VEGF基因修饰兔骨髓间充质干细胞的研究

背景:血管内皮生长因子的应用是组织工程组织血管化的有效手段.但是,血管内皮生长因子价格昂贵,并且半衰期非常短.很难在体内保持有效的作用浓度.故本实验将其转入组织工程的种子细胞--骨髓间充质干细胞中,使骨髓间充质干细胞能够有效地分泌血管内皮生长因子,从而达到促进血管生成的目的.目的:探讨应用人血管内皮生长因子165进行基因修饰的可行性,为血管化组织工程组织的构建及缺血性疾病的治疗奠定实验基础.设计:观察对比实验.单位:吉林大学第一医院和吉林大学再生医学科学研究所.材料:实验于2003-06/2004-08在吉林大学再生医学科学研究所吉林大学重点实验室(生物安全实验室二级)完成.健康新西兰大耳白兔由吉林大学实验动物中心提供.4.0～5.0月龄,体质量215～315 kg,雌雄兼用,实验过程中对动物处置均在麻醉状态,无菌条件下完成,符合动物伦理学标准.实验药品及试剂:Ham F12培养基(GIBCO公司),噻唑蓝(MTT)(Sigma公司),pLXSN-KDRp-VEGF165与pcDNA3.0载体由本实验室自备,ELISA检测试剂盒(深圳晶美生物公司),感受态大肠杆菌DH5α、限制性内切酶BamH Ⅰ、Xho Ⅰ、HandⅢ、EcoR Ⅰ和标准DNA分子(Promega公司).方法:分离、培养新西兰大白兔骨髓间充质干细胞.构建并鉴定pcDNA3.0-VEGF165真核表达载体,利用脂质体介导其转染骨髓间充质干细胞.采用ELISA方法检测转基因骨髓间充质干细胞的人血管内皮生长因子蛋白表达,MTT法检测转基因骨髓间充质干细胞表达物对血管内皮细胞增殖活性的影响,设单纯培养骨髓间充质干细胞及pcDNA3.0转染骨髓间充质干细胞组为对照组.主要观察指标:①重组质粒双酶切和基因测序分析.②ABC.ELISA法观察质粒转染后的骨髓间充质干细胞的人血管内皮细胞生长因子蛋白表达.③通过MTT法检测人血管内皮生长因子165基因转染骨髓间充质干细胞培养上清对血管内皮细胞增殖的影响.结果:①构建的质粒用Hind Ⅲ和Xho Ⅰ双酶切、琼脂糖凝胶电泳鉴定结果与预期完全相同,PcR和酶切鉴定正确后行测序鉴定,测序结果正确,证明所构建的质粒为pcDNA3.0-VEGF165重组质粒.②ABC-ELISA法检测结果表明,人血管内皮生长因子165基因重组载体转染骨髓间充质干细胞后24 h,即有较高水平的人血管内皮生长因子165蛋白表达,48及72 h呈下降趋势,但与对照组比较,差异显著(P＜0.05).③MTT法检测人血管内皮生长因子165基因转染骨髓间充质干细胞培养上清对血管内皮细胞增殖的影响,结果显示,含2%,4%,8%,16%和32%转染人血管内皮生长因子165基因的骨髓间充质干细胞培养上清促血管内皮细胞增殖率均高于对照组(P＜0.05).结论:人血管内皮生长因子165基因可成功地转染至骨髓间充质干细胞中,并可进行有效地表达.

Study of VEGF transfection on rabbit mesenchymal stem cells

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.