| Pancreatic carcinoma remains one of the most malignant tumor,which is easy to metastasize to nerves,blood vessel,lymph vessel.Current screen modalities are unable to identify it for inaccessible location,proximity to other vital organs,inherently aggressive pattern of growth,and atypical symptom,which make most patients have local or metastasis spread at the time of presentation that precludes surgical resection. Less than 30%of cases are constitute candidates for resection.Furthermore,pancreatic carcinoma is highly resistant to current available chemotherapy and radiation protocols. Therefore,new modalities in the treatment of this disease to improve its prognosis are required urgently.For the development of DNA recombinant technology,and the consent of accumulated abnormal gene resulting tumorigenesis,biotherapy is becoming one of the most means in oncotherapy.In theory,it can reconvert the malignant attribute of tumor cells by gene therapy.It has been improved in vitro that tumor growth, angiogenesis,and metastasis was inhibited,tumor cell apoptosis was increased,life span was significant prolongation of athymic mice bear to tumor by transfecting tissue inhibitor of metalloproteinase to Pane-1 cell.Human tissue factor pathway inhibitor 2(TFPI-2) gene,localizated to chromosome 7q22,is a broad-spectrum serine protease inhibitor that inhibits plasmin,trypsin,metrix metalloproteinase 1 and 3 in vitro,which is important to maintain the integrity of extracellular matrix and regulate the migration and invasion of tumor.TFPI-2 transcripts are abundant in various adult human tissue,such as liver,pancreas,kidney,heart, skeletal muscle and prostate gland.It has been discovered that TFPI-2 is related to tumorigenesis and development of multitude tumors such as neuroglioma,chorionic carcinoma,melanoma,and lung cancer. In this research,we construct the eukaryotic expression vector pEGFP-C1-TFPI-2 of TFPI-2 gene,and then transfect it to pancreatic carcinoma cell line Panc-1 cell. Afterwards,we study the influence of TFPI-2 expression on growth curve,invasion and migration in vivo/in vitro,and apoptosis of Pane-1 cell and its potential mechanism.All these studies will provide new experimental basis for prognosis and recidivation of pancreatic carcinoma.Objective:We observe the expression of TFPI-2 in pancreatic carcinoma cell line Panc-1 cell, construct the eukaryotic expression vector pEGFP-C1-TFPI-2 of TFPI-2 gene and then transfect it to pancreatic carcinoma cell line Pane-1 cell.Afterwards,we detect and compare the growth curve,invasion and migration and apoptosis in TFPI-2 expression and non-expression group by Boyden chamber,model of subcutaneous explantation tumor in athymic mice and flow cytometry,study the-influence of TFPI-2 expression on growth,invasion and migration in vivo/in vitro,and apoptosis of Panc-1 cell.Methods:Reverse transcriptase polymerase chain reaction(RT-PCR):Amplification of TFPI-2 gene and detection of TFPI-2 mRNA after transfection.Western blot:Detection of TFPI-2 protein before and after transfection.Cell culture and transfection:Lipofectamine2000 was used as a media.Invasion experiment of Boyden chamber:To observe the influence of TFPI-2 on invasion and migration in vitro.Modeling of subcutaneous explantation tumor in athymic mice:To observe the influence of TFPI-2 on invasion and migration in vivo.Detection of cell apoptosis:DNA Ladder electrophoresis and flow cytometry was used. Results:The eukaryotic expression vector pEGFP-C1-TFPI-2 of human TFPI-2 gene was constructed successfully.There was non-expression of TFPI-2 in Panc-1 cell,after transfection,it can express TFPI-2 mRNA and protein stably.TFPI-2 can inhibit invasion of pancreatic carcinoma cell in vitro.TFPI-2 can inhibit growth and invasion of pancreatic carcinoma.TFPI-2 can induce apoptosis of pancreatic carcinoma cell.Conclusion:TFPI-2 expression may obviously inhibit the invasion ability of pancreatic cancer cells in vitro and in vivo by maintaining the integrity of ECM or inducing cell apoptosis, which provides an experimental basis for curing human pancreatic cancer with gene-therapy.
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