Navneet Kaur, B V Sunil Kumar, Kanika Mahajan, Satparkash Singh
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引用次数: 3
摘要
据报道,组织金属蛋白酶抑制剂(TIMP)家族的所有四个成员都在体外乳腺癌细胞中过度表达。TIMP-4 表达失调预示着癌症的不良预后。本研究通过实时定量逆转录酶聚合酶链反应(qRT-PCR)测定了 TIMP-4 的表达水平与狗乳腺肿瘤发生的关系。乳腺组织样本采集自健康犬乳腺和肿瘤受试者。结果发现,在复杂犬乳腺癌中,TIMP-4 表达上调(5.856 倍)。此外,还在大肠杆菌中异源表达了 TIMP-4 成熟肽。重组蛋白通过 Ni- NTA 亲和层析法纯化,并进一步通过 Western 印迹法确认。研究发现rTIMP-4具有功能活性,能抑制基质金属蛋白酶11(MMP-11)的活性。用rTIMP-4免疫小鼠可提高抗原特异性血清抗体滴度,该血清可用于检测和定量乳腺肿瘤犬血清中的该蛋白。TIMP-4 可作为犬乳腺肿瘤的标记物。据我们所知,这是首次报道复杂犬乳腺癌异源表达 TIMP-4。
Expression and characterization of tissue inhibitor of metalloproteinase 4 from complex canine mammary carcinomas.
All four members of the tissue inhibitors of metalloproteinase (TIMP) family have been reported to be over-expressed in breast cancer cells in vitro. Dysregulation of TIMP-4 expression predicts poor prognosis in cancers. The present study evaluated the association of the expression levels of TIMP-4 with mammary tumor development in dogs, measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mammary tissue samples were collected from healthy canine mammary gland and from tumor subjects. TIMP-4 expression was found to be upregulated (5.856 times) in complex canine mammary carcinomas. Also, TIMP-4 mature peptide was expressed heterologously in E. coli. The recombinant protein was purified by Ni- NTA affinity chromatography and further confirmed by western blotting. The rTIMP-4 was found to be functionally active and could inhibit matrix metalloproteinase 11(MMP-11) activity. Immunization of mice with rTIMP-4 resulted in increased antigen specific serum antibody titer, and this serum could be suitably used to detect and quantify the protein in sera of dogs with mammary tumors. TIMP-4 could act as a marker of canine mammary tumors. To the best of our knowledge, this is the first report of heterologous expression of TIMP-4 from complex canine mammary carcinomas.