Study of VEGF transfection on rabbit mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Vascular endothelial growth factor (VEGF) is a very effective way to make tissue engineer bone vascularization.However, because of expensive and short half-life, VEGF cannot maintain effective concentration in blood after injection. To resolve the problem effectively, gene transfection technique is used in this experiment to transfer human VEGF into seed cells-mesenchymal stem cells (MSCs) of tissue engineer bone and to make it secrete VEGF which could vascularize bone.OBJECTIVE: To explore the possibility of human vascular endothelial growth factor 165 (VEGF165) to transfect rabbit MSCs, and establish the experimental foundation of angiogenesis tissue engineering organization and the treatment of ischemic disorders.DESIGN: Observation control trail.SETTING: First Hospital of Jilin University and Institute of Frontier Medical Sciences of Jilin University.MATERIALS The experiment was conducted in the Key Laboratory (BSL-2) of Frontier Medical Sciences of Jilin University between June 2003 and August 2004. Health New Zealand white rabbits, 4.0-5.0 months old, weighing 2.5-3.5 kg, half male and half female, were provided by Animal Center of Jilin University. The rabbits were handled under asepsis and anesthetized condition,corresponding to the animal ethical standard. Medicine and reagents Ham F12 culture media (Gibco, U.S), MTT (Sigma, U.S)PLXSNKDRp-VEGF165 and pcDNA 3.0 vectors were prepared in the present laboratory. ELISA detection kit (Jingmei company,Shenzhen), DH5 α, restriction endonucleases Barn H I, Xhol Ⅰ, Hind Ⅲ, EcoR Ⅰ and standard DNA molecule (Promega,U.S) were also used in this study.METHODS: Rabbits' MSCs were separated and cultivated. The pcDNA 3.0-hVEGF165 expression vector was constructed and identified, pcDNA3.0-VEGF165 eukaryotic expression vector was constructed, the vector was used directly to transfect MSCs. The cultural supernatant then was collected and the soluble protein of human VEGF gene expression was analyzed with ELISA method.The proliferation capability of human umbilical vein endothelial cells (HUVEC) stimulated by the supernatant was measured with MTT methods, untreated MSCs and pcDNA3.0 transfected MSCs were used as control groups.MAIN OUTCOME MEASURES: ① Result of restriction enzyme digestion and DNA sequencing of the recombinant plasmid pcDNA3.0-VEGF165;② the secretion of human VEGF165 proteins of the transfected MSCs analyzed by ABC-ELISA; ③ MTT method was used to detect the effects of MSCs culture supematant transfected with VEGF165 on HUVEC cells proliferation ability.RESULTS: ①Result of restriction enzyme digestion and DNA sequencing of the recombinant plasmid The constructed plasmid was digested with Hind Ⅲ and XHol Ⅰ, and then two pieces fragments were isolated with agarose gel electrophoresis, which was accordance with expected results. And sequencing results showed that PeDNA3.0-VEGF165 eukaryotic expression vector was successfully constructed. ② ABC-ELISA method: Compared with the control group, concentration of human VEGF protein in the supernatant of the cultured cells increased significantly after the MSCs were transfected with pcDNA3.0-VEGF165 for 24, 48, 72 hours (P＜0.05).③ MTT method was used to detect the effects of MSCs culture supernatant transfected with VEGF165 on HUVEC cells proliferation ability. The results showed MSCs supematant transfected with VEGF165 (2%, 4%,8%, 16%, and 32%) had statistical significance in promoting HUVEC cells proliferation rate compared with the normal control (P＜0.05).CONCLUSION: Human VEGF gene can be successfully transfected into MSCs and expressed effectively.