重组慢病毒转染兔骨髓间充质干细胞与脱钙骨基质构建转基因组织工程材料

Rabbit bone marrow mesenchymal stem cells transfected with recombinant lentivirus and decalcified bone matrix to construct transgenic tissue engineering materials

背景:缺损组织的修复重建受限于自体或异体可替代移植材料的来源问题而导致临床应用受限,转基因干细胞和组织工程材料研究开辟了新的治疗思路。目的:探究增强型绿色荧光蛋白重组慢病毒转染兔骨髓间充质干细胞在体外的生物学特性以及与脱钙骨基质体外构建转基因组织工程材料的生物矿化特性。方法:细胞贴壁及密度离心法获得兔骨髓间充质干细胞,增强型绿色荧光蛋白重组慢病毒以感染复数为100转染第5代兔骨髓间充质干细胞,体外观察转染细胞与未转染细胞的增殖能力、细胞表型、细胞周期以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达的差异;增强型绿色荧光蛋白重组慢病毒转染骨髓间充质干细胞与脱钙骨基质在体外构建转基因组织工程材料,对其进行扫描电镜观察及元素能谱分析。结果与结论:增强型绿色荧光蛋白重组慢病毒成功转染骨髓间充质干细胞后,在转染24,48 h细胞增殖较未转染细胞缓慢(P <0.05);在转染72 h后,细胞表型未发生变异,细胞周期、细胞增殖能力以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达量与未转染细胞无明显差异(P> 0.05);增强型绿色荧光蛋白标记的骨髓间充质干细胞在脱钙骨基质支架上有较好的生物相容性,根据荧光表达强度推测目的基因在2周左右发挥最大生物学功能,且出现了钙磷矿化物沉积,体现出优越的生物矿化特性。

BACKGROUND:The use of autologous or allogeneic transplantation materials for tissue repair and reconstruction is limited and carries limitations in clinical applications.Transgenic stem cells and tissue engineering materials offer new therapeutic possibilities.OBJECTIVE:To investigate the in vitro biological characteristics of enhanced green fluorescent protein recombinant lentivirus transfected into rabbit bone marrow mesenchymal stem cells and the biommineralization characteristics of transgenic tissue engineering materials constructed using decalcified bone matrix.METHODS:Rabbit bone marrow mesenchymal stem cells were obtained through adherent culture and density gradient centrifugation.The fifth-generation bone marrow mesenchymal stem cells were transfected with enhanced green fluorescent protein recombinant lentivirus at the optimal multiplicity of infection(MOI=100).The differences in cell proliferation capacity,cell phenotype,cell cycle,alkaline phosphatase expression,osteogenic transcription factor(Runx2)expression,and osteocalcin gene expression after osteogenesis were observed between the transfected cells and non-transfected cells in vitro.The micromorphology and elemental energy spectrum analysis of the transgenic tissue engineering materials constructed from cells and decalcified bone matrix scaffold were also observed.RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells transfected with enhanced green fluorescent protein recombinant lentivirus showed that cell proliferation at 24 and 48 hours after transfection was slower than that of untransfected cells(P<0.05).After 72 hours,the cell phenotype remained unchanged,and the expressions of alkaline phosphatase,Runx2,and osteocalcin after osteogenic induction were not significantly different from those of untransfected bone marrow mesenchymal stem cells(P>0.05).The transgenic tissue engineering materials constructed in vitro using enhanced green fluorescent protein recombinant lentivirus transfected bone marrow mesenchymal stem cells and decalcified bone matrix showed good biocompatibility on the decalcified bone matrix scaffold.Based on fluorescence expression intensity,it was estimated that the target gene would exert its maximum biological function in about 2 weeks,and calcium phosphate deposits would continue to appear,demonstrating superior biomineralization characteristics.