组织因子/活化凝血Ⅶ因子复合物对大肠癌LoVo细胞系表达基质金属蛋白酶-7的调控作用  

Regulation of promatrilysin expression by tissue factor/actived coagulation factor Ⅶ complex in LoVo colon cancer cell line

作　　者：汤坚强[1] 万远廉[1] 刘玉村[1] 戎龙[1] 汪欣[1] 吴涛[1] 潘义生[1] 朱静[1] 

机构地区：[1]北京大学第一医院普通外科,北京100034

出　　处：《北京大学学报（医学版）》2007年第5期453-457,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30471683);教育部科学技术研究重点项目(02003);教育部教育振兴行动计划特殊专项("九八五"工程)资助~~

摘　　要：目的:观察活化凝血Ⅶ因子(actived factorⅦ,FⅦa)对大肠癌LoVo细胞系表达基质金属蛋白酶-7(ProMMP-7)的影响,探讨其可能的组织因子信号转导通路。方法:(1)用Western blot检测100nmol/L FⅦa刺激LoVo细胞系不同时间点及不同浓度FⅦa刺激LoVo细胞系12h后细胞ProMMP-7蛋白水平;用Western blot检测5mg/L组织因子(tissue factor,TF)抗体预孵育LoVo细胞系0.5h后再给予100nmol/L FⅦa刺激12h后细胞ProM-MP-7蛋白水平。(2)观察用100nmol/L FⅦa刺激时丝裂原激活蛋白激酶(MAPKs)各亚通路信号蛋白(包括细胞外信号调节激酶ERK1/2,丝裂原激活蛋白激酶P38及c-Jun-N末端蛋白激酶JNK)磷酸化水平变化。(3)在FⅦa刺激前0.5h分别加入ERK1/2,P38和JNK信号通路特异阻断剂PD98059,SB203580和SP600125,培养12h后检测细胞ProMMP-7蛋白的变化。结果:(1)FⅦa可刺激LoVo系细胞表达ProMMP-7并呈时间依赖性和剂量依赖性,在刺激的12h左右ProMMP-7达峰值,是正常对照组的5.5±0.6倍(P=0.006);用TF抗体预处理的LoVo细胞系,其FⅦa上调ProMMP-7表达的作用被完全阻断。(2)用100nmol/L FⅦa刺激LoVo细胞5min时,MAPKs磷酸化活性蛋白p-ERK1/2和p-P38表达水平开始增加,至10min时达峰值(分别为对照组的2.2±0.3倍和3.9±0.5倍,P值分别为0.02和0.01),存在时间效应关系,而p-JNK活性无改变(为对照组的1.1±0.1倍)。(3)ERK1/2通路阻断剂PD98059及P38通路阻断剂SB203580可部分减少FⅦa上调LoVo细胞系表达ProMMP-7的效应,分别降低了32%±5%(P=0.01)和61%±10%(P=0.009),JNK通路特异性阻断剂SP600125对ProMMP-7的表达无明显影响。结论:FⅦa通过与LoVo细胞表面的TF结合诱导ProMMP-7的表达,并呈时间依赖性和剂量依赖性;ERK1/2和P38MAPKs亚通路参与LoVo细胞TF信号转导通路并与TF/FⅦa调控ProMMP-7表达有关。Objective：To assess the expression of Promatrilysin in LoVo colon cancer cell by FⅦa stimulation,and to investigate the effect of MAPKs signal transduction pathway on up-regulation of Proatrilysin.Methods：（1）The expression of ProMMP-7 was detected by Western blot at different time points（0,2,4,6,9,12 and 24 h）and with different doses of（0,0.1,1,5,10,25 and 100 nmol/L）FⅦa stimulation.The change of ProMMP-7 expression was observed with 5 mg/L tissue factort（TF）antibody prior to 100 nmol/L FⅦa.（2）The activation of MAPKs（ERK,p38,JNK）signaling pathways were assessed at different time points after being stimulated with 100 nmol/L FⅦa and the changes of ProMMP-7 expression were detected after the special signal pathway inhibitors（PD98059,SB203580,SP600125）were applied,respectively.Results：（1）The expression of ProMMP-7 in LoVo cells was up-regulated by FⅦa in a time-effcet dependent and dose-effcet dependent manner,and markedly reached the peak level at h12,5.5 folds that of the control group（P=0.006）.The up- regulation of ProMMP-7 was completely inhibited by blockade with TF antibody.（2）A time-dependent phosphorylation of ERK1/2 and P38 in LoVo cells was induced with FⅦa incubation,reached the peak at min10,2.2 folds and 3.9 folds those of the control groups respectively,but not JNK.（3）The upregulation effect of ProMMP-7 was partially blocked after incubation of ERK1/2 inhibitors PD98059 and P38 inhibitors SB203580 prior to FⅦa,The expression of ProMMP-7 decresaed by 32%±5% and 61%±10% respectively（P〈0.05）.whereas JNK inhibitors SP600125 did not have the effect.Conclusion：FⅦa induces tissue factor-dependent up-regulation of ProMMP-7 in LoVo cells.ERK1/2 and p38 signal pathways are not only involved in TF/FⅦa mediated signaling,but also related to the upregulation of MMP-7 in LoVo cells.

关 键 词：因子Ⅶa 结直肠肿瘤 基质金属蛋白酶类 丝裂原激活蛋白激酶类 

分 类 号：R735.3[医药卫生—肿瘤]