检测hPTTG1mRNA在肿瘤细胞的表达水平 ;(3)构建pCMX GFP hPTTG1表达质粒并转染HeLa等6种细胞 ,用荧光共聚焦显微镜观察GFP hPTTG1的细胞内分布 ;(4)构建pIRESneo hPTTG1表达质粒并转染NIH3T3细胞 ,经G4 18选择后检测hPTTG1是否有直接的致肿瘤作用。结果 (1)hPTTG1与大鼠PTTG的同源性为 79%。开放可读框架由 6 0 9bp组成 ,编码 2 0 2个氨基酸 ;(2 )hPTTG1在血液系统肿瘤细胞表达水平较其它肿瘤细胞为高 ,其表达水平由强到弱依次为THP 1,Raji,CEM ,AR2 30 ,DLD 1,H12 99,HeLa ,HepG2和A5 4 9肿瘤细胞 ;(3)hPTTG1的细胞内分布在HeLa、Cos 7和DU14 5细胞主要位于细胞核 ,在A5 4 9、DLD 1和NIH3T3细胞则呈胞浆和胞核的弥漫性分布 ;(4)hPTTG1明显抑制NIH3T3细胞的生长 ,降低〔3 H〕胸腺嘧啶脱氧核苷的掺入率。hPTTG1单独不具有细胞肿瘤转化作用。结论 (1)hPTTG1在血液系统肿瘤细胞表达水平较其它肿瘤细胞为高 ;(2 )hPTTG1的细胞内分布与细胞类型有关 ;(3)hPTTG1单独不具有细胞肿瘤转化作用。
Objective To clone human pituitary tumor transforming gene 1 (hPTTG1) cDNA and determine its distribution in different cells and whether it has direct tumor transformation in vitro. Methods (1) cloning hPTTG1 cDNA by rapid amplification (RACE); (2) detecting the expression level of hPTTG1 mRNA in tumor cells by Northern blotting; (3) constructing pCMX GFP hPTTG1 expression plasmid and transfection of 6 kinds of cells such as HeLa, (4) The pIRESneo hPTTG1 expression plasmid was constructed and transfected into NIH3T3 cells. After the selection of G418, hPTTG1 was directly tested for tumorigenicity. Results (1) The homology between hPTTG1 and rat PTTG was 79%. The open reading frame consisted of 6 0 9bp encoding 220 amino acids. (2) hPTTG1 expression in hematological malignancies was higher than that in other tumor cells. The expression level of hPTTG1 from high to low was THP1, Raji, CEM , AR2 30, DLD 1, H12 99, HeLa, HepG2 and A549 tumor cells; (3) The intracellular distribution of hPTTG1 was mainly located in the nucleus in A549, DLD 1 and NIH3T3 cells Cells showed diffuse distribution of cytoplasm and nucleus; (4) hPTTG1 significantly inhibited the growth of NIH3T3 cells and reduced the incorporation rate of [3H] thymidine. hPTTG1 alone does not have cell tumor transformation. Conclusions (1) The hPTTG1 expression in hematological malignancies is higher than that of other tumor cells. (2) The intracellular distribution of hPTTG1 is related to cell types. (3) hPTTG1 alone does not have the effect of cell tumor transformation.