The Effect Of Antisense VEGF Vector On Cell Proliferation And Angiogenesis In Pancreatic Cancer

In western world, pancreatic cancer now ranks the fifth leading causes of cancerdeath, is responsible for over 20% of deaths due to gastrointestinal malignancies.Although the resection rate is improved due to the advances in surgery techniques, theprognosis is still poor. This is attributable in large part to the difficulties in diagnosis,the aggressiveness of pancreatic cancers, early lymphatic and blood metastasis, and thelack of effective systemic therapies. Up to now, chemical therapy of pancreatic canceris mainly restricted to cytotoxic drugs, those drugs are also harmful for normal cells.Because of the genetic instability, chemical therapy becomes drug resistance selectionof pancreatic neoplasia.In 1971, Folkman brought up a hypothesis of anti-angiogenesis for the first time.When tumor grows to certain extent, new vasculature is needed to access so as toprovide nutrition and facilitate metastasis. Vascularization of tumor is the key step for atiny local neoplasia turning to an enlarged tumor capable of metastasis. Evidencereveals that in the absence of access to an adequate vasculature, tumor will berestricted to a diameter of approximately 0.4mm. In this course, a series ofproangiogenic factors are concerned, they are bFGF, PDGF, PDEGF, FGF,Angiopoietin-1, TGF-β1, TGF-α, EGF and so forth. Perhaps the best characterizedproangiogenic factor is Vascular Endothelial Growth Factor(VEGF), which isrelatively unique among growth factors in terms of its specificity for the vascularendothelium. To aim for VEGF, a lot of protocols are put out to inhibit angiogenesis,including VEGF antibody, VEGF-toxin combiner, low-molecular VEGF antagonist, etc.Antisense VEGF protocol is regarded as an effective gene treatment method to inhibitVEGF produced by tumor.In our research, VEGF121 cDNA was amplified by PCR from plasmidpcDNA3-VEGF121. Eco RⅠand Bam HⅠrestriction enzyme site were introduced tothe 5' and 3' of VEGF121 cDNA respectively. PCR product was digested by restrictionenzyme Eco RⅠand Bam HⅠ, then inversely inserted into polylinker site of plasmidpcDNA3, constructed human antisense VEGF121 eukaryotic expression vectorpcDNA3-ASVEGF121. Antisense VEGF121 vector was transfected into humanpancreatic cancer cell BxPC-3 by liposome. Positive clone was obtained by theselection of G-418. Plasmid neoR DNA was amplified from genome DNA oftransfected cells by PCR to test the integration of exogenetic plasmid. PCR resultreveals that exogenetic plasmid has been integrated into the cell genome. 5In vitro, we tested VEGF121 expression at transcriptional level by RT-PCR. VEGFprotein in culture media was tested by ELISA. We also tested VEGF, Flk-1 and Bcl-2expression in culture cells by immunohistochemistry. Colony forming test and growthcurve MTT assay were carried out to assess the proliferation potency. Culture cellswere labeled by Annexin Ⅴ-FITC and PI, then apoptosis was tested by flowcytometry. Proliferation effect of Culture media on vascular endothelial cell wasevaluated by testing growth curve of ECV-304. In Vitro Angiogenesis Assay wasperformed to test angiogenesis potency of culture media.In vitro research reveals that antisense VEGF121 vector can significantly reduceVEGF121 mRNA expression in pancreatic cancer cell, and VEGF protein is alsoinhibited in culture media. Immunohistochemical stain reveals that antisense VEGF121vector can also inhibit VEGF expression in cell surface, and the expression of VEGFreceptor, Flk-1, is found significantly elevated. Pearson correlation analysis revealsthat VEGF positive rate is reversely correlated with that of Flk-1(P=0.017). We alsofound that inhibiting VEGF leads decreased expression of Bcl-2 in pancreatic cancer,but no statistical correlation was found(P=0.074). Colony forming test reveals thatcells transfected with antisense VEGF121 vector have low colony forming rate, growthcurve reveals that cell...