The Experimental Research On Extracellular1-3domain Of VEGF Receptor KDR Gene Therapy For Glial Brain Tumor

Glioma, the most common malignant tumor in central nervous system, withcharacteristics of high incidence rate, high recurrence rate, high mortality rate,low cure rate, etc, seriously threatening human health, is one of the intractableand refractory tumors in neurosurgery treatment. Since no practically effectivetreatment method has been found at present, it is urgent to seek new treatmentmethod.Glioma is the typical solid tumor of vascular dependence, and its growth,development, infiltration and metastasis are closely related to the formation ofnew vessels, and the formation process of new vessels is strictly regulated by theangiogenesis regulatory factor. Vascular endothelial growth facto(rVEGF)is thepresently known proangiogenic factor with the strongest and the most specificeffect, which play the role of promoting vascularization mediated by thereceptor of vascular endothelial membrane. VEGF exhibits high affinity bindingto two distinct endothelial cell receptor tyrosine kinases, the fms-like tyrosinekinase Flt-1and the kinase insert domain containing receptor KDR. Recentfindings suggest that KDR is the key mediate of mitogenic proliferationmigration differentiation and microtubule generation. Blocking the VEGF-KDRpathway may inhibit angiogenesis. KDR belongs to receptor tyrosine kinasesfamily and is expressed almost exclusively in endothelial cells. KDR has atypical tyrosine kinase receptor structure with7immunoglobulin (Ig)-likedomains in the extracellular region, as well as a long kinase insert in the tyrosine kinase domain. Evidence shows that domain1-3is the essential domain tocombine with the VEGF, and KDRn3is the gene order of these three domains, akind of dissoluble KDR.This research firstly constructs the adenovirus vector pAd-KDRn3ofKDRn3and observes its expression in mammalian cells, to lay foundation forthe research on the effect of KDRn3in treating glioma. Then, through vitrotransfection of rat's C6glioma cell into pAd-KDRn3, observe the expression oftarget gene in C6glioma cell and its inhibited effect on the multiplication ofhuman's vein endothelial cells (ECV-304cell), and discuss the feasibility ofpAd-KDRn3gene transfection inhibiting the formation of new vessels. At last,conduct animal experiment research of treating glioma with pAd-KDRn3, andobserve the curative effect of KDRn3on resisting the angiogenesis of glioma tocontrol the growth of the glioma.The research work of this experiment:1. The construction and expression of adenovirus vector adenovirus vectorObjective: By using the second generation adenovirus vector constructionsystem to construct adenovirus vector pAd-KDRn3, observe the transfectionefficiency as well as the expression of foreign gene, to lay foundation forresearching the effect of KDRn3on treating glioma.Methods: Taking pcDNA3.1/KDRn3as a template, expand KDRn3genethrough PCR, and clone it into T-vector; adopt adenovirus backbone plasmidpAdeasy-1and shuttle plasmid pAdTrack-CMV to construct and recombineKDRn3adenovirus vector, to identify enzyme digestion; recombine theadenovirus vector transducer293cell of KDRn3gene; apply method of WesternBlot to analyze the protein expression of KDRn3.Results:①The sequence analysis shows that KDRn3gene is correct.②KDRn3gene can be successfully cloned into pAdTrack-CMV and achieves homologous recombination in E.Coli5183cell, and KDRn3gene inserts intoadenovirus genome of deficit form.③after recombining adenovirus vectortransducer293cell of KDRn3gene, recombinant virus can be produced, and therecombinant virus can highly actively transfect COS-7cell.④the cell transfectsthe recombinant virus can express high-level KDRn3protein.Conclusion: Adenovirus vector pAd-KDRn3is successfully constructed,and it can successfully transfect COS-7cell and express high-level KDRn3.2. pAd-KDRn3transfection of rat's C6glioma cell in vitro and its inhibitedeffect on the multiplication of vascular endothelial cellsObjective: Adopt adenovirus vector pAd-KDRn3to transfect rat's C6glioma cell in vitro, observe the expression of target gene in C6glioma cells andits inhibited effect on the multiplication of human's umbilical vein endothelialcells (ECV-304), and discuss the feasibility of pAd-KDRn3gene transfection ininhibiting new angiogenesis based on cell level.Methods: Observe the expression of EGFP in transfection group andcontrol group under the fluorescence microscope, and observe the number ofcells with green fluorescence in cytoplasm24hours,48hours, and72hoursafter transfection in transfection group; apply the method of ELISA to test theexpression level of KDRn3in the supernatant layer of pAd-KDRn3transfection;apply the method of MTT to test the inhibited effect of each group on themultiplication of human's umbilical vein endothelial cells (ECV-304).Results:①After24hours of transfection, the transfection group emitsgreen fluorescence in cell endochylema; after48hours of transfection, thenumber of cells emitting green fluorescence in endochylema increases; after72hours of transfection, the number of cells emitting green fluorescence inendochylema reaches more than80%. But no green fluorescence can be seen inthe control group.