稳定表达细胞周期蛋白D1反义基因逆转人胃癌细胞恶性表型

Stable overexpression of cyclin D1 antisense gene reverses the transformed phenotype of human gastric cancer cells

目的 探讨细胞周期蛋白D1对肿瘤生物学行为的影响及用于基因治疗的可能。方法 利用重组技术将人细胞周期蛋白D1cDNA全片段克隆至亚克隆载体Bluescript KS中。利用该载体内的T2 /T3成对启动子体系 ,体外转录法制备正、反义细胞周期蛋白D1单链RNA探针。同时利用该载体转换酶切位点的作用 ,构建了人细胞周期蛋白D1反义RNA真核表达载体pDORD1AS ,阳离子脂质体介导下转染胃癌细胞株 ,经G41 8筛选获得细胞周期蛋白D1反义RNA稳定表达的阳性细胞克隆。用免疫细胞化学、流式细胞仪、分子杂交、病理形态学和细胞生物学方法对该阳性细胞克隆进行检测 ,同时设未经转染和转染空载体胃癌细胞组作对照。结果 阳性细胞克隆细胞周期蛋白D1表达较对照组减少 (抑制率约为 3 6% ) ;细胞体积增大 ,核浆比例下降 ;细胞倍增时间延长 (4 2 .2h比 2 6.8h及 2 6.4h) ,生长饱和密度下降 (1 .9× 1 0 5 比 4.8× 1 0 5 及 4.8× 1 0 5 ) ,G1/G0 期细胞比例增加 (80 .9%比 64 .6%及63 .8% ) ,血清依赖性有所恢复 ,裸鼠成瘤率下降 (0 / 4比 4/ 4及 4/ 4 )。结论 稳定表达细胞周期蛋白D1反义RNA对胃癌细胞SGC790 1 /VCR的恶性表型有一定的逆转作用 ,进一步深入的研究是有意义的。

Objective To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. Methods A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So,it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector.PDORD1AS,an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1,was obtained by selection in G418.The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. Results Compared with control cell lines,SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%),increased cell size and cytoplasm to nucleus ratio, increased doubling time (42.2 h to 26.8 h and 26.4 h),decreased saturation density (1.9×10 5 to 4.8×10 5 and 4.8×10 5), increased percentage of cells in the G 1/G 0 phase (80.9% to 64.6% and 63.8%),reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). Conclusion Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.