难愈性创面修复局部人血管内皮生长因子165真核表达载体的构建与评价

Construction and evaluation of human vascular endothelial growth factor 165 eukaryotic expression vector in repair of refractory wound

目的:克隆人血管内皮生长因子165cDNA并构建其真核表达载体,为放创复合伤的难愈性创面的促愈修复提供实验基础。方法:实验于2001-09/2003-05在第三军医大学创伤、烧伤与复合伤国家重点实验室完成。从白血病HL-60细胞提取总RNA,经过反转录-聚合酶链反应扩增编码人血管内皮生长因子165的cDNA序列,将其克隆至pMD18-T载体后进行测序鉴定,然后将该序列插入双顺反子的真核表达载体pIRES2-EGFP的多克隆位点中。观察以下指标①反转录-聚合酶链反应扩增人血管内皮生长因子165cDNA。②阳性重组质粒pMD18-T-hVEGF165的筛选。③DNA测序鉴定。④阳性重组质粒pIRES2-EGFP-VEGF165的筛选。结果:反转录-聚合酶链反应扩增得到约600bp的目的DNA片段,酶切pMD18-T-hVEGF165得一与目的片段大小一致的条带,且测序分析其与GeneBank中报道的编码人血管内皮生长因子165cDNA序列完全一致。pIRES2-EGFP-hVEGF165的酶切电泳显示目的条带成功插入真核表达载体pIRES2-EGFP中。结论:成功构建了含有人血管内皮生长因子165cDNA的真核表达载体,如果通过转基因技术可提高血管内皮生长因子165在放创复合伤难愈性创面的局部的表达,有望为修复难愈创面开辟一条新的途径。

AIM:To clone cDNA encoding human vascular endothelial growth factor 165(hVEGF165) and construct the recombinant eukaryotic plasmid of hVEGF165 so as to provide experimental bases for promoting refractory wound healing in combined irradiation-wound injury. METHODS:The experiment was conducted in the State Key Laboratory of Trauma,Burns and Combined Injury,Third Military Medical University from September 2001 to May 2003.The total RNA extracted from leukemia HL-60 cells was amplified into the sequence of cDNA encoding hVEGF165 by reverse transcription-polymerase chain reaction(RT-PCR),and then inserted into pMD18-T vector for sequencing analysis.Next,the identified cDNA fragment was inserted into the multiple clone sites of eukaryotic expression bicistron plasmid pIRES2-EGFP.The observation indexes were as follows:①cDNA encoding hVEGF165 amplified by RT-PCR,②screening of positive recombinant plasmid pMD18-T-hVEGF16,③DNA sequencing analysis and ④screening of positive recombinant plasmid pIRES2-EGFP-VEGF165. RESULTS:A DNA fragment about 600-bp in length was obtained by RT-PCR amplification,and a strip accordant with the target fragment in size was also obtained with pMD18-T-hVEGF165 enzyme.The sequence analysis validated that it was identical to the sequence of cDNA encoding hVEGF165 reported in GeneBank.The target strip was successfully inserted into eukaryotic expression vector pIRES2-EGFP by pIRES2-EGFP-hVEGF165 enzyme electrophoresis. CONCLUSION:The eukaryotic expression vector containing cDNA encoding hVEGF165 was successfully constructed.It is hopeful to employ a new way for improving the refractory wound healing if the expression of hVEGF165 is increased in the locally healed refractory wound in combined wound-irradiation injury by transgenic technique.