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目的探讨重组腺病毒介导的肠富集Krüppel样因子(Krüppel like factor 4,KLF4)基因转染后对小鼠成骨细胞成骨分化的影响。方法采用酶消化法分离培养小鼠原代成骨细胞,经成骨诱导培养21 d后行茜素红染色。利用pHBAd-EF1-MCS-GFP系统构建携有KLF4的重组腺病毒载体(Ad-KLF4)。不同感染复数(multiplicity of infection,MOI)Ad-KLF4转染成骨细胞后流式细胞仪检测转染效率。将成骨细胞随机分为对照组(未转染组)、KLF4组(Ad-KLF4转染)和GFP组(Ad-GFP转染)。Western blot检测小鼠成骨细胞中KLF4蛋白的表达;MTT检测细胞增殖;Real-time PCR检测成骨相关基因碱性磷酸酶(alkaline phosphatase,ALP)、骨涎蛋白(bone sialoprotein,BSP)、核心蛋白结合因子2(runt-related transcription factor 2,RUNX2)的表达。结果倒置显微镜下可见成骨细胞呈多边形、立方形等,茜素红染色示大量红色钙化结节的形成。经酶切鉴定、基因测序成功构建携带KLF4基因的重组腺病毒载体(Ad-KLF4)。成骨细胞经最佳MOI(100 PFU/m L)处理后,与对照组及GFP组比较,KLF4组可检测到KLF4蛋白及mRNA水平显著上升(P<0.01);成骨细胞增殖明显上升(P<0.05,P<0.01);成骨分化相关基因ALP、RUNX2、BSP的表达显著下降(P<0.05,P<0.01)。结论腺病毒介导的KLF4基因转染成骨细胞后可促进成骨细胞增殖,抑制其成骨分化,从而抑制骨形成。
Objective To investigate the effect of recombinant adenovirus-mediated Krüppel-like factor 4 (KLF4) gene transfection on the osteoblastic differentiation of mouse osteoblasts. Methods Mouse primary osteoblasts were isolated and cultured by enzymatic digestion method and stained with alizarin red after 21 days of osteogenic induction. The recombinant adenoviral vector carrying KLF4 (Ad-KLF4) was constructed using the pHBAd-EF1-MCS-GFP system. Transfection efficiency was determined by flow cytometry after transfecting osteoblasts with different multiplicity of infection (MOI) Ad-KLF4. Osteoblasts were randomly divided into control group (untransfected group), KLF4 group (Ad-KLF4 transfected) and GFP group (Ad-GFP transfected). Western blot was used to detect the expression of KLF4 protein in mouse osteoblasts; MTT was used to detect cell proliferation; alkaline phosphatase (ALP), bone sialoprotein (BSP), core Expression of runt-related transcription factor 2 (RUNX2). Results The inverted microscope showed that the osteoblasts were polygonal and cuboid. Alizarin red staining showed the formation of a large number of red calcified nodules. After restriction enzyme digestion, the recombinant adenovirus carrying KLF4 gene (Ad-KLF4) was constructed successfully by gene sequencing. Compared with control group and GFP group, the level of KLF4 protein and mRNA in KLF4 group increased significantly (P <0.01) after osteoblasts were treated with optimal MOI (100 PFU / m L); the proliferation of osteoblasts increased significantly P <0.05, P <0.01). The expressions of ALP, RUNX2 and BSP in osteoblast were significantly decreased (P <0.05, P <0.01). Conclusion Adenovirus-mediated KLF4 gene transfection of osteoblasts can promote osteoblast proliferation, inhibit osteogenic differentiation, thereby inhibiting bone formation.