重组共表达质粒pIRES-hVEGF_(121)cDNA/hBMP-4转染骨髓间充质干细胞对其成骨分化的影响

Expression of recombinant coexpression plasmid of pIRES-hVEGF_(121)cDNA/hBMP-4 in bone marrow mesenchymal stem cells and its effects on osteogenic differentiation

背景:在许多情况下,人体组织修复是多因子共同协调作用的结果,因而共表达多基因载体的构建能够满足多因子基因治疗的需要,是近年来基因治疗的新思路。目的:观察重组pIRES-hVEGF121cDNA/hBMP-4共表达质粒转染大鼠骨髓间充质干细胞后的表达及其对大鼠骨髓间充质干细胞向成骨细胞分化的影响,为骨缺损的联合基因治疗奠定基础。方法:使用全骨髓培养法分离、培养大鼠骨髓间充质干细胞;流式细胞仪检测骨髓间充质干细胞表面标志抗原的表达;重组pIRES-hVEGF121cDNA/hBMP-4共表达质粒经酶切鉴定后,在脂质体介导下将其转入大鼠骨髓间充质干细胞,提取总RNA和总蛋白,进行RT-PCR和Western blot检测;茜素红染色检测转染后大鼠骨髓间充质干细胞的成骨分化能力。结果与结论:流式细胞仪检测显示CD90表达强阳性,CD45表达阴性;重组pIRES-hVEGF121cDNA/hBMP-4共表达质粒成功转染至大鼠骨髓间充质干细胞中,并获得有效共表达;转染后第14天,茜素红染色阳性,能够诱导大鼠骨髓间充质干细胞向成骨细胞分化,为骨缺损的联合基因治疗奠定了基础。

BACKGROUND：Under some conditions,human body tissue repair is the result of common coordination of many factors.Thus,the construction of coexpression of many genetic carriers can meet the requirement of gene therapy of multiple factors,and is the new idea of recent gene therapy.OBJECTIVE：To observe the expression of recombinant coexpression plasmid of pIRES-hVEGF121cDNA/hBMP-4 in transfected rat bone marrow mesenchymal stem cells（BMSCs） and its effects on the differentiation of rat BMSCs into osteoblasts,and provide a basis for combination gene therapy of bone defects.METHODS：BMSCs were isolated and cultivated from the bone marrow of rats by the whole bone marrow culture method.BMSCs were analyzed by the flow cytometry to detect the surface antigens.Recombinant coexpression plasmids of pIRES-hVEGF121cDNA/hBMP-4 were confirmed by restriction enzymolysis and then transfected rat BMSCs by lipofectamine.The total RNA and total proteins extracted from the resulting combination were detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot assay.Alizarin red staining was utilized to detect the differentiation of rat BMSCs after transfection.RESULTS AND CONCLUSION：Flow cytometry analysis showed that BMSCs were strong positive for CD90,but did not express CD45.Recombinant coexpression plasmid of pIRES-hVEGF121cDNA/hBMP-4 was transfected successfully and the plasmid could coexpress hVEGF121cDNA and hBMP-4 efficiently in rat BMSCs.Alizarin red staining presented strong positive for 14 days after transfection.Human VEGF121 and BMP-4 gene can induce rat BMSCs to differentiate into osteoblasts,which provides a basis for combined gene therapy of bone defects.