The Package Of RAAV-hVEGF165 And Its Expression In Mesenchymal Stem Cell And Cardiomyocyte

Cardiovascular disease, accounting for 35% percentage of mortality in human beings, has been the first rank of causing-death diseases, 50% of which is due to coronary heart disease(CHD). Epidemic materials show that there are 225 patients with chronic heart failure(CHF) per 1000,000 people, and its annul mortality is 35%. With the increasing of living level and the improving of medical environment in China, the incidence of CHD is increasing too. The surviving rate of CHD is elevating by the methods, including PTCA, PCI, CABG and so on, but the core question, the losing of functional cardiomyoctes, is not solved effectively because current medical methods couold not break the dead recycle from myocardial injury/necrosis to adaptation and deadaptation of normal myocardium for the extra load to the death of more normal cardiomyocytes, but just slow down the pace of the disease which is the root reason why CHD is self-developing. So the cellular therapy to rebuild myocardium and gene therapy to restore blood supply of ischemic area are coming to the focus.The concept of gene therapy is to substitute normal gene for abnormal gene or correct it, including: target gene cloning, gene transferring, target cell selection and clinical experiments. Target gene and gene transferring are key steps.Vascular endothelial growth factor(VEGF) is a multifunctional growth factor with molecular weight 34-46KD, secreted by several cells, whose biological functions are to promote angiogenesis, enhance vascular permeability, sustain the normal structure of vessels and so on. VEGF is popularly accepted as one of pro-angiogenesis growth factors, and applied to treat ischemic diseases widely.The perfect vector used in gene transferring should be characteristics of targetspecificity, high stability, innocuity, high efficiency of gene transfer and long duration of expression. Usually, transferring vectors are divided into two kinds: non-viral vector and viral vector. The latter gradually becomes mainstream because of the former's shortcomings which is low efficiency of gene transferring, short duration of expressing, and difficulty for clinical application. Adeno-associated virus vector(AAV) is becoming the focus gradually because of its more advantages than other viral vectors, such as: safety with no pathogenesis, low immunogenesis, physical stability, more transfecting cells, and longer duration expression. The recombined-AAV is acknowledged as one of the most promising vectors in gene therapy and used widely as the gene transfer vector in metabolic disease and genetic disease.This study used the package platform of AAV: rAAV-Helper free system, to construct rAAV-hVEGF165 vector and rAAV-EGFP vector, and observed the expression of rAAV-hVEGF165 in mesenchymal stem cells(MSCs) and myocardium, both of which are based for the next experiments.Parti The package of rAAV-hVEGF165 vectorObjective: Constructing AAV vector plasmids with gene engineering andmolecular biological, packing rAAV-hVEGF165 and rAAV-EGFP with three plasmids co-transfection techniques.Methods: 1. Plasmids were constructed by standard molecular biology techniques. 516bip,Bamh I and Xba I fragment containing the full-length cDNA for human VEGF165 was isolated from a plasmid pcDNA3-hVEGF165. The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with hVEGF165 cDNA to construct interim plasmid pCMV-hVEGF165. E.coliDH5awas transformed with the constructed plasmid pCMV-hVEGF165 by electroporation method and selected on ampicillian LB agar plate. The structure of plasmid pCMV-hVEGF165 was verified by restriction enzyme digested, PCR and sequencing using the primers which were both part of the pCMV-MCS plasmid sequence, beta-globin intron and hGH pA. After verified the plasmid pCMV-hVEGF165, to carry out next step experiment.2. To avoid the lost of ITR which could cause decrease the yield of viral vector, the pCMV-hVEGF165 was digested by Not I and the open read frame containing hVEGF165 cDNA was cloned into ITR contained plasmid pAAV-MCS which was also digested by Not I . The plasmid pAAV-hVEGF165 was constructed and transformed into E.coli DH5aby electroporation method, then the plasmid was harvested and verified by PCR, the sequence of the sense primer and antisense were part of L-ITR and R-ITR of pAAV-hVEGF165 plasmid, the product of PCR was 2976bp. 140bp ITR part of pAAV-hVEGF165 plasmid could cut out by double digested using Not I and Pst I . Primer used to sequence the pAAV-hVEGF165 plasmid were the same of sequencing primer of pCMV-hVEGF165.3. The process of construction pAAV-EGFP plasmid was similar to that of pAAV-hVEGF165.The EGFP cDNA sequence was derived from polymerase chain reaction amplification of plasmid pEGFP-Nl using a pair of primers. The sequence of the sense primer was 5'-AGAGTCGACAGATGGTGAGCAAGGGCGAGGAG-3'(leaded-inSal I site), and the antisense primer was5'-GAGAGATCTTTACTTGTACAGCTCGTCCATG-3'(leaded-in Bglll site).The product of PCR was 720bp EGFP sequence which have Sal I and BglW site in each terminals. The EGFP sequence was double digested by Sal I and Bgl II .The plasmid pCMV-MCS was also double digested by the same enzymes and ligatedwith EGFP to construct plasmid pCMV-EGFP which was verified by PCR and sequence. The plasmid pCMV-EGFP and pAAV-MCS were digested by Not I , and the open read frame containing EGFP was cloned into ITR containing pAAV-MCS fragment to construct plasmid pAAV-hVEGF165.The plasmid was verified by restriction enzyme digested, PCR and sequencing.4. HEK293 cells were grown to 80%~90% confluence, trypsinized and counted, then co-transfected by electroporation using pAAV-hVEGF165(or pAAV-EGFP),pRC and pHelper to package rAAV-hVEGF165 and rAAV-EGFP. 72 hours later, the cells were under Chloroform-NaCl sediment-Chloroform extract to dissociate and purify rAAV. Viral particle of purified rAAV were assayed by AVSachTM ELISA. Used SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. rAAV-hVEGF165 was also identified with electronic microscope.Results: In this study we used the technique of gene engineering andmolecular biology to constructed pCMV-hVEGF165 and pCMV-EGFP plasmid, then cloned the open read frame containing interested gene into pAAV-MCS to construct pAAV-hVEGF165 and pAAV-EGFP. Successfully avoid the lost of ITR in pAAV plasmid which could cause decrease the yield of viral vector. All constructed plasmid verified by PCR and sequencing. HEK293 cells was co-transfected by electroporation using three plasmid, dissociate and purify rAAV-hVEGF165 and rAAV-EGFP. The viral particles of rAAV were 2xlOn, Under SDS-PAGE and Coomassie brilliant blue staining could find three notable viral capsid protin strap of AAV. Observed the viral particle under electron microscope, rAAV uniformity and the diametere were about 20-25 ran, the figure were polyhedron. The successful packaging of rAAV establish a groundwork for later experiment using this vector in vivo and in vitro.Part 2The culture and identification of MSCs of Wistar rats and myocardium of neonate ratsObjective: to establish the cultivating and purifying conditions of MSCs and myocardium.Methods: 1. the culture of MSCs: The bone marrow samples were collectedfrom 4 Wistar male rats'(weight from 120-150g) femurs and cleaned twice with 10% FBS culture medium. Then the cells in bone marrow aspirates were sent to the culture flask by the density 4*105 / cm2 and cultivated in incubator under the conditions of 37°C> 5%CC>2. The cells were observed with inverted microscope. 72 hours later the culture medium was exchanged, and then should be exchanged once three days.2. the culture of cardiomyocytes: The hearts were collected from 10 neonate rats(born within 3 days), and cut into pieces under the sterile environment. The pieces were put into a small triangular flask and trypsinized. The supernatant of trypsinized mixture was collected together except for the first supernatant and put into another triangular flask semi-full of 10%FBS culture medium to stop trypsinization. Next, wash the mixture twice with 10%FBS culture medium and cultivate it in culture flask for pre cultivation of 90 mins in incubator. After that put the medium mixture into another culture flask by cell density 3xlO5cells/ml. 12 hours later the culture medium was exchanged, and then should be exchanged once three days.3. using Hematoxylin and eosin(HE) stain and immunocytochemical stain to identify MSCs and myocardium.Results: 1. We have successfully cultivated MSCs and the purity of the thirdgeneration is up to over 95% through HE stain and immunocytochernical stain.2. Neonate myocardium has also been cultivated successful with the purity more than 95% by HE stain and immunocytochemical stain.Part 3Study on the expression of rAAV-hVEGF165 in MSCs andmyocardium in vitroObjective: to observe . rAAV-hVEGF165 expressing in MSCs and myocardium in vitroMethods: 1. the method of MSCs and myocardium culture is the same as before.2. MSCs and myocardiums were transfected with rAAV-EGFP under different dose (1 x 10\ U\05, 1 x 106, 1 x 10\ 2x 106v.p./cell), and 96hours later the cells were observed under fluorescence microscope.3. MSCs were transfected with rAAV-hVEGF165 or rAAV-EGFP (lxl07v.p./cell), used un-transfected cells as control group, while myocardiums withlxio6v.p./cell.4. Using ELISA to measure the concentration of VEGF in supernatant at 4th, 7th, 10th day.Results: 1. 96 hours after transfecting MSCs and myocardium, greenfluorescence protein was found to be expressed under fluorescence microscope. The percentage of emission of green fluorescent cell of MSCs was 3%,29%,38%,53%,57% when cells were infected with rAAV-EGFP at different viral particle (1 x 104? 1 x 105 ? 1 x 106. 1 x 107? 2x 106v.p./cell), while myocardium's was 7%,33%,55%,57%,59%.2. The concentration of VEGF in supernatant of MSCs transfected withlxl07v.p./cell rAAV-hVEGF165 and myocardium transfected with 1 x 106v.p./cell rAAV-hVEGF165 respectively is increasing gradually at 4th, 7th, 10th day, and both are significantly higher than bare AAV group and PBS control group.Conclusions: We have constructed rAAV-hVEGF165 and rAAV-EGFP andcultivated MSCs and myocardium successfully. Experiment in vitro proved that interested gene could be expressed in MSCs and myocardium stably.