失神经支配在大鼠骨折愈合过程中降钙素基因相关肽对骨保护素／破骨细胞分化因子影响的实验研究

Denervation affects the influence of CGRP on OPG/ NKL system in rite fracture healing process： an experimental study in rats

目的通过建立选择性切断大鼠感觉／运动神经联合胫骨骨折的动物模型，研究感觉／运动神经损伤后对骨折愈合的影响，并检测骨折愈合过程中降钙素基因相关肽（calcitonin gene related pepfide，cGRP）对骨保护素（osteoprotegerin，OPG）／砬芝骨细胞分化因子（receptor activator nuclear factor kappa B ligand，RANKL）体系的影响，初步探讨周围神经调节骨折愈合的机制。方法取60只Wistar大鼠随机分为3组：前根（运动神经）切断＋胫骨骨折组（anterior rhizotomy group，ART组）；后根（感觉神经）切断＋胫骨骨折组（posterior rhizotomyg roup，PRT组）；单纯胫骨骨折组（sham operatedgroup，SO组）。分别于骨折术后7、14、21、28d处死大鼠，在骨折处上、下5mm部位取骨痂标本。免疫组织化学检测CGRP、OPG、RANKL的表达；采用软件Image-proplus 6．0进行图片分析；采用SPSS16．0进行统计学分析。结果CGRP在SO组各时间点均呈强阳性表达，术后14d和21d，SO组、ART组和PRT组比较差异均有统计学意义（P〈0．05）。OPG在SO组术后7d呈强阳性表达，以后逐渐下降但保持较高水平；术后14d，PRT组和SO组、ART组比较差异有统计学意义（P〈0．05）；术后21d，SO组和PRT组、ART组比较差异有统计学意义（P〈0．05）。RANKL在SO组21d为表达高峰，以后逐渐下降；术后14d，PRT组和ART组、S0组比较差异有统计学意义（P〈0．05）；术后21d，SO组和ART组、PRT组比较差异有统计学意义（P〈0．05）。结论在骨折愈合过程中，感觉神经纤维对骨折愈合的影响较运动神经纤维显著。CGRP能够调节OPG／RANKL的表达量的比值，从而影响骨折的愈合过程。失神经支配（尤其是感觉神经）导致CGRP对OPG／RANKL表达的调节作用降低，这不利于骨折的愈合。完整的神经支配是正常骨折愈合的必要条件之一。

Objective To study the influence of sensory/motor nerve injury on fracture healing and the irfluaence of ealeitonin gene teiated peptide （CGRP） on osteoprotegcrln （OPG）/reeeptor activator nuclear factor kappa B ligand （RANKL） system during the fractttre healing process through the establishment of selective transection of sensory/motor nerve in rats with tibia fracture as an animal model and to elucidate the peripheral nerve injury regulation mechanism during fracture healing. Methods Sixty Wistar rats were divided into 3 groups randomly： motor nerve （ventral root） transection ＋ tibia fracture group （anterior rhizotomy group, ART group） ; sensory nerve （dorsal root） transection ＋ tibia fracture group （posterior rhizotomy group, PRT group） ; simple tibia fracture group （sham operated group, SO group）. The transverse tibia fracture was treated by intrameduUary nailing. The rats were sacrificed at postoperative day 7, 14, 21 and 28 respecfivdy to harvest the bony calluses 5 mm proximal and 5 mm distal to the fracture site. The expression of CGRP, OPG and RANKL was detected by immtmohistochemistry and analyzed by Image-proplus 6.0 software. The comparison among the groups was analyzed using SPSS 16.0. Results Strong positive expression of CGRP was seen at each time point in SO group. At postoperative day 14 and 21, the differences of CGBP expression between SO group, ART group and PRT group were significant （ P 〈 0.05）. OPG expression was strongest at postoperative day 7 in SO group, while remaining high even with gradual decrease. The differences in OPG expression were significant （ P 〈0.05） at postoperative day 14 between PRT group and SO group and between PRT group and ART group, while that difference was seen between SO group and PRT group and between SO group and ART group at postoperative day 21. RANKL expression in the SO group peaked at postoperative day 21, and later gradually decreased. At postoperative day 14 the differences between PRT group and ART group and between PRT group and SO group were significant （ P 〈 0.05）, while at postoperative day 21 the differences between SO group and ART group and between SO group and PRT group were significant （ P 〈 0.05）. Conclusion During the fracture healing process, the influence of sensory nerve fibers was significantly higher than that of the motor nerve fibers. CGRP can regulate the OFG/RANKL expression ratio, thus affecting the healing process. Denervafion （especially the sensory nerves） causes reduced CGRP regulation on OPG/RANKL expression, which is not favorable for fracture healing. Complete irmervation is a necessary condition for the normal healing of fractures.