反义细胞周期素D1基因对肝癌细胞株HepG2的作用

目的通过基因反义封闭技术体外抑制细胞周期素D1(cyclin D1)的表达,研究其对肝癌细胞cyclin D1蛋白表达和细胞增殖的影响。方法以肝癌细胞株HepG2为研究对象,通过转染可表达cyclin D1反义互补脱氧核苷酸(AScDNA)的质粒后,观察cyclin D1反义cDNA对HepG2细胞cyclin D1基因表达及体外增殖活性的影响。结果四甲基偶氮唑盐法检测细胞增殖活性显示转染表达反义cyclin D1的质粒后, HepG2细胞的增殖受到抑制,抑制作用在48 h左右最强;逆转录聚合酶链反应检测显示cyclin D1 mRNA基因的表达明显被抑制;免疫组织化学检测结果显示cyclin D1蛋白表达明显降低; 流式细胞仪检测结果显示G0/G1期的细胞比例增高,G2+M和S期的细胞比例下降,HepG2细胞周期在G1期被阻滞。结论cyclin D1反义cDNA可以特异性的抑制肝癌HepG2细胞株cyclin D1蛋白的表达,从而调控细胞周期,抑制肝癌细胞增殖。利用cyclin D1反义cDNA进行细胞周期调控对于肝细胞癌的生物治疗具有一定的应用前景。

Inhibitory effect of cyclin D1 antisense cDNA on human hepatocarcinoma cell line HepG2

Objective To investigate the effect of antisense cDNA of cyclin Dl on the cyclin Dl gene expression and cell proliferation of human hepatocarcinoma HepG2 cells in vitro. Methods Plasmids containing cyclin Dl antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation were examined by MTT method, RT-PCR, immunohistochemical means, and flow cytometry. Results Cyclin Dl antisense cDNA significantly inhibited the growth of HepG2 cells. The inhibition peaked at 48 hour after transfection by MTT method. RT-PCR analysis showed that cyclin Dl antisense cDNA down-regulated cyclinD1 at the mRNA levels. Expression level of cyclin Dl protein was also decreased as shown by immunohistochemical studies. Cell-cycle analysis by flow cytometry showed that transfected HepG2 cells were arrested at the G1 phase of the cell cycle. Conclusions Our data suggest that cyclin Dl antisense cDNA could specifically inhibit the expression of cyclin Dl mRNA and protein and regulate cell cycle and cell proliferation of HepG2 cells. Cyclin Dl antisense cDNA may serve as a potential antitumor strategy in regulating cell-cyclein treating advanced HCCs.