【作者】 张伶；

【导师】 魏于全；

【作者基本信息】 四川大学 ， 肿瘤学， 2005， 博士

【摘要】 背景与目的:细胞周期蛋白B1(Cyclin B1)为有丝分裂期(M期)细胞周期蛋白,与细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK1)结合形成成熟促进因子(muturior promoting factor,MPF),MPF的激活为真核细胞启动有丝分裂所必需,在肿瘤的发生发展中起重要作用;Cyclin B1在许多肿瘤细胞系均高表达,其高表达被视为肿瘤具有恶性潜能的标志之一。反义cDNA是反义技术中一种简便有效的下调靶基因的方法,但目前尚未见应用该方法靶向Cyclin B1基因抗肿瘤治疗的相关报道。 方法:构建含有小鼠Cyclin B1反义全长cDNA的重组质粒(pAS-mCLB1)并将pAS-mCLB1及空载体pcDNA3.1(+)分别转染小鼠Lewis肺癌(LL/2)和CT26结肠癌细胞系(CT26),建立了AS-mCLB1及pcDNA3.1(+)稳定转染子(Ac及Pc细胞);通过流式细胞仪检测Ac、Pc及未经转染(Nc)细胞的细胞周期分布及凋亡;分别用两步法半定量RT-PCR及Western blot比较上述三种细胞中mCyclin B1的mRNA及蛋白含量;MTT及细胞生长实验用于检测细胞增殖活性;在小鼠体内分别接种上述三种细胞,观察细胞在体內的成瘤性及荷瘤小鼠的生存时间;然后对Ac与Nc组的细胞及动物血清进行蛋白组学差异分析;免疫组化检测更多还原

【Abstract】 Background and Objective: Cyclin B1, a cell cycle protein in mitosis phase, connecting with p34 kinase (CDK1) to form muturior promoting factor (MPF), is necessary for mitotic initiation in eukaryotic cells and it plays an important role in cancer development; cyclin B1 is over-expressed in a variety of tumors and is thought to be an indicator of the malignant potential of tumors. Antisense cDNA is a simple, easy and influential method to down-regulate the expression of target genes. It remains to be clarified whether down-regulation of cyclin Bl with full-length antisense cDNA of cyclin B1 (AS-CLB1) in tumor cells is an effective strategy for cancer therapy.Methods: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin Bl (pAS-mCLBl) and pcDNA3.1 empty vector were constructed and introduced into LL/2 and CT26 cells to establish AS-mCLB1 (Ac) and pcDNA3.1 (Pc) stable transfectants respectively. The cell cycles and apoptosis of Ac, Pc and untransfected (Nc) cells were determined using flow cytometry. mRNA and protein content of cyclin Bl were tested by two-step semi-quantitative RT-PCR and western blot assay respectively. The activity of cell proliferation was measured by MTT and cell growth assay.Tumorigenicity and survival were observed in the mice which were implanted with Ac, Pc and Nc cells respectively. The proteomics of cells and animal serums were compared between the AC and NC groups. In the tumor tissues, the expression of mCyclin B1 was detected with immunohistochemistry and DNA Fragmentation Detection Kit was used to test DNA fragmentation associated with the apoptotic cells.Result: Prominent G1 arrest and apoptosis were shown and abnormal morphology with inhibition of cell growth appeared in the Ac cells in which persistent and evident down-regulation of mCyclin B1 expression was induced. Moreover, after implanting, tumors in the Ac group developed on day 9 versus day 5 in the controlled groups (Nc and Pc) in LL/2-bearing mice. In CT26-loaded mice, tumors in the Ac group developed on day 6 versus day 3 in the controlled after inoculation. Therefore, survival benefits might be achieved through the inhibition of tumorigenicity in the mice of Ac groups. Both in cells and animal serums, the differences of proteomics were apparent. The expression of mCyclin B1 protein was decreased and cell apoptotic rate was increased in the tumor tissues of Ac groups.Conclusion: To our understanding, this is the first report concerning the biological activities of cyclin Bl knockdown tumor cells using AS-mCLB1. These results suggested that the suppression of cyclin Bl by AS-CLBl could induce G1 arrest and apoptosis of tumor cells obviously, which involved in inhibiting the activity of tumor cells in vitro and in vivo efficiently. So AS-CLB1 might be a viable method for tumor biotherapy.更多还原