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目的:将携带增强型绿色荧光蛋白(EGFP)基因标记和Netrin-1基因的腺病毒(Ad5-Netrin-1-CMV-EGFP)转染骨髓间充质干细胞(BMSCs),观察其对BMSCs分泌细胞因子、自身分化和小管形成的影响。方法:体外分离培养大鼠BMSCs,转染病毒后,real-time PCR检测BMSCs表面受体,RT-PCR检测细胞因子的表达,ELISA法检测Netrin-1的表达情况,并通过小管形成实验和Transwell迁移实验观察Netrin-1联合BMSCs对小管形成能力和细胞迁移的影响。结果:腺病毒感染的BMSCs表达多种Netrin-1受体并分泌血管内皮生长因子(VEGF)、成纤维细胞生长因子(bFGF)、脑源性神经营养因子(BDNF)和神经生长因子(NGF)等细胞因子。Netrin-1具有促进HUVEC形成小管能力,但比VEGF差(P<0.05);Netrin-1联合VEGF促进HUVEC形成小管能力比单独Netrin-1或VEGF更强(P<0.05);Transwell迁移实验显示Netrin-1具有与VEGF相当的促进BMSCs迁移能力,单独Netrin-1或VEGF迁移能力不及Netrin-1联合VEGF(P<0.05)。结论:Ad5-Netrin-1-CMV-EGFP成功转染BMSCs并分泌Netrin-1,Netrin-1联合VEGF促进小管形成能力更强,Netrin-1具有促进BMSCs迁移能力,联合VEGF更能促进BMSCs迁移。
OBJECTIVE: To transfect bone marrow mesenchymal stem cells (BMSCs) with Ad5-Netrin-1-CMV-EGFP carrying enhanced green fluorescent protein (EGFP) gene and Netrin-1 gene to observe its effect on BMSCs-secreting cells Factors, self-differentiation and tubule formation. Methods: BMSCs were isolated and cultured in vitro. Real-time PCR was used to detect the surface receptors of BMSCs. The expression of cytokines was detected by RT-PCR. The expression of Netrin-1 was detected by ELISA. The expression of Netrin- Effect of Netrin-1 combined with BMSCs on tubule-forming ability and cell migration in Migration assay. Results: Adenovirus-infected BMSCs expressed a variety of Netrin-1 receptors and secreted vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) Other cytokines. Netrin-1 had the ability of promoting the formation of tubules in HUVEC but was less than that of VEGF (P <0.05); Netrin-1 combined with VEGF enhanced the ability of HUVEC to form tubules more than Netrin-1 or VEGF alone (P <0.05); Netrin- -1 had the same ability to promote the migration of BMSCs as VEGF. The ability of Netrin-1 or VEGF alone was less than that of Netrin-1 combined with VEGF (P <0.05). Conclusions: Ad5-Netrin-1-CMV-EGFP can successfully transfect BMSCs and secrete Netrin-1. The combination of Netrin-1 and VEGF can promote tubule formation. Netrin-1 can promote the migration of BMSCs. Combined with VEGF can promote the migration of BMSCs.