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中文摘要：
目的探究海参粘多糖（hGAG）对MDA-MB-231乳腺癌细胞介导的凝血过程的影响及其作用机制。方法采用MDA-MB-231乳腺癌细胞为模型,观察经不同浓度的hGAG处理后的肿瘤细胞对血浆复钙时间的影响;采用S-2222发光底物检测凝血因子Xa（FXa）的生成;以Fluo-4/AM为荧光探针,在荧光酶标仪下检测细胞Ca2＋的变化;流式细胞仪检测细胞表面组织因子（TF）,采用real-time PCR和Westernblot的方法分别对TF的mRNA和蛋白水平进行测定;并观察hGAG对AKT、MAPK、NF-κB等信号通路的影响。结果hGAG 0.01～1μmol.L-1可以呈剂量依赖性的方式延长肿瘤细胞诱导的血浆复钙时间,并降低FXa的生成,对Ca2＋无明显影响,而能够下调TF的mRNA和蛋白表达,抑制p38的磷酸化而对其它信号通路无明显影响。结论 hGAG通过下调TF的表达及p38的磷酸化而抑制MDA-MB-231细胞诱导的凝血过程。
英文摘要：
Aim To investigate the effects of holothurian glycosaminoglycan（hGAG） on the coagulation process mediated by MDA-MB-231 breast cancer cells and its possible mechanisms.Methods MDA-MB-231 breast cancer cells were used as models,the effect of tumor cells treated with different concentrations of hGAG on the plasma recalcification time was observed.Coagulation factor Xa（FXa） generation was examined with chromogenic substrate S-2222.The changes of Ca 2＋ labeled with fluorescent probe Fluo-4/AM were measured by fluorescence plate reader.Flow cytometry was used to detect tissue factor（TF） on the surface of tumor cells.Real-time PCR and western blot were used to measure the mRNA and protein expression of TF,respectively.AKT,MAPK,NF-κB and other signaling pathways were also examined after treated with hGAG.Results hGAG at concentrations of 0.01μmol·L-1～1μmol·L-1 extended the plasma recalcification times mediated by tumor cells in a dose dependent manner and decreased the FXa generation but had no obvious effect on Ca 2＋ changes.hGAG decreased the mRNA and protein expression of TF,and reduced the phosphorylation of p38 rather than other signaling pathways.Conclusions hGAG inhibits the coagulation process mediated by MDA-MB-231 breast cancer cells through downregulating the expression of TF and phosphorylation of p38.
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