| The study of combined gene therapy to glioma withangiastain and wild type p53 induced bythe rAAV-LyoVec vectorObjectiveSince 1992, when NIH of the United States authorized the fast clinical pro-ject of molecular neurosurgery, which is a clinical project to cure glioma withHSR-tk/GeV system induced by retronituse, the worldwide attention of themedical field to the gene therapy of malignancy has been caused. Up to now,232 clinical testing programs of gene therapy have been authorized going on, a-mong which there are 105 programs of gene therapy for tumor, and 14 items ofthem is aiming at glioma. At present, though the effect is off at best, a brandnew pattern will be provided to the cure of glioma due to the continual develop-ment of basic and clinical study of the gene therapy of glioma, and the establish-ment of increasing combined treatment programs in particular, This study con-struct a new type of vector of safe , no assistant pollute virus , without viral gene,and validate transfecting rate of them. We validate effect of vector to carry angi-astain and wild type p53 purpose gene to combined treatment C6 glioma in vivoand in vitro, and find safe and effective gene treatment ways about treatment C6glioma.This study was carried out in three steps:1. construction and the evaluation of rAAV-LyoVec vector.This study Construct the rAAV-GFP vector without viral gene, new typeof vector rAAV-GFP-LyoVee. measure the transfecting rate of the vector toquantitative rat C6 cells, and compare the transfecfing rate of them, and confirmthe high transfecfing rate vector, consummate the construction manner of hightransfecting rate vector according to the industrialization standard, preparing for the vector to carry angiastain and wild type p53 purpose gene to combined treat-ment C6 glioma.2. The establishment of the nude mouse C6 glioma modelNude mouse C6 glioma model is a typic testing model for the study of thebiological characteristics and the treatment of human tumor. At present, nudemouse tumor model are usually used as the most important animal model of genetherapy because it can keep the morphological characteristic of the primarytumor itself. This experiment made the nude mouse model of C6 glioma for thestudy of the gene therapy of glioma, examine the expression of protein level ofGFAP in vivo and in vitro by immunohis-tochemistry, authenticate its heredi-tary, and studied the histopathology characteristic of the model.3. combined gene therapy to glioma with angiastain and wild type p53 in-duced by the rAAV-LyoVec vectorThe experiment studied of combined gene therapy to glioma with angiastainand wild type p53 induced by the rAAV-LyoVec vector. Construct the nudemouse glioma model by the method of C6 Cell suspension vaccination, Observechanges of glioma volume in groups according to different time, measure expres-sion of protein level of angiastain and wild type p53 by immunohistochemistry,conf'rrrn apoptosis, virus transfection and tissue necrosis of C6 glioma cells ofeach group by electron microscope after different gene therapy, confirm that ang-iastain was expressed in transfected C6 glioma cells at molecular level throughRT-PCR, and studied the effects of combined gene therapy program to cure C6glioma from the tissue, cell, protein and molecular level.Materials and Methods1. construction and the evaluation of rAAV-LyoVec vector1.1 The construction of the viral vector without viral gene:Construction of rAAV-LyoVec vector pSNAV and vector plasmid withGFP gene used method of literature, the name of plasmid was pSNAV-GFP.The name was 293 SG1 after pressure selection of G418. 293 cell at 37℃with5％CO2 for 72 hours, collect the cells, centrifuge brachytely, discard superna- tant, freeze thawing the cell cliques three times quickly, heat the lysate at 50-60℃for 30 min to inactive the remained AAV-GFP, centrifuge brachytely toremoval the cell debris, purify the crude extract of rAAV-GFP, according tothe method of chloroform treatment-PEG/NaCI precipitation-chloroform ex-traction and concent-ration, collect the cells and culture fluid, add 10％vol-ume of chloroform, shaking intensely, add solid NaCl to achieve the final con-centration of 1mol/L, centrifuge, collect supernatant, add PEG to achieve thefinal concentration of 10％, centrifuge, discard supernatant, heavy suspenseprecipitate using PBS, add DNase to achieve the final concentration of 1μg/ml,extract it with chloroform, collect the phase which was purified rAAV-GFP.1.2 The construction of the new type of vector of rAAV-GFP-LyoVec:Prepare rAAV-GFP 80ul, add RPMI 1640, positive ion liposome 2ml,got (TMAG) (DLPC) (DOPE) with the molar ratio of 1:2:2 (the total quantitywas 1 umol), dissolved it in 0.5ml chloroform, evaporated the solvent, soddenthe adipose membrane in 0.2ml PBS containing 1.5×108rAAV-GFP particle,mixed it by turbine agitator for 2 min, adjusted the suspension volume to 0.5mlwith PBS, the vectors which had not catch rAAV-GFP will displace to Ficollgradiant, and this 0.5ml rAAV-GFP conjugating liposome LyoVec, which con-tained 1.5×108 rAAV-GFP granula/micromole hpid. The amount of hposomealso increased to 15nmol lipid (1.5×108 granula/ml) accordingly, and 0.5mlrAAV-GFP and LyoVec new type of vector was put into incubator at 37℃with5％CO2.1.3 Transfect C6 cells in vitro with three groups of vectors of rAAV-GFP,LyoVec-GFP, and rAAV-GFP-LyoVec, and MOI(multiplicity of infection)is 105v. g/cell. It means that ratio of the amount of viral