细胞周期素D1反义cDNA治疗肝癌的研究

目的 通过基因反义封闭技术抑制细胞周期素D1（CydinD1）的表达，研究其对肝癌细胞增殖以及成瘤性的影响。方法 以肝癌HepG2细胞株为研究对象，通过转染可表达CyclinD1反义互补脱氧核苷酸（AScDNA）的质粒后，观察CyclinD1反义cDNA对肝癌细胞CyclinD1基因表达、体外增殖活性及裸鼠体内成瘤性的影响。结果 噻唑蓝（MTT）法检测细胞增殖活性显示转染表达反义CydinD1的质粒后，HepG2细胞的增殖受到抑制．抑制作用在48h左右最强；逆转录-聚合酶链反应（RT-PCR）检测显示CyclinD1 mRNA基因的表达明显被抑制；间接免疫荧光检测结果显示CyclinD1蛋白表达显著降低；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；裸鼠成瘤试验显示肝癌HepG2细胞的成瘤性受到明显抑制。结论 CyclinD1反义cDNA可以特异性的抑制肝癌HepG2细胞株CyclinD1蛋白的表达，从而调控细胞周期，抑制肝癌细胞增殖及体内成瘤性。CyclinD1反义cDNA对于肝细胞癌的生物治疗具有一定的应用前景。

Antitumor effects of Cyclin D1 antisense cDNA on human hepatocellular carcinoma

Objective To investigate the effect of antisense cDNA of Cyclin D1 on the cell proliferation and tumorigenicity of human hepatocarcinoma HepG2 cell line. Methods Plasmids containing Cyclin D1 antisense cDNA were constructed and transfected into HepG2 cells. Their effects on cell proliferation and gene expression were examined by MTT method, RT-PCR method, immunofluorescent assay and flow cytometry. To assess the effect of antisense Cyclin D1 on tumorigenicity, the tansfected cells were injected subcutaneously in nude mice. Results Cyclin D1 antisense cDNA could significantly inhibit the growth of HepG2 cells. The inhibitory effecr peaked at 48 h after transfection by MTT method. RT-PCR analysis revealed that Cyclin D1 antisense cDNA could down-regulate the expression of Cyclin D1 mRNA. The expression level of Cyclin D1 protein was also decreased by immunofluorescent assay. Cell-cycle analysis by flow cytometry indicated that the transfected HepG2 cells were arrested at the G1 phase of the cell cycle. Antisense expressing cells displayed complete loss of tumorigenicity in nude mice. Conclusion Cyclin D1 antisense cDNA could specifically inhibit the expression of Cyclin D1 mRNA and protein and regulate cell cycle. Furthermore, the cell proliferation and tumorigenicity of HepG2 cells were significantly inhibited. This therapeutic potential of Cyclin D1 antisense cDNA may lead to new cell-cycle-based antitumor strategies for human hepatocarcinoma.