论文部分内容阅读
观察fascaplysin对人宫颈癌HeLa细胞体外增殖的抑制作用及其分子机制。MTT法显示fascaplysin对HeLa细胞体外增殖有显著的抑制作用,其IC50为1.79μmol·L-1;流式细胞周期检测显示,fascaplysin未能诱导HeLa细胞发生G1期阻滞,但出现浓度依赖性的sub-G1期峰;Annexin V/PI染色证实fascaplysin能诱导HeLa细胞发生凋亡,且以早期凋亡为主;Western blotting分析显示,fascaplysin能下调CDK4和cyclin D1的蛋白表达,并引起CDK4/cyclinD1特异性pRb位点Ser795的蛋白磷酸化水平下降;caspase-3、-9活性检测以及Western blotting结果证明,fascaplysin能显著增加活性caspase-3、-9的水平,下调pro-caspase-8的蛋白表达,使Bid表达减少,促进胞浆中细胞色素c释放增多,同时还能下调Bcl-2的蛋白表达。研究结果表明,fascaplysin虽然抑制了CDK4/cyclin D1复合物的活性,但并未能使HeLa细胞阻滞于G1期。该化合物对HeLa细胞体外增殖的抑制作用是通过剂量依赖性地诱导细胞凋亡实现的,其机制可能与激活caspase-8前体的活性,Bid切割为tBid数量的增加,抑凋亡因子Bcl-2表达的下调,胞浆中细胞色素c含量的增多,以及活性caspase-3、-9水平的增加有关。
To observe the inhibitory effect of fascaplysin on the proliferation of human cervical carcinoma HeLa cells in vitro and its molecular mechanism. MTT assay showed that fascaplysin significantly inhibited the proliferation of HeLa cells in vitro with an IC50 of 1.79 μmol·L -1. Flow cytometry showed that fascaplysin failed to induce G1 phase arrest in HeLa cells in a concentration-dependent manner sub-G1 peak. Annexin V / PI staining confirmed that fascaplysin induced apoptosis in HeLa cells and predominated by early apoptosis. Western blotting analysis showed that fascaplysin down-regulated the protein expression of CDK4 and cyclin D1 and caused CDK4 / cyclinD1 Caspase-3, -9 activity and Western blotting results showed that fascaplysin can significantly increase the level of active caspase-3, -9, and down-regulate pro-caspase-8 protein expression , Reducing the expression of Bid and promoting the release of cytochrome c in the cytoplasm, meanwhile, it also down-regulated the protein expression of Bcl-2. The results showed that although fascaplysin inhibited the activity of CDK4 / cyclin D1 complex, it failed to arrest HeLa cells in G1 phase. The inhibitory effect of this compound on HeLa cell proliferation in vitro was achieved through apoptosis-inducing in a dose-dependent manner. The mechanism may be related to the activation of caspase-8 precursor, the increase of the number of tBid by Bid cleavage, the increase of the amount of anti-apoptotic factor Bcl- 2 expression, cytoplasmic cytochrome c content increased, and the activity of caspase-3, -9 levels increased.