氨氯地平对小鼠黑色素瘤B16 MMP-9和TIMP-1表达的调节作用

Regulatory Effect of Amlodipine on Expressions of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Murine Melanoma B16

目的观察氨氯地平对小鼠黑色素瘤B16基质金属蛋白酶-9(Matrix metalloproteinase-9,MMP-9)和MMPs组织抑制因子(Tissue inhibitor of metalloproteinase-1,TIMP-1)表达的调节作用,并探讨其抑制肿瘤转移的作用机制。方法采用MTT法检测氨氯地平对B16细胞增殖的影响;建立C57BL/6小鼠黑色素瘤B16的自发性肺转移模型,随机分为阴性对照组(生理盐水)、阳性对照组(环磷酰胺20 mg/kg)、氨氯地平高[10 mg/(kg.d)]、中[3 mg/(kg.d)]、低[1 mg/(kg.d)]剂量组,采用明胶酶谱法检测肿瘤组织中MMP-9的活性;RT-PCR法检测肿瘤组织中MMP-9和TIMP-1基因mRNA的表达;免疫组化SP法检测肿瘤组织中MMP-9和TIMP-1蛋白的表达。结果氨氯地平呈时间和剂量依赖性抑制小鼠黑色素瘤B16细胞的生长,作用48 h的IC50为11.56μmol/L;氨氯地平可下调肿瘤组织中MMP-9的活性及MMP-9基因mRNA和蛋白的表达,上调TIMP-1基因mR-NA和蛋白的表达。结论氨氯地平通过调节MMP-9和TIMP-1基因的表达发挥其抑制肿瘤转移的作用。

Objective To observe the regulatory effect of amlodipine on expressions of matrix metalloproteinase-9（MMP-9） and tissue inhibitor of metalloproteinase-1（TIMP-1） in murine melanoma B16 and investigate the mechanism of amlodipine in inhibiting the metastasis of tumors.Methods The effect of amlodipine on proliferation of B16 cells was determined by MTT method.C57BL / 6 mouse model of spontaneous pulmonary metastasis of melanoma B16 was established.The model mice were randomly divided into negative control（physiological saline）,positive control[cytoxan（CTX） 20 mg / kg] and amlodipine at high [10 mg /（kg·d）],moderate [3 mg /（kg·d）] and low [1 mg /（kg·d）] groups.The MMP-9 activity in tumor tissue was determined by gelatin zymography,while the expressions of MMP-9 and TIMP-1 mRNAs by RT-PCR,and expressions of MMP-9 and TIMP-1 proteins by immunohistochemical assay with SP staining.Results Amlodipine showed time-and dose-dependent inhibitory effect on the growth of B16 cells,of which the IC50 48 h after treatment was 11.56 μmol / L.Results Amlodipine down-regulated the MMP-9 activity as well as expressions of MMP-9 mRNA and protein in tumor tissue,while up-regulated the expressions of TIMP-1 mRNA and protein.Conclusion Amlodipine inhibited the metastasis of tumor by regulating the expressions of MMP-9 and TIMP-1 genes.