基质金属蛋白酶组织抑制剂-3对喉癌细胞顺铂化疗敏感性提高的机制研究

Mechanism of Tissue Inhibitor of Metalloproteinases-3 in Improving the Chemotherapy Sensitivity of Cisplatin in Laryngeal Carcinoma Cells

目的探讨基质金属蛋白酶组织抑制剂-3(TIMP-3)提高喉癌Hep-2细胞顺铂化疗敏感性的作用机制。方法试验分为空白对照组(A组)、顺铂组(B组)和顺铂+TIMP-3组(C组),A组和B组均为10 m L Hep-2细胞液溶于10%胎牛血清中,分别加入0和5μmol/L顺铂5 m L,C组为10 m L转染TIMP-3的Hep-2细胞液加入顺铂(5μmol/L) 5 m L,置CO2培养箱中,于37℃、5%CO2、20%O2条件下培养72 h。以噻唑蓝(MTT)比色法检测Hep-2细胞活力及细胞单克隆形成数目,以流式细胞仪检测Hep-2细胞凋亡情况,以荧光共聚焦显微镜检测线粒体膜电位(MMP)水平,以实时荧光定量聚合酶链反应(PCR)法检测TIMP-3 mRNA表达水平,以酶联免疫吸附法检测细胞色素酶C、胱天蛋白酶-3(Caspase-3)、胱天蛋白酶-9(Caspase-9)蛋白表达水平。结果与A组比较,B组和C组细胞活力、存活率水平、细胞单克隆形成数目、MMP均显著降低,细胞凋亡率、TIMP-3 mRNA和细胞色素酶C、Caspase-3、Caspase-9蛋白表达水平均显著升高(P <0. 01);C组上述指标均优于B组(P <0. 01);TIMP-3与细胞色素酶C、Caspase-3、Caspase-9呈显著正相关(P <0. 01)。结论 TIMP-3能提高喉癌Hep-2细胞顺铂化疗敏感性,其机制与上调TIMP-3,促进MMP去极化和线粒体细胞色素C释放,进而诱导Caspase-3和Caspase-9过表达引起细胞凋亡有关。

Objective To investigate the mechanism of tissue inhibitor of metalloproteinases-3( TIMP-3) in improving chemotherapy sensitivity of cisplatin in laryngeal carcinoma Hep-2 cells. Methods The experiment was divided into the blank control group( group A),cisplatin group( group B) and cisplatin + TIMP-3 group( group C). 10 m L Hep-2 cell solution was dissolved in 10% fetal bovine serum in group A and group B,5 m L 0 and 5 μmol/L cisplatin were added in group A and group B respectively. 10 m L Hep-2 cell solution transfected TIMP-3 was added to 5 m L cisplatin( 5 μmol/L) in group C. All of them were placed in a CO2 incubator,cultured at 37 ℃,5% CO2 and 20% O2 for 72 h. MTT assay was used to detect the activity of Hep-2 cells and the number of monoclonal formation. Flow cytometry was used to detect the apoptosis of Hep-2 cells. Fluorescence confocal microscopy was used to detect the level of mitochondrial membrane potential( MMP) and real-time fluorescence quantitative polymerase chain reaction( PCR) was used to detect the expression of TIMP-3 mRNA. The enzyme-linked immunosorbent assay was used to detect the expression levels of cytochrome C,Caspase-3 and Caspase-9 protein. Results Compared with group A,the cell viability,survival rate,number of monoclonal formation and MMP in group B and group C were significantly decreased,while the apoptosis rate,TIMP-3 mRNA and the expression levels of cytochrome C,Caspase-3 and Caspase-9 protein in group B and group C were significantly increased( P < 0. 01),the above indexes in group C were better than those in group B( P < 0. 01). TIMP-3 was positively correlated with cytochrome C,Caspase-3 and Caspase-9( P < 0. 01). Conclusion TIMP-3 can promote the chemotherapy sensitivity of cisplatin in laryngeal carcinoma Hep-2 cells. Its mechanism is related to the up-regulation of TIMP-3,promotion of MMP depolarization and release of mitochondrial cytochrome,and then inducing overexpression of Caspase-3 and Caspase-9 to induce apoptosis.