| The incidence and mortality rate of cancer continues increasing these years.Tumor dissemination and metastasis is a main cause of cancer-related death.With the rapid development of molecular biology,the molecular mechanisms of tumor metastasis became one of the hottest field in experimental oncology.Although produced major development,the information of tumor metastsis is fragmented and is not yet exploited in clinical practice.Tumor progression and metastasis can be considered both as a cascade of events as well as a continuous selection process.This process include extracellular matrix(ECM) recognition,matrix degredation,migration,haematogenous dissemination and organ selection metastasis.It is accepted that the malignant cell-ECM interactions must be considered as the key feature of malignancy .The malignant cell must recognize and degradation ECM before invasion and metastasis,so It is important to reasearch the matrix degrading enzymes for understanding and controlling the metastasis.The serine protease family is one member of these ECM-degrading enzymes families, includes uPA,elastse,plasmin and cathepsin G.A new sub-family of the serine protease family was recently identified,and it was named as type II transmembrane serine protease(TTSPs).The members of TTSPs were extensively expressed on many kinds of cell membrane,and took part in diverse cellular activities,such as ECM degradation, cell surface proteolysis,interaction with cell cytoskeleton,cell signal transduction and invasion.The SNC19/ST14/Matriptase/MT-SPl was recently found as a new member of TTSPs.SNC19 gene was isolated and identified from a substractive cDNA library of colorectal cancer by Zhejiang University CancerInstitute,and was uploaded in Genbank in 1995,and denoinated as ST14(suppression of tumorigenicity 14) by Gene Nomenclature Committee in 1998.From then on,many homolegue production of SNC19 was found in many species , many types of tissue cells,cell lines,and milk,such as Matriptase,MT-SP 1 ,Epithin Xmt-spl,Matriptase-2 and M-matriptase-2.SNC19/ST14 protein was denominated as ST14/MT-SPl/Matriptase /TADG-15,EC 3.4.21.In vitro and/or in vivo,it could efficiently activated the proteinase activated receptor-2(PAR-2),single-chain uPA(sc-uPA), and the hepatocyte growth(scattering) factor(HGF/SF).The uPA(urokinase-type plasminogen activator) and HGF/SF were closely correlated with ECM degradation , growth,movement and metastasis of cancer cells.The SNC19/ST14 protein had a serine-proteinase domain and trypsin-like activity,so it could also directly degradated ECM.In situ hybridization showed that the positive rate of SNC19 mRNA expressed in the mucosa adjacent to colorectal carcinaoma of the lymph node metastasis group(64.7%) was higher than that in non-metastasis group(15.4%)(P<0.05).And others study showed that ST14/MT-SPl/Matriptase/TADG-15 had highly expressed in prostate cancer,breast cancer, and cervical cancer tissue,and could interact with cytoskeleton ptotein(F-actin).These results suggested that SNC19/ST14/MT-SPl/Matriptase may be a metastasis-related gene,but we didn't know the detail information that how the ST14/MT-SPl/Matriptase took part in the tumor metastasis,and whether it promoted or inhibited metastasis.To further know the function of SNC19/ST14/MT-SPl/Matriptase during the tumor invasion and metastasis process,this dissertation was development as follows:Part l:To establish a colorectal cancer cell line which stably expressed the full length Open Reading Frame(ORF) of SNC19/ST14 gene-RKO-SNC19/ST14In this part,we constructed and amplified the eukaryotic expression recombination vetcor pSecTag2a-SNC19/ST14.The recombination and null vector were transfected into the the colorectal cancer cells -RKO cells(not expressed SNC19/ST14) by liposome,and the transfected RKO cells were screened by Real-Time PCR,Western Blot andImmunohistochemical technique. We harvested two cell clones one was the transfected population cell clone-RKO-SNC 19-1, and the other was a monoclonal cell clone-RKO-SNC 19-2.Both...
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