摘要
目的体外研究金属蛋白酶(MMPs)抑制剂对外源Rac1基因表达介导的HT1080纤维肉瘤细胞侵袭胶原屏障的影响。方法分别转染显性负调控突变体Rac1V12N17(HN)、持续活化型Rac1V12(HV)或空载体(HW)于纤维肉瘤HT1080细胞,G418筛选。蛋白印迹分析检测外源性Rac1基因表达,并在含胶原蛋白凝胶的3D基质中培养,用得克萨斯红结合的鬼笔环肽染色显示细胞肌动蛋白骨架构型。在含胶原蛋白凝胶覆盖滤膜的Transwell小室进行细胞侵袭胶原屏障实验,并观察MMPs抑制剂和抑肽酶对上述细胞侵袭实验的影响。结果HV细胞伸出明显的突起,HN细胞形态紧凑,HV细胞侵袭胶原屏障的能力大于HW细胞,而后者又强于HN细胞,这种差别在应用广谱MMPs抑制剂后消失,而抑肽酶则无影响。结论外源活化型Rac1基因在纤维肉瘤HT1080细胞内稳定表达可诱发肌动蛋白纤维聚集,并可增加HT1080纤维肉瘤细胞侵袭胶原屏障的能力,但这种细胞侵袭胶原蛋白能力的增强可被MMPs抑制剂阻断。
Objective To investigate the effect of MMPs inhibitor on HT1080 fibrosarcoma cell invasion across collagen barrier mediated by exogenous Racl gene expression, Methods HT1080 fibrosarcoma cell line that stably expressed transfected dominant negative （Rac1V12N17-HN）, constitutively active Racl（Rac1V12-HV） and vector（HW） were used. Exogenous Racl gene expression was detected by western blot analysis. Structure of actin cytoskeleton was stained with Texas Red-conjugated phalloidin to show the morphological containing collagen protein. Cell invasion assay characters of the cells cultured in 3D medium across collagen barrier was performed on a thin layer of medium gel containing collagen covered membrane of transwell chamber, and MMP in- hibitor and Aprotinin were used to observe their effects on above-mentioned invasive assay. Results HT1080 cells stably expressing Racl mutant exhibited distinct morphological and invasive properties, and the increased invasive ability could be eradicated after using MMPs inhibitor. Conclusion The stable expression of exogenous Racl gene in HT1080 fibrosarcoma cell line could induce aggregation of actin fiber in cells, enhance its invasive properties and these effects could be blocked by MMPs inhibitor, and these results may suggest MMPs activity is required in Racl mediated HT1080 fibrosarcoma cell invasion across collagen barrier.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2008年第2期181-184,F0002,共5页
Suzhou University Journal of Medical Science
基金
教育部高等学校博士学科点专项科研基金资助项目(2004-0161-005)
