去甲斑蝥素通过下调VE-Cadherin和MMP-2/9活性抑制结肠癌血管拟态形成的体外研究  

Inhibition of Norcantharidin on Vascular Mimicry Formation of Colon Cancer by Down-regulating VE-cadherin Expression and MMP-2/9 Activity in Vitro

作　　者：侯风刚[1,2] 李文[2] 刘见荣[2] 石齐[2] 吕祥[2] 任建琳[2] 花宝金[1] 

机构地区：[1]中国中医科学院广安门医院肿瘤科,北京100053 [2]上海中医药大学附属市中医医院肿瘤科,上海200071

出　　处：《上海中医药大学学报》2014年第4期64-69,共6页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：上海市教委科研创新基金项目(12YZ057)

摘　　要：目的:探讨去甲斑蝥素(NCTD)对结肠癌中血管生成拟态(VM)形成的作用及其可能的机制。方法:采用MTT法检测结肠癌细胞HCT116对NCTD的敏感性,筛选无毒剂量;在三维培养基础上观察NCTD作用后VM的形成情况;采用实时荧光定量PCR(qRT-PCR)方法检测在NCTD干预后细胞内血管内皮钙黏蛋白(VE-Cadherin,VE-Cd)的mRNA表达水平;Western Blot检测NCTD作用后VE-Cd蛋白表达水平;采用明胶酶谱法检测NCTD对三维培养的HCT116细胞MMP-2和MMP-9活性的影响。结果:三维培养下无毒剂量的NCTD能有效地抑制VM形成;VE-Cd mRNA表达在药物作用后出现明显下调;而HCT116细胞的胞浆和胞膜中VE-Cd蛋白均呈下调趋势,胞膜中VE-Cd蛋白下调更明显;三维培养的HCT116细胞MMP-2和MMP-9活性随着NCTD药物浓度的增加而逐渐降低。结论:体外实验研究中,NCTD可以有效抑制结肠癌VM的形成,其机制可能与下调VE-Cd表达和降低MMP-2、MMP-9的活性有关。Objective： To discuss the action and its mechanism of norcantharidin（ NCTD） on the formation of vasculogenic mimicry（ VM） in colon cancer in vitro. Methods： MTT method is adopted to detect the sensitivity of the colon cancer cells HCT116 to NCTD,and screen the nontoxic dose; the formation of VM after treatment of NCTD was observed on the basis of 3D culture; qRT-PCR was used to detect the expression of intracellular VE-Cd mRNA after intervention of NCTD;Western Blotting was used to detect the expression of VE-Cd protein after treatment of NCTD; Gelatin Zymography was used to detect the influence of NCTD on MMP-2 and MMP-9 activities in HCT116 cells with 3D culture. Results： NCTD at non-toxic dose can effectively restrain the formation of VM in 3D culture; expression of VE-Cd mRNA is significantly decreased after drug treatment; while VE-Cd proteins either in cytoplasm or cell membrane of HCT116 cells were both declined,especially in cell membrane; MMP-2 and MMP-9 activities of HCT116 cells in 3D culture were gradually decreased with the increase of NCTD concentration. Conclusion： This study shows that NCTD can effectively inhibit the formation of colorectal VM,and the main mechanism is related with the down-regulation of VE-Cd expression and the reduction of MMP-2 and MMP-9 activities in vitro.

关 键 词：去甲斑蝥素 结肠癌 HCT116细胞株 血管生成拟态 血管内皮钙黏蛋白 基质金属蛋白酶 

分 类 号：R285.5[医药卫生—中药学]