CAJ | 학술논문

目的研究钠氢交换蛋白1(NH1)特异性抑制剂Cariporide对人胆囊癌细胞株GBC-SD体外转移能力的影响,初步探讨其作用机制.方法采用双羧乙基碳氧荧光素四乙酰氧甲酯(BCECF-AM)荧光染料和酚红检测细胞内外氢离子浓度(pH)值.采用细胞-基质黏附实验、细胞划痕实验和细胞迁移侵袭(Transwell)实验分别检测Cariporide对GBC-SD细胞的黏附、迁移和侵袭能力的影响;实时PCR、蛋白印迹法分别检测Cariporide对GBC-SD细胞基质金属蛋白酶(MMPs) mRNA及蛋白表达的影响.结果 Cariporide处理后GBC-SD细胞的细胞内pH和NHE1活性明显下降,胞外pH明显上升.细胞-基质黏附实验结果显示,Cariporide处理后的GBC-SD细胞体外黏附率降低至对照组的80％和61％,差异有统计学意义(P＜0.05).细胞划痕实验和Transwell侵袭实验结果显示细胞损伤愈合的速度和穿过滤膜的细胞数量均少于对照组.实时PCR及蛋白印迹结果表明Cariporide可显著降低MMP-2和MMP-9 mRNA和蛋白水平的表达量(P均＜0.05).结论 Cariporide可有效抑制人胆囊癌细胞株GBC-SD的体外转移能力,其作用机制可能与细胞内外pH值改变降低MMPs的表达有关.
목적 연구납경교환단백1(NH1)특이성억제제Cariporide대인담낭암세포주GBC-SD체외전이능력적영향,초보탐토기작용궤제.방법 채용쌍최을기탄양형광소사을선양갑지(BCECF-AM)형광염료화분홍검측세포내외경리자농도(pH)치.채용세포-기질점부실험、세포화흔실험화세포천이침습(Transwell)실험분별검측Cariporide대GBC-SD세포적점부、천이화침습능력적영향;실시PCR、단백인적법분별검측Cariporide대GBC-SD세포기질금속단백매(MMPs) mRNA급단백표체적영향.결과 Cariporide처리후GBC-SD세포적세포내pH화NHE1활성명현하강,포외pH명현상승.세포-기질점부실험결과현시,Cariporide처리후적GBC-SD세포체외점부솔강저지대조조적80％화61％,차이유통계학의의(P＜0.05).세포화흔실험화Transwell침습실험결과현시세포손상유합적속도화천과려막적세포수량균소우대조조.실시PCR급단백인적결과표명Cariporide가현저강저MMP-2화MMP-9 mRNA화단백수평적표체량(P균＜0.05).결론 Cariporide가유효억제인담낭암세포주GBC-SD적체외전이능력,기작용궤제가능여세포내외pH치개변강저MMPs적표체유관.
Objective To study the effects of sodium hydrogen exchange protein 1 (Na+/H+ exchange 1 ; NHE1)-specific inhibitor Cariporide on the metastasis of GBC-SD cells in vitro and to preliminarily explore the potential anti-metastatic mechanism of Cariporide.Methods The regulatory effect of Cariporide on intracellular pH and extracellular pH of GBC-SD cells were analyzed by BCECF and phenol red.The effect of Cariporide on the adhesion,migration,and invasion of GBC-SD cells were determined using cell-matrix adhesion assay,wound healing assay,and cell invasion assay.Real-time PCR (RT-PCR) and Western blot assay were also used to detect the effects of Cariporide on the levels of MMP-2 and MMP-9 mRNA and on the protein expression in the GBC-SD cells,respectively.Results Cariporide can significantly decrease the intracellular pH and the NHE1 activity,but increase the extracellular pH in a dose-dependent manner.The cell-matrix adhesion assay showed that the adhesive ratio of the GBC-SD cells treated with Cariporide was reduced to 80％ and 61％ compared with the controls.The wound healing and cell invasion assays showed that after the Cariporide treatment,the healing speed of GBC-SD cells injury and the amount of the cells passing through the filter membrane were less in the treatment group than that in the control group.RT-PCR and Western blot results showed that Cariporide produced a distinguished inhibition of MMP-2 and MMP-9 expression in GBC-SD cells,both on the mRNA and on the protein levels.Conclusions Cariporide can inhibit the in vitro metastasis of human GBC-SD cells effectively.The mechanism may be related to the downregulated expression of MMPs.