神经生长因子增强骨形态蛋白-9诱导鼠胚胎成纤维细胞成骨分化的作用  

Nerve growth factor potentiates bone morphogenetic protein-9-induced osteogenic differentiation in mouse embryonic fibroblasts

作　　者：皮昌军 邹翔[1] 张然熙[1] 吴念[1] 何百成[2] 邓忠良[1] 陈亮[1] 

机构地区：[1]重庆医科大学附属第二医院骨科,400010 [2]重庆医科大学药学院药理肿瘤实验室

出　　处：《中华创伤杂志》2016年第2期171-175,共5页Chinese Journal of Trauma

基　　金：国家自然科学基金项目（31000434）;重庆市自然科学基金项目（cstc2011jjzt0063）

摘　　要：目的研究神经生长因子（NGF）对骨形态发生蛋白-9（BMP-9）诱导鼠胚胎成纤维细胞（MEFs）成骨分化的影响。方法构建重组腺病毒转染MEFs，分为NGF组、BMP-9组及NGF联合BMP-9组（联合组），以绿色荧光蛋白（GFP）处理为对照组。采用细胞化学染色以及读数检测各组第3，5天碱性磷酸酶（ATP）活性变化，RT—PCR检测第9天骨桥素（OPN）mRNA表达水平，Westernblot、免疫细胞化学检测各组第9天OPN蛋白表达水平；茜素红染色检测各组第14天钙盐沉积水平。结果BMP-9组ALP活性升高，但NGF组ALP活性无明显变化，联合组ALP活性明显强于NGF组或BMP-9组。对照组、BMP-9组、NGF组及联合组OPNmRNA表达水平分别为0．92±0．03，1．28±0．04，0．94±0．03，1．62±0．04（F=214．60，P〈0．01）．与BMP-9组比较，联合组OPNmRNA相对表达水平明显升高（t=10．569，P〈0．05）。Westernblot检测结果显示，对照组、BMP-9组、NGF组及联合组OPN蛋白相对表达水平分别0．604-0．05，0．844-0．03，0．55±0．05，1．274-0．05（F：162．50，P〈0．01），与BMP-9组比较，联合组OPN蛋白相对表达水平明显升高（t=11．778，P〈0．05）。免疫组织化学染色结果显示，对照组、BMP-9组、NGF组及联合组OPN蛋白相对表达水平相对密度分别为3．63±0．17，6．27±0．30，3．86±0．18，10．16±0．18（F=602．60，P〈0．01），与BMP-9组比较，联合组OPN蛋白相对表达水平明显升高（t=22．280，P〈0．05）。联合组钙盐沉积水平明显高于BMP-9组或NGF组。结论NGF能协同增强BMP-9诱导MEFs的成骨分化作用。Objective To investigate the effect of nerve growth factor （NGF） on osteogenesis induced by bone morphogenetic protein-9 （BMP-9） in mouse embryonic fibroblasts （MEFs）. Methods MEFs were respectively transfected with adenovirus-mediated NGF （NGF group）, BMP-9 （BMP-9 group） and NGF ＋ BMP-9 （combined group） and green fluorescence protein （GFP） （control group）. Cytochemical staining was used to test the activity of alkaline phosphatase （ALP） 3 d and 5 d after treatment. Level of osteopontin （OPN） mRNA was detected by RT-PCR 9 d after treatment. Level of OPN protein was assayed by Western blot and immunocytochemistry 9 d after treatment. Mineralization was detected by Alizarin red staining 14 d after treatment. Results ALP activity in MEFs was elevated in BMP-9 group rather than in NGF group, but a significant increase in ALP activity was noted in combined group. In control group, BMP-9 group, NGF group and combined group, level of OPN mRNA was 0. 92 ±0. 03, 1.28 ±0.04, 0.94± 0.03 and 1.62 ± 0.04 respectively （ F = 214.60, P 〈 0.01 ） ; level of OPN protein was 0. 60± 0.05, 0.84 ±0. 03, 0. 53±0.05 and 1.27±0. 05 respectively （ F = 162.5, P 〈 0. 01 ）. In comparison, OPN mRNA and protein were significantly up-regulated in combined group than in BMP-9 group （t = 10.569 and 11. 778,P 〈 O. 05）. In control group, BMP-9 group, NGF group and combined group, relative density of OPN protein was 3.63 ±0.17, 6.27 ±0.30, 3.86 ±0. 18 and 10.16±0. 18 respectively （ F = 602.6, P 〈 0. 01 ）, with a significant higher level in combined group than in BMP-9 group （t =22. 280,P 〈0.05）. Level of mineralization was significantly higher in combined group than in BMP-9 or NGF group. Conclusion NGF can potentiate the osteogenesis induced by BMP-9 in MEFs.

关 键 词：神经生长因子 骨形态发生蛋白类 骨生成 间充质干细胞 

分 类 号：R68[医药卫生—骨科学]