基因移植人血管内皮细胞生长因子斜备小鼠新生血管模型  

Establishment of new vessels model by implantation of VEGF165 in mouse

作　　者：谢芳[1] 杨力[1] 汤苏阳[2] 郑岩[1] 易成刚[1] 夏炜[1] 潘勇[1] 马戈甲[1] 杨阳[1] 

机构地区：[1]第四军医大学西京医院全军整形外科研究所,西安710032 [2]武警总医院皮肤再生医学科

出　　处：《中华整形外科杂志》2011年第5期373-375,共3页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金面上项目（30772260）

摘　　要：目的探讨血管内皮细胞生长因子基因用重组慢病毒为载体移植制备新生血管动物模型的可行性。方法构建重组慢病毒介导的人血管内皮细胞生长因子（humanvascularendothelialgrowthfactor，hVEGF165）基因，并转染小鼠成纤维细胞（NIH／3T3）。利用流式细胞仪挑选转染成功的细胞，将转染成功的表达人VEGF165的NIH／3T3细胞植入C57小鼠的骨骼肌，建立小鼠的新生血管模型。在新生血管模型上，用ELISA的方法进行小鼠血清VEGF测定。观察瘤体组织形态学。用免疫组化的方法观察是否有表达CD31的细胞。结果经酶切鉴定、聚合酶链反应（PCR）及基因测序证实Lenti—VEGF165-GFP构建成功，包装后测定滴度为6．19×10^7TU／ml。用基因移植的方法成功建立了稳定的小鼠新生血管模型，并证实小鼠外周血中有VEGF的分泌。14d后细胞植入处局部肿胀。44d后HE染色发现细胞植入部位有血管组织的新生，免疫组化显示有围成规则或不规则管状表达CD31的细胞。结论将表达人VEGF165的NIH／3T3细胞植入C57小鼠的骨骼肌，可以产生稳定有效的新生血管模型。Objective To investigate the feasibility of establishing a murine new vessels model with Lentiviral vector （LVs） mediated human vascular endothelial growth factor-165 （pcDNA3.1/ VEGF165） gene. Methods Lentivirus plasmid carrying a human VEGF165 was constructed and was used to transfect mouse＇ s NIH/3T3 cells. The NIH/3T3 cells with high secretion of VEGF were injected into the skeletal muscle of mouse to establish a mouse new vessels model by implantation of vascular endothelium growth factor （VEGF） gene. The external secretion of VEGF was measured with ELISA. Histological examination was carried out after injection. The expression of CD31 was assessed with immunohistochemical method. Results The lenti-VEGF165-EGFP was correctly constructed and confirmed by restriction endonuclease analysis, polymerase chain reaction and DNA sequencing analysis. Lentivirus plasmid carrying a human VEGF165 was constructed, lenti-VEGF165-EGFP was used to transfect mouse ＇ s NIH/ 3T3 cells, and human VEGF165 gene was assessed. NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model. The external secretion of VEGF in peripheral blood was measured with ELISA. The legs became swollen in experimental group 14 d after injection. It found the cells expression of CD31 44 d after injection, and histological analysis showed the swollen tissue was composed of small new vessels. Conclusions NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model.

关 键 词：模型 动物 血管内皮细胞生长因子 小鼠 成纤维细胞 

分 类 号：R-332[医药卫生]