构建稳定表达血管内皮细胞生长因子的人间充质干细胞

Establishment of human mesenchymal stem cells transferred by vascular endothelial growth factor

背景:血管内皮细胞生长因子可有效治疗缺血性心脏病,但其在体内难以保持持续的有效浓度。目的:构建稳定表达血管内皮细胞生长因子的人骨髓间充质干细胞株,检测经基因转染后外源基因的表达。设计、时间及地点:观察实验,于2006-03/2007-04在上海胸科医院完成。材料:人骨髓间充质干细胞株、血管内皮细胞生长因子由上海市胸科医院胸部肿瘤研究所基础实验室提供。方法:扩增血管内皮细胞生长因子片断,构建pcPGK-hVEGF 165真核表达质粒,利用脂质体介导转染人骨髓间充质干细胞,然后进行阳性细胞克隆筛选。主要观察指标:以RT-PCR、PCR、Western Blot、ELISA方法检测在稳定转染了pcPGK-VEGF165-IRES-GFP的3株细胞和稳定转染pcPGK-IRES-GFP的3株细胞中,人血管内皮细胞生长因子165在人骨髓间充质干细胞中的表达情况。结果:扩增血管内皮细胞生长因子后,成功了构建质粒pcPGK-hVEGF165,并利用脂质体顺利转染了人骨髓间充质干细胞,并筛选出稳定表达的细胞株。RT-PCR、PCR检测结果显示,在稳定转染血管内皮细胞生长因子骨髓间充质干细胞中表达的血管内皮细胞生长因子165信使RNA明显高于对照及未转染的人骨髓间充质干细胞,WesternBlot、ELISA检测结果显示,稳定转染血管内皮细胞生长因子人骨髓间充质干细胞及培养上清中的血管内皮细胞生长因子蛋白明显高于对照及未转染的人骨髓间充质干细胞。结论:采用脂质体介导基因转移技术可将人血管内皮细胞生长因子165顺利转染人骨髓间充质干细胞中,并获得稳定表达血管内皮细胞生长因子165的细胞株。

BACKGROUND： Vascular endothelial growth factor （VEGF） is able to effectively treat the ischemic heart disease, but in vivo VEGF cannot be maintained effective concentration. OBJECTIVE： To detect the expression of VEGF mRNA and protein in human bone marrow mensenchymal stem cells transferred by VEGF-165 gene. DESIGN, TIME AND SETTING： The empirical study was conducted from March 2006 to April 2007 at Shanghai Chest Hospital. MATERIALS： Human bone marrow mesenchymal stem cell line and VEGF were offered by Basic Laboratory, Thoracic Tumor Institute, Shanghai Chest Hospital. METHODS： hVEGF165 gene was reconstructed in pcPGK-vector and transferred into human bone marrow mensenchymal stem cells （BMSCs） by liposome-mediated method, clone screening by G418. MAIN OUTCOME MEASURES： The mRNA and protein of VEGF gene in transferred cells was detected by reverse transcription-polymerase chain reaction （RT-PCR）, Real time PCR, Western Blot, enzyme linked immunosorbent assay （ELISA） in 3 stem cells of pcPGK-VEGF165-1RES-GFP and pcPGK- IRES-GFP, respectively. RESULTS： pcPGK-hVEGF165 vector was reconstructed and transferred into hMSCs successfully. The expression of hVEGF165 in the transfected hMSCs was demonstrated with RT-PCR and Real time PCR. Western Blot and ELISA demonstrated that the expression of hVEGF165 in the transfected hMSCs and VEGF protein in supernatant were significantly more than untransfected hMSCs. CONCLUSION： hVEGF165 can be successfully transfected into BMSCs by using liposome mediated gene transfer. Stably expressed VEGF165 cell line can be obtained.