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
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摘要
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CO_2培养箱中,于37℃、5%CO_2、20%O_2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 CO_2 incubator,cultured at 37 ℃,5% CO_2 and 20% O_2 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.
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 CO_2 incubator,cultured at 37 ℃,5% CO_2 and 20% O_2 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.
引文
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[2]HIRONO J,SANAKI H,KITADA K,et al.Expression of tissue inhibitor of metalloproteinases and matrix metalloproteinases in the ischemic brain of photothrombosis model mice[J].Neuroreport,2017,29(3):1-3.
[3]DEKKER S,BANK J,SOONAWALA D,et al.Fp749 urinary tissue inhibitor of metalloproteinases-2(timp-2)and insulinlike growth factor-binding protein 7(igfbp7)in acute and chronic kidney dysfunction[J].Nephrology Dialysis Transplantation,2018,33(suppl_1):i298.
[4]JIN R,XU S,LIN X,et al.MiR-136 controls neurocytes apoptosis by regulating tissue inhibitor of metalloproteinases-3 in spinal cord ischemic injury[J].Biomedicine&Pharmacotherapy,2017,94(3):47-54.
[5]TIMMERMANS AJ,LANGE CA,DE BOIS JA,et al.Tumor volume as a prognostic factor for local control and overall survival in advanced larynx cancer[J].Laryngoscope,2016,126(2):E60-E67.
[6]何亚,刘晓颖,邓庆华,等.丙泊酚与水合氯醛对亚低温大鼠海马神经元凋亡的影响比较[J].中国药业,2017,26(6):12-15.
[7]DUELLMAN T,DOLL A,CHEN X,et al.dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases[J].Metalloproteinases Med,2017,4(3):63-73.
[8]XUE W,ZHANG Z,ZENG S,et al.Expression and Clinical Significance of Tissue Inhibitor of Metalloproteinases-1(TIMP-1)and a Disintegrin and Metalloproteinase with Thrombospondin Type 1Motif 1(ADAMTS1)in Post-Kidney-Transplant Bladder Tumors[J].Annals of Transplantation,2017,22(3):622-623.
[9]KIM HS,VARGAS A,EOM YS,et al.Tissue inhibitor of metalloproteinases 1 enhances rod survival in the rd1 mouse retina[J].PLoS One,2018,13(5):322-323.
[10]LAO G,REN M,WANG X,et al.Human tissue inhibitor of metalloproteinases-1 improved wound healing in diabetes through its anti-apoptotic effect[J].Experimental Dermatology,2017,8(3):45-47.
[11]KORMI I,NIEMINEN MT,HAVULINNA AS,et al.Matrix metalloproteinase-8 and tissue inhibitor of matrix metalloproteinase-1 predict incident cardiovascular disease events and all-cause mortality in a population-based cohort[J].European Journal of Preventive Cardiology,2017,24(11):585-586.
[12]黄好武,黄欢文,姜一平,等.芹菜素对肺癌细胞的抗增殖作用和抗肿瘤药物的增敏作用[J].中国药业,2017,26(12):20-22.
[13]MA XH.Department of Clinical Laboratory,Heze Multiple Hospital Changes of Intramyocardial Matrx Metalloproteinases and Tissue Inhibitor of Metalloproteinase Protein Expression[J].China&Foreign Medical Treatment,2017,9(2):23-25.
[14]LI X,FU X,GAO Y,et al.Expression of tissue inhibitor of metalloproteinases-1 and B-cell lymphoma-2 in the synovial membrane in patients with knee osteoarthritis[J].Experimental&Therapeutic Medicine,2018,15(1):885-889.
[15]LAO G,REN M,WANG X,et al.Human Tissue Inhibitor of Metalloproteinases Improved Wound Healing in Diabetes through Its Anti-apoptotic Effect[J].Experimental Dermatology,2017,26(Suppl_2):89-91.