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目的:研究用人骨髓诱导培养内皮祖细胞(EPCs)的方法,探讨携带目的基因增殖缺陷的腺病毒对EPCs的转染情况、目的基因的表达情况和转染前后EPCs生物活性的变化。方法:用梯度离心法从人骨髓分离单个核细胞,加入人血管内皮生长因子(VEGF165)诱导培养,用流式细胞仪测定细胞表面标志CD14、CD34、CD45、CD166、CD29、CD44、HLA-DR、CD31、CD106、CD133和VEGFR2(KDR)。增强型绿色荧光蛋白基因(EGFP)作为报告基因,流式细胞仪检测腺病毒载体(Ad-EGFP)对EPCs的感染效率。用携带人干扰素γ(hγIFN)基因的腺病毒载体(Ad-hγIFN)感染EPCs(EPCs-hγIFN),ELISA检测细胞EPCs-hγIFN分泌hγIFN的情况。MTT实验观察EPCs、EPCs-EGFP和EPCs-hγIFN的增殖情况,流式细胞仪检测EPCs、EPCs-EGFP和EPCs-hγIFN细胞CD133、KDR和CD34的表达情况。结果:从骨髓分离的单核细胞经过诱导培养2周后,贴壁的单核细胞开始表达EPCs标志CD133、KDR和CD34。Ad-EGFP感染复数(MOI)为150pfu/EPCs时,感染率即可达90%以上。EPCs-hγIFN培养上清可以测得较高浓度而且浓度稳定的hγIFN。腺病毒转染后,EPCs的增殖活性和表面标志表达无显著性变化。结论:从人骨髓可以培养出EPCs,腺病毒可以把目标基因成功转入EPCs,转入的基因可以稳定地表达,在携带目的基因的腺病毒转染后,EPCs的细胞生物活性无显著性改变。
OBJECTIVE: To study the method of culturing EPCs with human bone marrow and investigate the transfection of EPCs, the expression of target gene and the biological activity of EPCs before and after transfection. Methods: Mononuclear cells were isolated from human bone marrow by gradient centrifugation and cultured with VEGF165. The cell surface markers CD14, CD34, CD45, CD166, CD29, CD44 and HLA-DR were determined by flow cytometry , CD31, CD106, CD133 and VEGFR2 (KDR). Enhanced green fluorescent protein gene (EGFP) as a reporter gene, flow cytometry Adenovirus vector (Ad-EGFP) on EPCs infection efficiency. EPCs (EPCs-hγIFN) were transfected with adenovirus vector (Ad-hγIFN) carrying human interferon gamma (hγIFN) gene and hγIFN secreted by EPCs-hγIFN was detected by ELISA. The proliferation of EPCs, EPCs-EGFP and EPCs-hγIFN were observed by MTT assay. The expressions of CD133, KDR and CD34 in EPCs, EPCs-EGFP and EPCs-hγIFN cells were detected by flow cytometry. Results: Adherent mononuclear cells began to express EPCs markers CD133, KDR and CD34 after induced by bone marrow mononuclear cells for 2 weeks. Ad-EGFP infection at a multiple (MOI) of 150pfu / EPCs, the infection rate can be up to 90%. EPCs-hγIFN culture supernatant can be measured at a higher concentration and concentration of hγIFN. After adenovirus transfection, EPCs proliferative activity and surface marker expression no significant change. CONCLUSION: EPCs can be cultured from human bone marrow. The adenovirus can transfer the target gene into EPCs successfully. The transfected gene can stably express. After transfection with adenovirus carrying the target gene, there is no significant change in the biological activity of EPCs .