摘要
目的为获得高表达生物活性血管内皮细胞生长因子VEGF165的皮肤种子细胞,拟将转基因治疗与真皮多能干细胞相结合,为难愈性创面的促愈治疗奠定基础。方法构建真核表达载体pIRES2-EGFP-VEGF165,经脂质体法介导转染真皮多能干细胞;半定量RT-PCR检测VEGF165 mRNA的表达,W estern b lot和酶联免疫ELISA法检测VEGF蛋白的表达;并检测了表达产物的生物活性及转染VEGF(derm almu ltipotential stem cells,DMSCs)增殖活性的变化。结果转染后VEGF165 mRNA和VEGF蛋白的表达均显著增强(P<0.01),约为对照组的1.6倍,且转染VEGF后DMSCs的培养上清显著提高hECV304细胞增殖活性(P<0.01)。MTT结果显示转染VEGF后DMSCs增殖活性显著增高,约为转染空载体组DMSCs的2倍(P<0.01)。结论成功获得了高水平表达并分泌具有生物活性的VEGF的基因治疗种子细胞DMSCs,为下一步动物在体进行基因治疗打下了基础。
Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165 （VEGF165） so as to promote refractory wound healing by gene therapy in combination with cell engineering. Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells （DMSCs） by lipofectin, the expression of VEGF mRNA was detected by RT-PCR, VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay （ELISA） and Westen blotting respectively. To evaluate the activity of VEGF secreted by transfected DMSCs, hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays. Meanwhile, the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTr. Results The results of RT-PCR, ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs. The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs. Conclusion The plasmid pIRES2-EGFP- hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection, and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第18期1854-1856,共3页
Journal of Third Military Medical University
基金
国家重点基础研究发展规划资助项目("973"项目)(G1999054205)
全军医学科研"十五"计划面上项目(01MA166)~~
关键词
VEGF165
DMSCs
基因疗法
vascular endothelial growth factor 165
refractory wound healing
dermal mesenchymal stem cells
cell engineering
gene therapy
