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目的:构建携带增强型绿色荧光蛋白报告基因的人血管内皮细胞生长因子121的真核质粒,检测所制备的表达hVEGF121/EGFP融合蛋白的骨髓间充质干细胞在不同时长缺氧后血管内皮细胞生长因子的水平。方法:设计携带酶切位点的扩增引物,通过PCR技术扩增人血管内皮细胞生长因子121编码基因全序列,以粘端连接克隆法将其编码片段定向连接入质粒载体pEGFP-N2的多克隆位点中,构建pEGFP-N2-hVEGF121重组质粒,采用酶切法、PCR以及DNA序列测定法鉴定所构建的重组质粒。全骨髓差速贴壁法分离培养骨髓间充质干细胞,体外扩增传代,通过形态学观察及表面标记物进行细胞鉴定。通过阳离子脂质体介导pEGFP-N2-hVEGF121重组质粒转染体外培养的骨髓间充质干细胞,使用荧光显微镜检测hVEGF121/EGFP融合蛋白的表达。将制备好的表达hVEGF121/EGFP融合蛋白的骨髓间充质干细胞在体外缺氧环境下分别培养6,12,24h,另设相同缺氧干预下的空白骨髓间充质干细胞组,以转染空载pEGFP-N2质粒的骨髓间充质干细胞组作为对照,采用RT-PCR法检测血管内皮生长因子mRNA的表达量。结果:酶切鉴定法、PCR鉴定法、DNA序列测定皆证实pEGFP-N2-hVEGF121重组质粒构建成功。使用pEGFP-N2-hVEGF121重组质粒转染骨髓间充质干细胞后,荧光显微镜检测提示hVEGF121/EGFP蛋白在骨髓间充质干细胞中存在表达。在体外模拟心肌缺氧的环境下分别培养6,12,24h后,RT-PCR检测示转染重组质粒的骨髓间充质干细胞组各时间段缺氧后的血管内皮生长因子mRNA表达量均高于对照组(P<0.01),且不同时间段间差异无显著性意义(P>0.05);空白骨髓间充质干细胞的血管内皮生长因子mRNA表达量随缺氧时程的延长而增加。结论:①成功构建携带人VEGF121/EGFP融合蛋白表达基因的真核表达质粒,并在骨髓间充质干细胞中获得表达。②证实表达hVEGF121/EGFP融合蛋白的骨髓间充质干细胞能在24h以内的不同时长缺氧下,维持较空白骨髓间充质干细胞更强、更稳定的血管内皮生长因子表达。
OBJECTIVE: To construct eukaryotic plasmid of human vascular endothelial growth factor-121 carrying enhanced green fluorescent protein (EGFP) reporter gene and to detect the effect of hVEGF121 / EGFP fusion protein on the growth of vascular endothelial cells Factor level. Methods: The amplification primers carrying restriction enzyme sites were designed and the full-length sequence of human vascular endothelial growth factor-121 gene was amplified by PCR. The coding sequence of human VEGF gene was amplified by PCR and cloned into plasmid pEGFP-N2 In the cloning site, recombinant plasmid pEGFP-N2-hVEGF121 was constructed. The constructed recombinant plasmid was identified by restriction enzyme digestion, PCR and DNA sequencing. Bone marrow-derived mesenchymal stem cells were isolated and cultured by differential bone marrow adhesion and proliferation in vitro. The morphological and surface markers were used for cell identification. BMSCs cultured in vitro were transfected with recombinant plasmid pEGFP-N2-hVEGF121 by cationic liposome, and the expression of hVEGF121 / EGFP fusion protein was detected by fluorescence microscopy. Bone marrow-derived mesenchymal stem cells expressing hVEGF121 / EGFP fusion protein were cultured in hypoxic environment for 6, 12 and 24 hours respectively. Blank bone marrow-derived mesenchymal stem cells under the same hypoxia intervention were transfected with empty vector The bone marrow-derived mesenchymal stem cells containing pEGFP-N2 plasmid as a control, the expression of vascular endothelial growth factor mRNA was detected by RT-PCR. Results: The recombinant plasmid pEGFP-N2-hVEGF121 was successfully constructed by restriction enzyme digestion, PCR and DNA sequencing. After transfection of bone marrow mesenchymal stem cells with recombinant plasmid pEGFP-N2-hVEGF121, the expression of hVEGF121 / EGFP protein in bone marrow mesenchymal stem cells was detected by fluorescence microscopy. After cultured for 6, 12 and 24 hours respectively in vitro simulated myocardial hypoxia environment, RT-PCR results showed that the expression of vascular endothelial growth factor mRNA in hypoxia group was higher than that in the mesenchymal stem cells transfected with recombinant plasmid (P <0.01), and there was no significant difference between different time points (P> 0.05). The expression of vascular endothelial growth factor mRNA of blank BMSCs increased with the prolongation of hypoxia. Conclusion: ①Eukaryotic expression plasmid carrying human VEGF121 / EGFP fusion protein gene was successfully constructed and expressed in bone marrow mesenchymal stem cells. ② The bone marrow mesenchymal stem cells expressing hVEGF121 / EGFP fusion protein were confirmed to maintain stronger and more stable expression of vascular endothelial growth factor than blank bone marrow mesenchymal stem cells under hypoxia for 24 hours.