【作者】 李刚；

【导师】 夏照帆； 路卫；

【作者基本信息】 第二军医大学 ， 外科学， 2002， 硕士

【摘要】 目的：（1）体外扩增人血管内皮细胞生长因子165基因全长序列并构建真核质粒表达载体；（2）用重组载体转染原代培养的人胎儿成纤维细胞，得到转基因细胞株；（3）对转基因细胞进行初步研究，探讨其直接应用于创面移植及用于复合人工皮的构建的可行性并为其提供原料。方法：（1）设计合成引物，抽提肝癌组织总RNA为模板，用RT-PCR方法体外扩增hVEGF₁₆₅全长基因，并以穿梭质粒pcDNA3为骨架，构建真核表达载体pcDNA3-hVEGF₁₆₅；（2）用FeGENE6转染试剂转染原代培养的人胎儿成纤维细胞，通过G418进行筛选，得到单克隆细胞，继续培养扩增得到转基因细胞株；（3）通过一系列实验对转基因细胞进行初步研究：①将转基因细胞消化收集，抽提总RNA，用RT-PCR方法检测转基因细胞VEGF基因的转录水平，并以正常细胞作对照；②用ELISA方法检测转基因细胞培养上清液中VEGF表达的浓度和持续时间，评价外源基因翻译水平和表达蛋白的分泌状况；③用转基因细胞培养上清液培养血管内皮细胞，通过测定血管内皮细胞细胞生长曲线的改变，来检测其表达蛋白的生物学活性；④采用MTT方法对转基因细胞的细胞增殖曲线进行测定；⑤Miles实验，通过检测培养上清液对血管通透性的影响，检测表达蛋白的生物学活性。结果：（1）成功扩增hVEgF₁₆₅全长基因，并以穿梭质粒pcDNA3为骨架，构建了真核表达载体pcDNA3-hVEGF₁₆₅；（2）构建的载体成功转染了人成纤维细胞，并通过G418进行筛选，得到转基因细胞株；（3）初步研究表明，转基因细胞有mRNA水平表达，且明显高于正常细胞；ELISA方法检测到培养上清液中有VEGF表达，但浓度较低，而正常成纤维细胞培养上清液中检测不出VEGF；转基因成纤维细胞与同代正常细胞相比生长曲线无明显变化；转基因细胞培养上清液可促进体外培养的人脐静脉血管内皮细胞的生长；Miles实验表明转基因细胞培养上清液可增加体内血管的通透性。结论：利用分子生物学方法，成功构建了人血管内皮细胞生长因子165真核表达载体pcDNA3-hVEGF₁₆₅；该载体转染原代培养的人胎儿成纤维细胞，获得的转基因成纤维细胞能够表达、分泌VEGF蛋白，且表达的VEGF能够促进体外培养的人脐静脉血管内皮细胞的生长，能够增加体内血管通透性，具有一定的生物学活性，从而为改善 人工皮的性状和功能，应用“治疗性血管形成”促进创面愈合提供了可行性依据。更多还原

【Abstract】 Background: Closure of full-thickness skin defects is very importan to thetreatInent of patients with severe burns. As these patients with severe bams isconcemed, the donor site is far from enough, which results in many seriouscomplications, even death. The artificial skin can partly solve the problem.ReconstrUction of composite skin is the ultimate aim of skin tissueengineering. The composite skin is mainly composed of tIiree layers: epidermallayer, dermal analog layer and fibroblast layer. Many ldnds of artificial skinhave been used in clinical work, but they are not very satisfying. The artificialskin, or the dermal analog often has slow vascularization or poor take rate. It isthe key to improve the angiogenesis of the artificial skin fOr improving itspractical value.The use of biological agents to stimulate or augment angiogenesis is anexciting field of scientific research and there is optimism that it will become animportant therapeutic modality. The potential of exogenous agents to stimulatethe development of new blood vessels into ischaemic tissue has recently arousedconsiderable inierest.The result of angiogeneses is better oxygenation, provision of nutrients andremoval of waste products from tissues. To be able to augment this ntaalphysiological process with exogenous biological theraPies is an exciting conceptand would represent a major advance in medicine. The optimal agents, vectors,sites of administration and dosage strategies will take time to define. There isjustifiable optimism that theraPeutic angiogenesis could revolutionise thetTeatment of ischemia, bllt many questiol1s remain unanswered. More research isessential.Objcctivcsl. To amplify hVEGF,,, gene in vitro and constructing an eukaryoticexpression vector pcDNA3- hVEGF,,,;2. To transfer the recombinant vector into human fibroblast, and obtainingthe fibroblast cells transferred with pcDNA3- hVEGF,,,;3. To do some basic research about the transferred fibroblast cells.Material and Methodsl. Reversible transcription polymerase chain reaction/RT-PCR has beenaPplied to arnplify hVEGF,,, gene Which was then constructed to aneukaryotic expression vector pcDNA3;2. The recombinant vectoT, pcDNA3- hVEGF,,, was then trandferred intohuman fibrobIast celIs by FeGENE transfection regent. SeIected byGeneticin/G418, the fibroblast cells transferred with the recombinamvector were obtained,3. Some basic research were done on the trandferred cells f?To demonstrate VEGF mRNA expression in the transferred fibroblastcells by RT-PCR method (2) To demonstrate the secreted VEGF protein concentration in thesupernatant by Enzyme-Linked Immunosorbent Assay/ELISA method (D To draw the cell proliferation curve of the transferred cells by MTTmethod ?To determine the biological activity of the secreted protein byculturing the human umbilical vein endothelial cells/HUVEC with thesupernatant of the transferred fibroblast cells (5) To determine the biological activity of the secreted protein byVascular Permeability Assay. Conditioned medium obtained from thetransferred cells was tested in guinea pigs for its permeability activityin a Miles assayResults:l.The hVEGF165 gene has been amplified in vitro and an eukaryotic expression vector pcDNA3- hVEGF165 was successfully constructed;2. The fibroblast cells transferred with the recombinant vector were obtained;3. It was proved that the transferred cells demonstrated VEGF mRNA expression and the expression level was more than normal fibroblast celis. It was also proved that VEGF protein was secreted into the supernatant and the concentration was about lOpg/ml. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo and improved the growth of the cultured HUVECs.Conclusions:The hVEGF165 gene was successfully cloned and transferred into fibroblast cells. The transferred cells demonstrated VEGF mRNA expression and protein secret更多还原