Yang Jiajia,Liu Ping,Xu Qian,et al.Effects of adenovirusmediated KLF4 overexpression on osteogenic differentiation of osteoblasts in vitro[J].J Third Mil Med Univ,2017,39(07):628-634.

过表达KLF4对小鼠成骨细胞成骨分化的影响(/HTML )




Effects of adenovirusmediated KLF4 overexpression on osteogenic differentiation of osteoblasts in vitro
Yang Jiajia Liu Ping Xu Qian Liu Zhihua Li Xiaoming Wang Chao Zhang Bo Deng Manjing

Department of Stomatology, State Key Laboratory of Trauma, Burns and Combined Injury, Department 4, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042; Department of Critical Medicine, Key Laboratory of Child Development and Disorders of Ministry of Education, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China

Krüppel like factor 4 adenovirus vector osteoblasts mice
R322.71; R329.28; R394.2

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/mL)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 determine the effect of recombinant adenovirus-mediated Krüppel like factor 4 (KLF4) transfection on the osteogenic differentiation of osteoblasts in vitro. Methods     Primary mouse osteoblasts were isolated from newborn C57 mice by enzymatic digestion and then cultured and identified. Alizarin Red S staining was carried out after osteogenic induction for 21 d. The recombinant adenoviral vector carrying the KLF4 gene (Ad-KLF4) was constructed by pHBAd-EF1-MCS-GFP system. The transfection efficiency was detected by measuring the different multiplicity of infection (MOI) with flow cytometry. Osteoblasts were randomly assigned into control group (non-transfection), GFP group (Ad-GFP transfection) and KLF4 group (Ad-KLF4 transfection). The expression of KLF4 protein was detected by Western blotting. MTT assay was used to analyze cell proliferation. Realtime PCR was used to detect the expression of osteogenic genes alkaline phosphatase (ALP), bone sialoprotein (BSP) and runt-related transcription factor2 (RUNX2).  Results     Primary osteoblasts were triangle and polygon in shape under inverted phase contrast microscope. A large number of red calcified nodules were found after Alizarin Red S staining. Restriction enzyme digestion and gene sequencing confirmed Ad-KLF4 was constructed successfully. At the optimal multiplicity of infection (MOI, 100 PFU/mL), Ad-KLF4 transfection resulted in significant increases in protein and mRNA levels of KLF4 (P<0.01), obviously increase in osteoblasts proliferation (P<0.05, P<0.01), and decreases in the mRNA levels of osteogenic genes ALP, BSP and RUNX2 (P<0.05, P<0.01), when compared with the control group and GFP group. Conclusion      Adenovirus-mediated KLF4 over-expression can promote osteoblast proliferation and inhibit osteoblast osteogenic differentiation, and thus inhibit bone formation.


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更新日期/Last Update: 2017-04-07