VEGF165过表达对BMP2成骨细胞分化影响的实验研究  

Effect of overexpression of vascular endothelial growth factor 165 on the osteogenic potential of bone morphogenetic protein

作　　者：张聪[1,2] 刘洪美[2] 李庆伟[2] 陈国武[2] 梁啸[2] 孟纯阳[2] 

机构地区：[1]新乡医学院研究生院,河南新乡453000 [2]济宁医学院附属医院脊柱外科,山东济宁272000

出　　处：《中国矫形外科杂志》2015年第8期727-732,共6页Orthopedic Journal of China

基　　金：山东省自然科学基金项目(编号:ZR2010HM090);山东省高等学校科技计划项目(编号:J14LL04)

摘　　要：[目的]探讨腺病毒转染血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)对骨形态发生蛋白(bone morphogenetic protein 2,BMP2)促成骨细胞分化的抑制性作用研究。[方法]采用密度梯度离心法分离兔骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs),取第3代BMSCs进行细胞表型鉴定并作为细胞实验对象,使用Ad-BMP2和Ad-BMP2-VEGF165载体体外转染BMSCs,倒置荧光显微镜下观察GFP表达变化;同时ELISA和Western blot检测BMP2和VEGF165蛋白的表达。然后,应用成骨细胞诱导培养液定向诱导BMSCs向成骨细胞分化。实验分3组:Ad-BMP2-VEGF165转染BMSCs组(Ad-BMP2-VEGF165组),AdBMP2转染BMSCs组(Ad-BMP2组),BMSCs组(对照组)。分别在成骨细胞诱导培养后7、14、21 d,通过Real-time PCR分析ALP和OC mRNA相对表达水平和碱性磷酸酶(alkaline phosphatase,ALP)活性测定、骨钙素(Osteocalcin,OC)免疫组化染色来评价各组BMSCs成骨分化潜能的影响。[结果]第3代细胞表面高表达CD29(99.82%)、CD44(94.14%),低表达CD14(3.11%)、CD34(0.34%);腺病毒转染后第5 h BMP2和h VEGF165蛋白水平表达最高;成骨细胞诱导培养14和21 d后,Ad-BMP2组成骨相关基因表达、OC免疫组化和ALP活性表达最高,结果与其他两组相比较差异具有统计学意义(P<0.05);而对照组表达最弱,与Ad-BMP2-VEGF165组比较差异具有统计学意义(P<0.05)。[结论]腺病毒载体Ad-BMP2与Ad-BMP2-VEGF165转染BMSCs后,均具有明显促进BMSCs体外诱导成骨细胞分化潜能,但Ad-BMP2诱导作用更为显著,同时说明VEGF165可能对BMSCs成骨细胞分化起抑制作用。[Objective]To determine the influence of adenoviral transfection with genes for bone morphogenetic protein 2（BMP2） and vascular endothelial growth factor 165（VEGF165） on the potential of bone marrow stem cells（BMSCs） to differentiate into osteoblasts.[Method]Density gradient centrifugation was used to separate mesenchymal stem cells from bone marrow.These BMSCs were cultured for three generations,and third-generation cells were phenotypically identified and used for the experiments.We observed differences in the expression of green fluorescent protein（GFP） with an inverted fluorescence microscope in BMSCs adenovirally transfected with genes for BMP2 or both BMP2 and VEGF165 in vitro; these genes were linked to the gene for GFP.Enzyme-linked immunosorbent assay and western blot were also performed to detect BMP2 and VEGF165 protein expression.The BMSCs were cultured in osteoblast-induction medium and divided into 3 groups： the BMP2-VEGF165 group,the BMP2 group,and the control group.At 7,14,and 21 days after the initiation of BMSC culture in osteoblast-induction medium,the potential of the BMSCs to differentiate into osteoblasts was analyzed using real-time polymerase chain reaction analysis of the transcription levels of alkaline phosphatase（ALP） and osteocalcin（OC） mRNA,an ALP activity assay,and immunohistochemical staining for OC.[Result]Flow-cytometric detection of cell surface antigens after the third passage showed thatthe BMSCs were under-expressing CD29（99.82%）,CD44（94.14%）,CD14（3.11%）,and CD34（0.34%）.Levels of BMP2 and VEGF165 protein peaked at 5 days after transfection.ALP and OC gene transcription,ALP activity,and OC distribution on immunohistochemical staining were significantly higher in the BMP2 group than in the other groups at 14 and 21 days of culture（P〈0.05）.ALP and OC gene transcription,ALP activity,and OC distribution were significantlylower in the control group than in the BMP2-VEGF165 group（P〈0.05）.[Conclusion]Adenoviral transfection with e

关 键 词：骨形态发生蛋白2 血管内皮舒张因子165 骨髓间充质干细胞 成骨细胞 腺病毒载体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]