Differential Effect Of Endogenous And Exogenous TIMP-1 Gene Expression For Invasive And Metastatic Characteristics Of Colorectal Cancer Cell

The crucial step of tumor invasion and metastasis is the degradation of the components of basal membrane and extracellular matrix. The MMP family has the important function for that. MMP-9 of the member in MMP family plays an important role in the growth of tumor, invasion, metastasis and angiogenesis. It is also the important factor to enhance the growth of tumor. TIMP-1 is the specific inhibitor of MMP-9. The decreasing expression of TIMP-1 is related with the increasing invasion of tumor. The overexpression of TIMP-1 is related with the decreasing growth and metastasis of tumor. However, there was another research to indicate that TIMPs also could promote the growth of tumor recently.Study hypothesis: There may be different effects for different resources of TIMP-lin biological conducts of tumor cells. In other words, it may be one of the factors for endogenous TIMP-1 expression from tumor cells themselves or exogenous TIMP-1 expression from the third kind of cells(basal cells or another host cells) to determine the differences of tumor biological conducts.Study purpose: To study the clinically pathological significances for MMP-9 and TIMP-1 on the expression of colorectal cancer and then identify the study hypotheses from the correlation between clinical-medicine and pathology. The models of colorectal cancer cells of the endogenous and exogenous TIMP-1 expression were established separately by use of colorectal cancer cell line TCH-8908 naturally expressing high MMP-9 but not expressing TIMP-1. To observe TIMP-1 expression from different resources resulting in the effects of invasive and metastatic characteristics in colorectal cancer. To make use of the differences of cell biological characteristics in tumor model and identify the study hypotheses.The study was divided by five parts:Part I : The study of clinically pathological significance of MMP-9and TIMP-1 expression in colorectal cancer. Method: Using the immunohistochemical method to detect the expression of MMP-9 and TIMP-1 of 70 post-operative samples in colorectal cancer and reviewing the clinically pathological characteristics at the same time. Analyzing the correlation between the expression of MMP-9 as well as TIMP-1 and clinical pathology. Result: MMP-9 expression could be as an effective indicator to determine invasion, metastasis and prognosis in colorectal cancer.Part II: Clone and identification of TIMP-1 gene. Method: UsingRT-PCR method to amplify TIMP-1 gene cDNA from Lovo cell RNA of colorectal cancer cell lines expressing TIMP-1. TIMP-1 gene amplified above would be inserted into pGEM-T vector by subcloning skill and performed the relative analyses of enzyme cutting and sequencing identification. Result: TIMP-1 gene was amplified successfully. This work could make the basis in following study.Part III: Construction and identification of tumor model expressingendogenous TIMP-1 gene in TCH-8908 cell of colorectal cancer. Method: Constructing eukaryotic expression vector with green fluorescent protein by use of cloned TIMP-1 gene. pcDNA3.1 was transformed into TCH-8908 cell of colorectal cancer cell line by electroporation. We would get stable TCH-8908( TIMP-1 ) subclone expressing TIMP-1 by selecting. Furthermore, using RT-PCR and immunohistochemical method for relative identification. Result: The colorectal cancer cell model stably expressing TIMP-1, endogenous TCH-8908( TIMP-1 ), was constructed successfully. This work could make the basis for further study about the effect between endogenous TIMP-1 expression and TCH-8908 cell biological conduct.Part IV: Construction and identification of exogenous TIMP-1 geneexpressing vector. Method: Using cloned TIMP-1 gene to construct adenoviral vector( pAdTIMP-1 ) for gene transfection. We could get adenovirus AdTIMP-1 by the pack of AD-293 cell and perform the relative identification. Result: The higher titer adenovirus was constructed successfully and could be used in the TIMP-1 gene transfection of eukaryotic cell. We would construct colorectal cancer mod...