细胞周期蛋白依赖激酶对肝癌细胞增殖及凋亡的影响

Influences of cyclin dependent kinase on proliferation and apoptosis in hepatic carcinoma cells.

目的:探讨细胞周期蛋白依赖激酶(cyclin dependent kinase,CDK)对原发性肝癌(肝癌)细胞增殖及凋亡的影响。方法:对体外培养的肝癌细胞株SMMC-7721分别予浓度为0μmol/L、2μmol/L、4μmol/L、8μmol/L、16μmol/L、32μmol/L CDK抑制剂rosco-vitine干预72 h,分别通过倒置显微镜、四甲基偶氮唑蓝法、流式细胞仪、逆转录PCR法观察SMMC-7721细胞的生长、增殖,细胞周期时相的分布、凋亡以及CDK2、半胱氨酸蛋白酶-3和BCL-2 mRNA的表达。结果:予roscovitine干预72 h后,光镜下可见随着roscovitine浓度的增加,部分细胞核固缩、胞体折光性减弱、胞浆内可见有颗粒和核碎片形成,其中以32μmol/L组的改变最为明显。四甲基偶氮唑蓝法结果显示,不同浓度的roscovitine对细胞生长均有一定的抑制作用,roscovitine对细胞的抑制作用呈剂量依赖性和时间依赖性。流式细胞仪检测发现,经roscovitine干预后肝癌细胞G0期、G1期及凋亡细胞的比例增加。逆转录PCR发现,随着roscovitine浓度的增加,CDK2 mRNA及凋亡相关基因BCL-2 mRNA水平降低,半胱氨酸蛋白酶-3 mRNA表达升高。结论:CDK表达降低可抑制增殖期肝癌细胞的增殖,诱导细胞的凋亡,凋亡机制可能与BCL-2、半胱氨酸蛋白酶-3的表达改变有关。CDK在肝癌细胞增殖及凋亡中起着重要作用。

Objective： To explore the influences of cyclin dependent kinase （CDK） on proliferation and apoptosis in hepatic carcinoma cells. Methods： After interfered by roscovitine of 0 μmol/L, 2 μmol/L, 4 μmoL/ L, 8 μmol/L, 16 μmol/L, 32 μmoL/L for 72 hours, inverted microscope, MTT, flow eytometry and reverse transeription-polymerase chain reaction （RT-PCR） were used to observe the effects of CDK on growth, proliferation, cell cycle, apoptosis and the mRNA expression of caspase-3, BCL-2 in SMMC-7721 cells cultured in vitro. Results ： After interfered by roseovitine for 72 hours, the microscope showed that a part of nucleus became pyknotic, the eytosome refractivity weakened, particles and nuclear debris appeared in cytoplasm with the increase of roscovitine concentration, and the effect of roscovitine in 32 μmol/L was the most remarkable. The results of MTT also showed that the roseovitine could inhibit the growth of cells with various dose, and the inhibition was dependent on dosage and time. Flow eytometry observed it also could increase the ratio of G0 , G1 and apoptotie cells. RT- PCR showed the mRNA expression of BCL-2 and CDK2 was reduced, and the mRNA expression of caspase-3 was increased by roseovitine. Conclusion： Roscovitine could restrain proliferation and induce apoptosis in hepatic carcinoma cells. The mechanism of apoptosis depends on the activity of BCL-2 and easpase-3. CDK may play an important role on the proliferation and apoptosis in hepatic carcinoma cells.