pIRES2-EGFP-hBMP2载体在人骨髓基质细胞中的表达及意义

Expression and significance of vector pIRES2-EGFP-hBMP2 in HFCL cells

目的 :研究人骨形成蛋白hBMP2真核表达载体pIRES2 -EGFP -hBMP2在人骨髓基质细胞系HFCL中的表达及意义。方法 :利用DNA重组技术 ,将hBMP2片段克隆到真核表达载体pIRES2 -EGFP中 ;EcoRΙ/BamHΙ双酶切鉴定 ;脂质体介导的方法转染HFCL细胞 ;荧光显微镜下观察转染细胞 ;免疫组化染色检测hBMP2基因的表达。结果 :经酶切鉴定显示 ,成功地构建了重组载体pIRES2 -EGFP -hBMP2。转染HFCL 4 8h后在荧光显微镜下观察到转染的HFCL细胞发绿色荧光。免疫组化染色显示转染hBMP2基因的HFCL细胞浆内有细的棕色颗粒 ,而转染空载体的HFCL细胞免疫组化呈阴性。结论 :所构建的pIRES2-EGFP -hBMP2真核表达载体在HFCL细胞中有良好的表达 ,为进一步研究转基因骨髓基质细胞对放射和化疗所致的造血和骨微环境破坏的改善奠定了基础。

Objective: To construct an eukaryotic expression vector pIRES2-EGFP- hBMP2 and to observe its expression in human bone marrow stromal cell line HFCL. [WT5HZ]Methods: The hBMP2 cDNA was inserted into an effective eukaryotic expression vector-pIRES2-EGFP. Restrictive enzyme EcoRΙ/BamHΙ digestion analysis was performed. Liposome-mediated gene transfer method was used to transfect HFCL cells. The fluorescence microscope and immunohistochemical staining was employed to observe the transfected cells and the expression of hBMP2 gene, respectively. Results: Restrictive enzyme EcoRΙ/BamHΙ digestion analysis showed that recombinant expression vector pIRES2-EGFP-hBMP2 had been constructed successfully. The recombinant vector pIRES2-EGFP-hBMP2 was transfected into HFCL cells. The transfected HFCL cells displaying green fluorescence were observed under fluorescence microscope. The immunohistochemical staining showed that positive reactant of brown-yellow could be seen in the cytoplasm of HFCL cells transfected by pIRES2-EGFP-hBMP2,while no hBMP2 expression was observed in HFCL cells transfected by empty vector. Conclusion: The results suggested that the recombinant expression vector pIRES2-EGFP-hBMP2 is proved to be expressed in HFCL cells successfully. The present study lay the foundation for the further test.