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初步研究了新合成的(顺)-3-(氯代亚甲基)-5-甲基-硫色满-4-酮(3-chloromethylene-5-fluoro-thiochroman-4-one,CMTK)对人乳腺癌MCF-7细胞增殖和凋亡的影响,分别用不同浓度的CMTK对细胞进行干预,采用改良的MTT法测定CMTK抑制细胞增殖的能力,台盼蓝染色法计数活细胞数,绘制生长曲线,流式细胞术检测细胞对细胞周期的影响,同时采用流式细胞术及TUNEL染色法(terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labelling)检测CMTK诱导凋亡,活性检测法测定细胞中人半胱氨酸蛋白酶8(caspase-8)和人半胱氨酸蛋白酶9(caspase-9)的活性,ELISA法测人死亡受体3(deathreceptor3,DR3)、人肿瘤坏死因子受体1(tumor necrosis factor receptor1,TNFR1)、人肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、人半胱氨酸蛋白酶3(caspase-3)的蛋白表达情况.结果表明,CMTK可明显抑制MCF-7细胞增殖,与顺铂(cisplatin,CDDP)相比,其抗肿瘤活性更明显,IC50为14.24±0.12molL1;生长曲线进一步说明CMTK抑制细胞增殖,其抑制作用呈时间-剂量依赖性;流式细胞仪检测分析显示,加药12和24h后可将细胞阻滞于G0/G1期(**P<0.05);TUNEL法和流式细胞仪分析CMTK可诱导凋亡的结果基本一致;与药物作用后,caspase-8和caspase-9的活性增高,并呈浓度剂量依赖性;ELISA法检测DR3蛋白量和TNF-细胞因子的量无变化,TNFR1和caspase-3的蛋白量显著增高,CMTK诱导肿瘤细胞凋亡的机制可能是:不仅可以通过死亡受体TNFR1介导凋亡途径,激活caspase-8;还可以通过线粒体凋亡途径,激活caspase-9;最终激活caspase-3凋亡通路.本研究为深入研究CMTK诱导肿瘤细胞凋亡的机制提供了依据.
A preliminary study of the newly synthesized 3-chloromethylene-5-fluoro-thiochroman-4-one (CMTK) The effect of different concentrations of CMTK on the cell proliferation was investigated by MTT assay. The ability of CMTK to inhibit cell proliferation was determined by MTT assay. The number of viable cells was counted by trypan blue staining and the growth of MCF-7 cells was observed Curvilinear and flow cytometry were used to detect the effect of cell cycle on cell cycle. CMTK induced apoptosis was detected by flow cytometry and terminal dexynucleotidyl transferase (TdT) -mediated dUTP nick end labeling. The activity of caspase-8 and caspase-9 were detected by ELISA. The expression of death receptor 3 (DR3), human tumor necrosis factor receptor 1 ( tumor necrosis factor receptor1 and tumor necrosis factor-α (TNF-α), and human caspase-3 protein.The results showed that CMTK could be significantly inhibited MCF-7 cell proliferation, compared with cisplatin (CDDP), its anti-tumor activity is more clear , The IC50 was 14.24 ± 0.12molL1. The growth curve further demonstrated that CMTK inhibited cell proliferation in a time-and dose-dependent manner. Flow cytometry analysis showed that cells were arrested at G0 / G1 (P <0.05). The results of TUNEL assay and flow cytometry analysis of CMTK inducing apoptosis were basically the same. After treatment with drugs, the activities of caspase-8 and caspase-9 were increased in a concentration-and dose- The amount of DR3 protein and the amount of TNF-cytokines did not change with the ELISA method, and the protein levels of TNFR1 and caspase-3 were significantly increased. The mechanism of CMTK-induced apoptosis of tumor cells may not only be mediated by the death receptor TNFR1 , Activates caspase-8, activates caspase-9 through mitochondrial apoptosis pathway and finally activates caspase-3 apoptosis pathway.This study provides the basis for further study on the mechanism of CMTK-induced apoptosis in tumor cells.