小鼠DOC-1R反义重组载体的构建及表达的研究  

Construction and Expression of Mouse DOC-1R Antisense Gene Vector

作　　者：周伟强[1] 姜莉[1] 生秀杰[1] 张学[1] 

机构地区：[1]中国医科大学卫生部细胞生物学重点实验室,辽宁沈阳110001

出　　处：《癌症》2002年第3期240-244,共5页Chinese Journal of Cancer

基　　金：国家自然科学基金高科技探索项目(编号:39980011)

摘　　要：背景与目的:DOC-1R(deletedinoralcancer-1related)基因是一个候选的抑癌基因,它与CDK2特异性结合后,抑制CDK2与cyclin形成复合物,进而对细胞周期进行调控。本研究拟构建DOC-1R反义重组质粒,并研究该基因表达受到抑制后对正常细胞增殖产生的影响。方法:小鼠DOC-1R基因筛选后构建pcDNA3-DOC-1R反义重组质粒,经细胞转染,通过细胞增殖能力测定、软琼脂培养观察DOC-1R对细胞生长状态的影响。结果:小鼠DOC-1R基因转染的NIH3T3细胞较空载体转染的细胞生长速度有较明显的差异。正义重组体pcDNA3-DOC-1R+可明显抑制细胞增殖,而反义重组体pcDNA3-DOC-1R-则促进细胞的增殖。在软琼脂培养中,正义重组体在软琼脂培养基中成集落能力下降,一方面克隆形成率下降,另一方面形成的集落也普遍较小;而反义重组体明显增加了成集落能力,克隆形成率也较正义重组体有明显的增加。结论:小鼠DOC-1R基因可明显抑制细胞的生长速度和成集落能力,这一作用有助于进行正常细胞生长、增殖规律和肿瘤治疗、预防方面的研究。Background &Objective:DOC-1R gene is a candidate tumor supp ressor gene that when connected specifically with CDK2can control t he course of cell cycle by restrainin g the reciprocity of CDK2and cyclin.The aim of this study was to construct the ant isense DOC-1R plasmid and to investi gate the effect of DOR-1R gene on the growth of normal cell.Method:The recombinant antisense plasmid w as constructed after screening the e xpression of DOC-1R gene.Following transfectio n,the effect of DOC-1R on cell growth was determined by assessing the abil ity of cell replication and observing soft agar culture.Result:The growth speed of NIH3T3transfected by mouse DOC-1R gene was of significant difference f rom that transfected by empty vector.The pcDNA3-DOC-1R + vector significantly inhibited the cell replication,whi le the pcDNA3-DOC-1R-vector stimul ated the cell replication.In soft agar culture,the colony formation capacity was decreased in the recombinant sense vector group.The clone formation rate w as decreased and the size of the colony f ormed was smaller as well.In contrast,the colony forming ability was rem arkably increased in the antisense vector group.The clone formation rate was increased significantly,compared to t hat in the sense group.Conclusion:Mouse DOC-1R gene can significantly inhibit cell growth a nd colony formation capacity.It will be helpful for the study on the mechanism of normal cell growth and replication as well a s for research in tumor treatment and prevention.

关 键 词：DOC-1R 细胞周期调控 反义核酸 小鼠 重组载体 肿瘤 

分 类 号：R730.2[医药卫生—肿瘤]