摘要
目的 探讨pVEGF165-IRES-Ang-1重组双顺反子质粒在大鼠骨髓间充质干细胞（bone marrow-derived mesenchymal stem cell,MSC）中表达的情况,为下一步的动物实验奠定基础.方法 构建插入人血管生成素-1（angiopoietin,Ang-1）和人血管内皮细胞生长因子165 （vascular endothelial growth factor 165,VEGF165）双顺反子的pIRES重组质粒,经脂质体转染大鼠MSC,以实时荧光定量PCR技术分析各基因人源、鼠源以及总mRNA的动态变化.结果 所构建的pVEGF165-IRES-Ang-1双顺反子重组质粒在大鼠MSC中成功表达了相应蛋白.人VEGF165得以高丰度表达,且在转染1d后mRNA检出的拷贝数最高;人Ang-1在4个检测时间点中的mRNA拷贝数比较稳定,但转录本的拷贝数较VEGF 165显著降低;转入人Ang-1和人VEGF 165后,各对应同源蛋白的总mRNA有显著增加,但大鼠MSC自身Ang-1与VEGF164的编码mRNA明显有下调趋势.结论 pVEGF165-IRES-Ang-1重组双顺反子质粒双基因表达量明显不一致,表现为受插入的位置关系影响,且可能受转染细胞内同源蛋白的总量调控.
Objective To construct bicistronic plasmid pVEGF165-IRES-Ang-1 and observe its expression in rat bone marrow-derived mesenchymal stem cell （MSC）. Methods The bicistronic plasmid pVEGF165-IRES-Ang-1 was constructed and then was transfected into rat MSC using liposome. The expression of human and rat derived mRNA of each gene as well as total mRNA was analyzed by real time quantitative polymerase chain reaction. Results The bicistronic plasmid pVEGF165-IRES-Ang-1 was successfully constructed and each gene was successfully expressed. The level of mRNA ex- pression of human derived VEGF165 was high and expressed highest after transfection for 24 hours. The level of mRNA expression of human derived Ang-1 was stable in 4 observed time points, but the copy number of mRNA transcripts was much less than VEGF165. After transfection of human derived Ang-1 and VEGF165 into rat MSC, the total mRNA expression was significantly increased, but mRNA encoded by rat MSC derived Ang-1 and VEGF164 was significantly downregulated. Conclusion Gene expression of Ang- 1 and VEGF 165 in pVEGF 165-IRES-Ang- 1 recombinant bicistronic plasmid is significantly inconsistent, which is influenced by the inserted position relationship and regulated by the total homologous protein in the transfected cells.
出处
《老年医学与保健》
CAS
2014年第6期384-388,426,共6页
Geriatrics & Health Care
基金
国家自然科学基金（81060155,81360292）
江西省自然科学基金（20132BAB205058）
江西省科技厅支撑计划（2010BSA10300）
江西省优势科技创新团队计划（20115BCB29023）
