沉默P75NTR联合hNGF过表达对BMSCs的影响及治疗骨折不愈合的实验研究

Nonunion is the difficulty of orthopedic treatment. Reduced blood supply and persistent fibrin deposition of fracture site can lead to nonunion. Some studies demonstrated that P75NTR is one of the receptors of NGF. Upregulated expression of P75NTR can inhibit angiogenesis and fibrin degradation, but enough expression of NGF can turn off this passage and rescue the damaged cells. P75NTR in nonunion studies have not been reported. Our previous studies shown that NGF promoted fracture healing and expression of P75NTR in nonunion tissue was significantly increased. Therefore, we hypothesize that silence of the expression of P75NTR and overexpression of NGF can promote the degradation of fibrin and angiogenesis, thereby treating nonunion. To verification this hypothesis, we treat radius nonunion model of rabbit through transplanting expression vector of P75NTR shRNA and pcDNA3-hNGF co-transfected BMSCs and observe the curative effects. The study is about to reveal the mechanism of nonunion and provide new ideas for the treatment of nonunion from the new perspective of P75NTR.

骨折不愈合是骨科治疗的难点。骨折部位的血液供应减少和纤维蛋白的持续沉积可导致骨折不愈合。研究表明P75神经营养因子受体（P75 Neurotrophin receptor，P75NTR）是神经生长因子（Nerve growth factor，NGF）的受体之一。P75NTR表达升高抑制血管再生和纤维蛋白降解，而过量的NGF关闭这一通道，挽救受损的细胞。P75NTR在骨折不愈合研究中未见报道。我们前期研究表明NGF促进骨折愈合，在骨折不愈合组织中P75NTR的表达明显增高。因此我们假设沉默P75NTR并联合hNGF过表达促进血管生成和纤维蛋白降解，能治疗骨折不愈合。为了验证这一假说，我们通过P75NTR shRNA和pcDNA3-hNGF表达载体共转染骨髓基质干细胞移植治疗兔桡骨骨折不愈合，观察其疗效。本研究将从P75NTR这个新视点阐明骨折不愈合的发生机制，为骨折不愈合治疗提供新思路。

骨折不愈合是骨科治疗的难点。研究表明P75神经营养因子受体（P75NTR）是神经生长因子（NGF）的受体之一。已证实P75NTR广泛存在于组织及细胞中，并对细胞分化、增殖以及凋亡有影响。P75NTR表达升高抑制血管再生和纤维蛋白降解，而过量的NGF关闭这一通道，挽救受损的细胞。P75NTR在骨折不愈合研究中未见报道。课题组前期实验发现在骨折不愈合的组织中P75NTR含量增高，呈现时间-量效关系。课题组通过全骨髓贴壁法培养的骨髓间充质干细胞（BMSCs），提取的细胞生长曲线符合正常细胞生长特征且生长活跃。前期构建大鼠P75NTR、NGF过表达质粒及慢病毒载体，双基因慢病毒共转染BMSCs后目的基因持续表达，传代后基因稳定。课题组进行单基因体外成骨实验,将过表达P75NTR转染大鼠BMSCs，成骨诱导分化后结果表明：P75NTR对调控BMSCs的增殖及成骨分化减弱。在前期实验的基础上，用慢病毒沉默P75NTR和过表达NGF共转染至大鼠BMSCs，成骨诱导分化后结果显示：共转染的BMSCs生长、分布良好；P75NTR蛋白表达明显下调，NGF蛋白表达明显增加；BMSCs增殖及成骨分化能力增强。在皮下异位成骨实验中，沉默P75NTR联合NGF过表达双基因共转染BMSCs 复合单纯脱钙骨基质（DBM）构建组织工程骨，其具有良好的异位成骨能力，细胞成活率高，成骨分化能力强。促进成骨的效果对比单基因更为明显。在大鼠骨折不愈合实验中，将沉默P75NTR联合NGF过表达双基因共转染BMSCs复合DBM构建组织工程骨移植到大鼠股骨外上髁骨缺损模型，通过对血管、纤维蛋白、骨痂的组织学检测，以及uCT的检查，结果表明沉默P75NTR和过表达NGF共转染BMSCs移植治疗骨折不愈合效果优良。本研究从P75NTR这个新视点阐明骨折不愈合的发生机制，提供新的治疗思路。为骨折不愈合的临床治疗奠定理论基础。