过表达P75神经生长因子受体负向调控骨髓间充质干细胞的成骨分化

文题释义： P75神经生长因子受体：即P75^NTR，神经生长因子的受体之一，以三聚体和单体的形式镶嵌于细胞表面，主要包含胞外区、跨膜区以及胞内区3个结构区域。低聚状态决定了其对细胞的主要调节功能，与配体结合后通过改变构象来进行信号传导，进而发挥调节细胞的作用。 成骨分化：多向分化干细胞向单能成骨细胞转变，并最终向骨组织发展的一系列生理过程称为成骨分化，对调节骨形成和骨再生具有重要意义，其成骨分化过程中，碱性磷酸酶的活力逐渐增加，细胞内或者胞外基质的钙等矿物质沉积，形成钙结节，骨钙素及Ⅰ型胶原蛋白表达增加。背景：P75^NTR广泛表达于神经组织及细胞中，并发挥着促进或抑制分化的双重作用；同时在骨折不愈合局部组织中也发现了P75^NTR存在着过表达，因此研究P75^NTR对骨髓间充质干细胞成骨分化的作用具有重要的意义，为临床上提高骨折不愈合修复的疗效提供重要靶点。 目的：观察P75^NTR过表达对大鼠骨髓间充质干细胞体外诱导成骨分化的影响。 方法：选取SD大鼠双侧股骨，用全骨髓分离贴壁法提取大鼠骨髓间充质干细胞并进行体外传代培养；构建表达EGFP的P75^NTR过表达质粒GV358-P75^NTR，并用空慢病毒载体包装收集P75^NTR过表达慢病毒载体；选取体外培养至原代10 d的大鼠骨髓间充质干细胞，消化后种板，加入P75^NTR过表达慢病毒及相关感染试剂进行感染实验，感染7 d后用荧光显微镜观察绿色荧光蛋白的表达，Western blot检测P75^NTR蛋白的过表达情况；感染后用常规培养基培养7 d，更换成骨诱导分化培养基培养，诱导培养后第7，10，14天用酶标法定量检测碱性磷酸酶活力，诱导培养后第7，14天用茜素红染色观察矿化结节形成情况。 结果与结论：①P75^NTR过表达慢病毒感染骨髓间充质干细胞后能表达出绿色荧光蛋白(感染效率约90%)，且P75^NTR蛋白表达量明显增多，与阴性病毒对照组比较差异有显著性意义(P < 0.05)，过表达P75^NTR细胞模型构建成功；②与阴性病毒对照组及未转染组相比，P75^NTR过表达组细胞诱导分化后相应时间点的碱性磷酸酶活力明显降低，矿化结节形成减少，细胞聚集分布减弱，差异有显著性意义(P < 0.05)；③结果表明，P75^NTR过表达负向调控了大鼠骨髓间充质干细胞的成骨分化。局部组织中P75^NTR过表达抑制周围骨髓间充质干细胞成骨分化可能是骨缺损或骨折不愈合的重要因素。 ORCID: 0000-0001-6923-3177(朱伦井) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Overexpression of P75 neurotrophin receptor negatively regulates osteogenic differentiation of bone marrow mesenchymal stem cells in vitro

BACKGROUND:P75 neurotrophin receptor(P75^NTR)is widely expressed in nerve tissues and cells,and plays a dual role in promoting or inhibiting differentiation.P75NTR is also overexpressed in local tissues with fracture nonunion.Therefore,P75NTR is studied for the osteogenic differentiation of bone marrow mesenchymal stem cells,which is of great significance to provide important targets for the clinical treatment of fracture nonunion.OBJECTIVE:To elucidate the effect of P75^NTR overexpression on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.METHODS:The bilateral femurs of Sprague-Dawley rats were selected,and the rat bone marrow mesenchymal stem cells were extracted by whole bone marrow separation and adherence method and subcultured in vitro.The P75NTR overexpression plasmid GV358-P75^NTR expressing enhanced green fluorescent protein was constructed,and the P75^NTR overexpression lentiviral vector was collected by empty lentiviral vector packaging.Rat bone marrow mesenchymal stem cells primary cultured in vitro for 10 days were selected,and seeded into culture plates after digestion.P75NTR overexpression lentivirus and related infection reagents were added for subsequent infection experiments.After 7 days of infection,the expression of green fluorescent protein was observed by fluorescence microscope and overexpression of P75^NTR protein was detected by western blot.Transfected cells were cultured for 7 days in a conventional medium,followed by culture in the osteogenic differentiation medium.Alkaline phosphatase activity was quantified by enzyme linked immunosorbent assay on the 7^th,10^th,and 14^th days after osteogenic induction.Formation of mineralized nodules was observed by alizarin red staining on the 7^th and 14^th days after osteogenic induction.RESULTS AND CONCLUSION:P75NTR overexpression lentiviral vector-infected bone marrow mesenchymal stem cells expressed green fluorescent protein(infection efficiency was about 90%),and the expression of P75^NTR protein was significantly increased,which was significantly different from that in the negative control group(P<0.05).Cell model of P75^NTR overexpression was successfully constructed.Compared with the negative control and blank control groups,the alkaline phosphatase activity of the P75NTR overexpression group was significantly decreased at the corresponding time point after osteogenic induction,the number of mineralized nodules was significantly reduced,and cell aggregation and distribution were significantly weakened(P<0.05).To conclude,P75^NTR overexpression negatively regulates the osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured in vitro.Overexpression of P75^NTR in local tissues inhibits the osteogenic differentiation of surrounding bone marrow mesenchymal stem cells,which may be an important factor for bone defects or fracture nonunion.