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目的构建人肝细胞生长因子(Human hepatocyte growth factor,hHGF)真核表达质粒,并检测其在人骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)中的表达及其对细胞生长的影响。方法采用密度梯度法从人骨髓中分离BMSCs,流式细胞术检测细胞表型,成脂及成骨诱导其分化。PCR扩增hHGF基因,定向克隆至pEGFP-N1载体中,构建重组表达质粒pEGFP-N1-hHGF,通过电穿孔法转染BMSCs,荧光显微镜下观察增强型绿色荧光蛋白的表达,RT-PCR及Western blot法检测hHGF基因mRNA的转录及蛋白的表达,MTT法检测hHGF对BMSCs增殖活力的影响。结果 BMSCs高表达CD29和CD44,不表达CD34和CD45;BMSCs在体外有向成脂及成骨细胞诱导分化的能力。酶切及基因测序证实,重组表达质粒pEGFP-N1-hHGF构建正确;转染后48 h观察到转染细胞中有绿色荧光蛋白表达;RT-PCR法和Western blot检测到hHGF基因在BMSCs中表达;转染hHGF基因的BMSCs增殖活力明显高于空白对照组和空载体转染组(P<0.05)。结论成功构建了hHGF基因真核表达质粒,转染人BMSCs后获得表达,表达的hHGF可促进BMSCs增殖。
Objective To construct eukaryotic expression plasmid of human hepatocyte growth factor (hHGF) and to detect its expression in human bone marrow mesenchymal stem cells (BMSCs) and its effect on cell growth. Methods BMSCs were isolated from human bone marrow by density gradient method. Cell phenotypes were detected by flow cytometry, and their differentiation was induced by adipogenesis and osteogenesis. The hHGF gene was amplified by PCR and cloned into pEGFP-N1 vector. The recombinant plasmid pEGFP-N1-hHGF was constructed and transfected into BMSCs by electroporation. The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescence microscope. blot assay hHGF gene mRNA transcription and protein expression, MTT assay of hHGF on BMSCs proliferation activity. Results BMSCs were highly expressed in CD29 and CD44 but not in CD34 and CD45. BMSCs could differentiate into adipocytes and osteoblasts in vitro. Enzyme digestion and gene sequencing confirmed that recombinant plasmid pEGFP-N1-hHGF was constructed correctly; expression of green fluorescent protein was observed in transfected cells 48 h after transfection; expression of hHGF gene in BMSCs was detected by RT-PCR and Western blot The proliferation activity of BMSCs transfected with hHGF gene was significantly higher than that of blank control group and empty vector transfected group (P <0.05). Conclusion The eukaryotic expression plasmid of hHGF gene was successfully constructed and transfected into human BMSCs. The expression of hHGF could promote the proliferation of BMSCs.