②After pAd-KDRn3transfecting C6glioma cell with different MOI, with the number of MOI increasing, the secretion level ofKDRn3also increases accordingly; after24hours of pAd-KDRn3transfection,KDRn3begins to express significantly, and it reaches the peak at72hours; afterthis, the expression presents gradual downtrend, and it lasts to96hours later.③The control group co-cultured with untransfected supernatant layer proliferatessignificantly, while cell proliferation of all groups co-cultured with transfectedsupernatant layer is significantly inhibited (p＜0.001), and with the number ofMOI increasing, its inhibition action becomes significantly increasing; besides,compared with the experimental group without VEGF, the inhibition action ofexperimental with VEGF on ECV304is more significant (p＜0.05).Conclusion: Recombined expression vector pAd-KDRn3can effectivelytransfect C6glioma cell and highly express in C6glioma cell. Recombinedexpression vector pAd-KDRn3can significantly inhibit the multiplication ofhuman's umbilical vein endothelial cells (ECV-304).3. Animal experimental research on treating glioma with pAd-KDRn3Objective: Observe the curative effect of KDRn3in inhibiting C6gliomaangiogenesis and the growth of the tumor.Methods: Through vaccinating C6glioma cell100μl (1×106/100μl/one) oflogarithmic phase under the lateral cutaneous of rat's thighbone of right hindlegs, observe the formation and growth condition of the tumor body. On theseventh day after the vaccination, select18rats that have grown subcutaneoustumor nodules with diameter of6~9mm, and divide them into three groups,6rats per group, including:①Group pAd-KDRn3;②Group pAdGFP;③GroupPBS (blank control). Measure the diameter and weight of the tumor28daysafter vaccinating the tumor cell, and calculate the weight of the tumor and itsvolume inhibition rate. Observe the change of routine pathomorphism of C6glioma; immunohistochemistry tests the expression of KDRn3protein in tumor tissue; observe the therapeutic action of pAd-KDRn3on C6glioma; test themicrovessel density of the tumor (MVD).Results:①The control group (Group pAdGFP and Group PBS): Theexternal appearance of the tumor presents irregular shape, with incompleteinvolucrum, tortuous blood vessels on the appearance, tough quality, solidtangent plane, liquifaction necrosis differing in size in the center, partialulceration of the surface of the tumor and partial tumor invading the muscle.Regular HE slice sees tumor cell in different sizes, with obvious atypia, and seesgiant cell tumor. With large cell nucleus, presenting circular shape or irregularshape, nucleoplasm increasing, karyokinetic division is common.Immunohistochemistry can't test the expression of KDRn3in tumor tissue.②Group pAd-KDRn3: The grown form of cell is similar to that of the controlgroup, with significant tumor necrosis, and great amount of inflammatory cellsaround the necrosis. Immunohistochemistry regards that positive staining ofclaybank can be seen in tumor tissue, located at endochylema. On the fourteenthday, the volume of the tumor in Group pAd-KDRn3is smaller than that of thecontrol group, with significant diversity (P<0.05); when on the21stday and28thday, the volume of the tumor in Group pAd-KDRn3in obviously smaller thanthat of other two groups, with significant diversity (P<0.05). On the28thday, themeasurement result of tumor samples shows that the weight of tumor in GrouppAd-KDRn3is significantly smaller than that of the other two groups, withsignificant diversity (P<0.05), and no significant diversity between Group pAdand Group PBS (P>0.05). The suppression ratio of the volume and the weight ofthe tumor in Group pAd-KDRn3is respective59.47%and60.67%. Comparedwith the control group, MVD of Group pAd-KDRn3is obviously less, withsignificant diversity (P<0.05). There is no statistical diversity between GrouppAd and Group PBS (P>0.05). Conclusion: Since pAd-KDRn3can effectively inhibit the new vesselsgrowth of glioma and the growth of the tumor, it is expected to be applied to thegene therapy of glioma.