The Package Of PAd-hTIMP1 And Its Protective Effect On Endothelial Cell Injury Induced By Homocysteine

Cardiovascular disease has been the first rank of causing-death diseases. According to the report of WHO, 1 of 3 death cases is caused by cardiovascular disease at present. With the increasing of living level and the improving of medical environment in China, the incidence of coronary heart disease(CHD) is increasing too. So far, although the traditional therapy, including drug treatment, interventional therapy and surgical therapy, has evidently elevated the surviving rate of CHD, it still couldn't cure CHD. Following the development of molecular biology, especially in the field of gene vector, technology of gene transfer and recognization of target genes, the young field of gene therapy promises major medical progress toward the cure of cardiovascular disease.Gene therapy is a form of molecular medicine that has the potential to influence significantly human health in this century. The basic concept gene therapy is: introduce into target cells a piece of genetic material that will result in either a cure for the disease or a slowdown in the progression of the disease, which includes target gene cloning, gene transferring and expression of target gene.A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Adenoviral Vector (AdV) is among the most promising gene transfer vehicles. AdV offer many advantages over other gene delivery systems. It could be produced in a highly concentrated form and is capable of targeting both dividing and nondividing cells, the adenovirus chromosome remains episomal in the transduced cell, thus avoiding the possibility of insertional mutagenesis, it also allows to accommodate heterologous DNA inserts of more than 8kb.Our target gene is the tissue inhibitor of metalloproteinases(TIMP1), and TIMP1 is the specific inhibitor of matrix metalloproteinase(MMPs), which is the main enzyme that can degradate the Extracellular matrix(ECM). It has been found close relationship between the disequilibrium of MMPs/TIMPs and cardiovascular diseases, such as atherosclerosis(AS), restenosis after angioplasty and heart failure.Hyperhomocysteinemia is recognized as an independent risk factor for the development of AS and other cardiovascular diseases. Homocysteine(Hcy) could influence the expression of MMPs and TIMPs in the endothelial cells, which decreases the stability of atherosclerotic plaque。Transfer TIMPs into cultured human umbilical vein endothelial cells by adenovirus could regulate the synthesis of ECM, which is considered to be the new method to cure CHD.In the study, recombinant pAd-hTIMP1 was constructed by the package platform of AdV: AdEasy system to infect the cultured Human Umbilical Vein Endothelial Cells(HUVEC) CRL-1730 interfered by homocysteine of different concentration, the expression level of TIMP1, MMP9 and MMP1 mRNA were observed by RT-PCR. Adenovirus-mediated overexpression of TIMP1 in vitro is the basic for the next experiments in vivo.Partâ… The Package of pAd-TIMP1 and pAd-TrackObjective: Constructing AdV vector plasmids with gene engineering and molemular biological technology.Methods: 1. PCR was used to amplify the 637bp fragment human TIMP1 (hTIMP1) DNA from the full-length cDNA in human cardiocyte, which was cloned into the plasmid of pUCm-T to construct the plasmid pUC- hTIMP1.2. After double digestion of plasmid pUC-hTIMP1 with BgιⅡand Notâ… , fragment was cloned into the pAdTrack-CMV which was also double digested by the same enzemes to create pAdTrack-CMV-hTIMP1. Orientation of the insert within the vector was assessed by diagnostic restriction enzyme analysis. The pAdTrack-CMV-hTIMP1 was linearized with Pemâ… .3. Linearized shuttle vector pAd-Track-hTIMP1 and pAd-Track was transferred into electrocompetent E.coliBJ5183 containing adenoviral backbone vector pAdEasy-1 by electroporation to create the plasmid pAd-hTIMP1, which was digested by BamHI and Pacâ… . Once confirmed, supercoiled plasmid DNA was transformed into DH5αfor large-scale amplification. The pAd-hTIMP1 and pAd-Track were linearized with Pacâ… .4. Linearized pAd-hTIMP1 and pAd-Track were transfected into 293T cells to generate recombinant adenovirus. Transfected cells were monitored for GFP expression and collected 7-10 days. The viral lysate was used to infect packaging cells 2-3 times .The resultant viruses were purified by CsCl banding. The pAd-hTIMP1 and pAd-Track were also identified with electronic microscope.Results: In this study we used the technique of gene engineering and molecular biology to construct shuttle vector with TIMP1 (pAdTrack-CMV-hTIMP1), then pAd-hTIMP1 was constructed by homologous recombination in E.coli BJ5183. All constructed plasmids verified by PCR and sequencing. 293T cells were transfected by the linearized pAd-hTIMP1, the process of viral production can be directly and conviently followed in the packaging cells by visualization of the GFP reporter. The final yields were generally 1.9×10¹²v.p./ml. TIMP1 fragment could be identified by PCR. Viral particles were observed by electronic microscope, the figure were polyhedron with a diameter of 80-90nm. The successful packaging of AdV settled a foundation for experiments in viro and in vitro.Partâ…¡Adenovirus-mediated overexpression of TIMP1 in cultured Endothelial Cells Objective: To study the effect of overexpression of TIMP1 on Cultured Human Umbilical Vein Endothelial Cells(HUVEC) interfered by homocysteine in different concentration, CRL-1730 were transfected with pAd-hTIMP1 and pAd-Track as a control.Methods: 1. Group. According to the interference, all the cells were divided into 3 groups: (1)blank group; (2)track group; (3)TIMP1 group. Then it was divided into subgroups due to the concentration of Hcy. The concentration were 0mmol/L, 0.01mmol/L and 0.1mmol/L. Cells were passaged to 6-well plates, each subgroup contained 8 wells, cells in 4 wells were collected to extract RNA after 6hr stimulated by Hcy. The conditioned media in left 4 wells was collected to detect MMP2 and MMP9 by zymography.3. Semiquantitate reverse transcription-polymerase chain reaction(SQRT-PCR) was used to detect the relative expression of hTIMP1, hMMP1 and hMMP9 to GAPDH respectively.Results: Cultured endothelial cells could express certain MMPs and TIMPs. After stimulated by Hcy, the expression of TIMP1 mRNA in cells could be increased at the concentration of 0.01mmol/l(p＜0.05), and the expression of MMP1 mRNA and MMP9 mRNA in cells could be increased at the concentration of 0. 1mmol/l(p＜0.05). When transfected with pAd-Track and pAd-hTIMP1, the expression of TIMP1 mRNA was elevated(p＜0.05), while MMP1 mRNA and MMP9 mRNA decreased.(p＜0.05), The MMP9 and MMP2 protein were detected by zymography, which showed decreased MMP9 protein while overexpression ofTIMP1 in EC.Conclusion: 1. Adenovirus Vector pAd-hTIMP1 and pAd-track were successfully packaged with AdEasy system.2. Hcy could improve the expression of MMP1 mRNA and MMP9 mRNA in HUVEC(CRL-1730), while overexpression pAd-hTIMP1 could inhibit the expression of MMP1 mRNA and MMP9 mRNA, which plays an important role in protecting the endothelial cells.