genome (v. g) and thecells transfected is 105. Counted with invert microscope, and compared thetransfecting rate.2. The construction of the nude mouse C6 glioma model2.1 Animal. six female nude rats weighting 15-20g were obtained fromthe Animal Department of China Medical University.2.2 Cell culture. The rat C6 glioma cells were cultured in the medium ofDMEM(penicillin 105U/L, streptomycin 100mg/L) containing 10％serum, and put into incubator at 37℃with 5％CO2 till logarithmic phase.2.3 The method of tumor cell suspension vaccination. Got the cells in loga-rithmic phase, digest them with 0.25％trypsin, centrifuged and discard the su-pernatant, then centrifuged and washed them with the DMEM without serum twotimes, counted the amount of the cells, adjusted the concentration of the cells,suspensed the cells in PBS(0.01mol/L, pH7.4), and the concentration of thecells was 5×106/ml. Then draw the cell suspension 200ul and vaccinated it tothe armpit of nude mouse. Six nude mouse were vaccinated in this group.2.4 The observation and measurement of tumor. After the nude mouse werevaccinated, culture them in the sterile cleaning barricade system with constanttemperature (25±2℃), constant humidity (45％-50％), observe conscious-ness, drink and eat and defecation of the nude mouse regularly, three diamen-sions snap gage measurement: V(cm3)=πD3/6, got the mean value and drewthe growing curve of tumor.2.5 The examination of histopathology. Kill the nude mouse, observe thebody appearance of the transplanted tumors carefully. Obtain tumor tissue andheld it in 10％formalin solution, embed it with paraffin wax, sect the tissue,regular HE dyeing, then did the examination of histopathology.2.6 Examined the expression of GFAP protein through immunohistochemis-try. Let the C6 glioma cells cultured in vitro creep onto slice and examine theexpression of GFAP protein in nude mouse transplanted tumor tissue by themethod of immunohistochemistry (method of ABC) regularly, the endochylemaof the positive cells was dyed with buffy, and the nucleus were conterstained byhaematoxylin.3. Combined gene therapy to cure C6 glioma with angiastain and wild typep53 induced by the rAAV-LyoVec vector3.1 Get 36 cultured nude mouse, randomize, and there were 6 groups inall.(1) PBS empty control group(2) LyoVec control group(3) rAAV-p53-LyoVeC group(4) rAAV-angiastain-lyoVeC group (5) rAAV-p53-LyoVeC+rAAV-angiastain-LyoVec group(6) Carboplafin group ( provided by Australia Keding company)After 4d of C6 cell inculability, injection cure begin, 1 group adds PBS50ul, 2,3,4,5 group adds 50ul/nude mouse, Measure the volume of tumor atthe 4d, 12d, 20d, 28d, 36d. Three diamensions snap gage measurement:V(cm3)=πD3/6, get the mean value and draw the growing curve of tumor. Dwas the mean value of the max diameter and minimum diameter of tumor.3.2 Immunohistochemistry examination and HE dyeing: get C6 transplan-ted tumor of each group, dehydrate it regularly, transparent, embed and cover itwith paraffin wax, sect, SABC method: confirm the level of express p53,VEGF, CD34.3.3 Another examinination of the expression of angiastain through RT-PCR.The primers were synthesized by Chinese Academy of Medical SciencesShanghai Biology engineering company: EcoRIPrimer 1 5′AGGAATTCATGGTGTTGCAGAC-CCAGGTCT-TCATTTCTCTGTTGCTCTGGATC-TCTGGTGCCTACGGGGCTGAAAACAG-GAAGTCCTC 3′Primer 2 5′AAGTCGACTTATTGTTCCGTG-GATACTGG 3′Extract RNA and poly (A)+RNA from (4) (5) groups C6 cell, synthe-sized cDNA, PCR extend, samples stored at 4℃and observed resules.Results1. construction and the evaluation of rAAV-LyoVec vector.The construction of the new type of vector of rAAV-GFP-LyoVec. Co-transfect C6 cells in vitro with three groups of vectors of rAAV-GFP, LyoVec-GFP, and rAAV-GFP-LyoVec, and MOI ( multiplicity of infection) was105 v. g/cell. It meaned that the ratio of the amount of viral genome ( v. g) andtransfected cells is 105. rAAV-GFP-LyoVec was higher than rAAV-GFPand LyoVec-GFP compared the transfecting rate.2. The construction of the nude mouse C6 glioma model Construct the nude mouse C6 glioma model, and the growing incubation pe-riod of armpit transplanted tumor of nude mouse was 4 days, establish the grow-ing curve of nude mouse.3. Combined gene therapy to cure C6 glioma with angiastain and wild typep53 induced by the AAV-LyoVec vector3.1 Establish the growing curve of tumor.3.2 The immunohistochemistry examination and HE dyeing: p53, VEGFand CD34 were expressed in each groups ceils with different degrees.3.3 Another examinination of the expression of angiastain at group (4) and(5) through RT-PCR. Gained a gene fragment in group (4), (5) by RT-PCR, which was confirmed as angiastain gene fragment by electrophoresis, butthis fragment was not seen in other group.Conclusions1. The efficiency of the new established vector rAAV-LyoVFec to transfectpurpose gene was much higher than the self-transfection of rAAV and LyoVec.2. Nude mouse tumor model was usually used as the most important animalmodel of gene therapy because it cuold keep the morphological characteristic ofthe primary tumor itself.3. rAAV-LyoVec high transfection rate vector induced the combined genetherapy to cure C6 glioma with vascular Chalone and wild type p53, and theeffect was much better comparing with the single gene therapy with angiastain orwild p53 gene. It was demonstrated that the combined gene therapy was an im-portant way to conquer the malignancy.
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