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| 1 | Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells显示文摘After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ. | Organ Grafting Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003 , China Department of Cardiothoracic Surgery, the General Hospital of Daqing Oil Field, Daqing 163001 , China and Department of Vascular Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China | 2005 | Hepatobiliary & Pancreatic Diseases International2005，4，3： | 2 |
| 2 | In vitro and in vivo mutational analysis of the 3'terminal regions of hepatitis e virus genomes and replicons显示文摘 | Graft J Nguyen H Kasorndorkbua C | 2005 | J Virol2005，79，2： | 1 |
| 3 | Inhalational exposure to carbonyl sulfide produces altered brainstem auditory and somatosensory-evoked potentials in Fischer 344N rats显示文摘 | Herr DW Graft JE Moser VC | 2007 | Toxicol Sci2007，95，1： | 1 |
| 4 | Distally hased venous flap:a new technique for the correction of syndaetyly without skin graft in adult patients 显示文摘 | End-of-skin grafts in flap for web Space defeets Cil Y Kocman AE Yapici AK | 2009 | Chir Orqani Mov2009，93，3： | 1 |
| 5 | Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands显示文摘 | Herman J G Graft J R Myohanen S | 1996 | Proceedings of the National Academy of Sciences of the United States of America1996，93，18： | 1 |
| 6 | Blood lipids and first- ever ischemic stroke/transient ischemic attack in the Bezafibrate Infarction Prevention (BIP) Registry: high tfiglycefides constitute an independent risk factor显示文摘 | Tanne D Koren - Morag N Graft E Goldbourt U | 2001 | Circulation2001，104，24： | 1 |
| 7 | Nonhaemon'hagic thalamic infarction : clinical, neuropsychological and electrophysiolog- ical findings in four anatomical groups defined by CT 显示文摘 | Graft Radford NR Damasio H Yamada T | 1985 | Brain1985，108，： | 1 |
| 8 | Automated registration of sequential breath-hold dynamic contrast-enhanced MR images : a comparison of three techniques 显示文摘 | Rajaraman S Rodriguez J J Graft C et at | 2011 | Magn Reson Imaging2011，29，： | 1 |
| 9 | Perioperative antibiotic prophylaxis in ureteroscopic stone removal显示文摘 | Knopf HJ Graft HJ Schulze H | 2003 | Eur Urol2003，44，1： | 1 |
| 10 | Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands显示文摘 | Herman JG Graft JR Myohanen S | 1996 | Proc Natl A- cad Sci USA1996，93，18： | 1 |
| 11 | Methylation-specific PCR : A novel PCR assay for methylation status of CpG islands 显示文摘 | Herman J G Graft J R Myohonen S | 1996 | Proc Natl Acad Sci U S A1996，93，18： | 1 |
| 12 | Targeting the eIF4F translation initiation complex for cancer therapy 显示文摘 | Konicek BW Dumstorf CA Graft JR | 2008 | Cell Cycle2008，7，16： | 1 |
| 13 | Methylation-specific PER:A novd PCR assay for methyl-ation status of CpG island显示文摘 | Herman JG Graft JR Myohanen S | 1996 | Proc NatAcad Sci USA1996，93，18： | 1 |
| 14 | False negative and false posi- tive errors in abdominal painevaluati on : failure to diagnose acute ap- pendieitis and unneeessary surgery 显示文摘 | Graft L Russell J Seashore J | 2000 | Aead Emerg Med2000，7，11： | 1 |
| 15 | Three-fixation combined with bone 显示文摘 | Goslings J C Schulrman H dimensional dynamic external grafting for Kienbock's disease Upper Extremmity Surgery 2000 A Broekhcizen A H | 2000 | Techniques in Hand and2000，4，2： | 1 |
| 16 | Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands显示文摘 | Herman JG Graft JR Myohanen S | 1996 | Proc Natl Acad Sci USA1996，93，18： | 1 |
| 17 | Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor 显示文摘 | Graft A A | 1997 | Microbiology1997，143，7： | 1 |
| 18 | eIF4E expression and its role in malignancies and metastases 显示文摘 | De Benedetti A Graft J R | 2004 | Oncogene2004，23，18： | 1 |
| 19 | Targeting the eu- karyotic protein translation initiation factor dE for cancertherapy 显示文摘 | Graft JR Konicek BW Carter JH | 2008 | Cancer Res2008，68，3： | 1 |
| 20 | Normal birth after singleembryo transfer in a patient with excessive polypronudear zygote formation following IVF and ICSI 显示文摘 | Matt DW Ingram AR GrafT DP | 2004 | Fertil Steril2004，82，6： | 1 |
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