【作者】 徐澄；

【导师】 熊昌源；

【作者基本信息】 湖北中医药大学 ， 中医骨伤科学， 2014， 博士

【摘要】 研究背景骨折是临床上常见的疾病。随着国内外学者对骨折愈合认识的深入，已经开始从各个方面探讨促进骨折愈合的方法。我们在临床工作和国内外文献中发现当四肢骨折伴随脑损伤时,骨折处往往可见骨痂过度生长，骨折愈合加速。目前主要认为是因为中枢神经损伤后引起，但其具体理论依据仍不明确。骨折局部电针刺激作为一种传统促进骨折、骨不连的治疗手段，在临床上有广泛的应用。能否通过在头部持续性的电针刺激来促使骨折愈合呢？查阅文献，还未见此类研究与报道。研究目的建立SD大鼠胫骨骨折模型，从形态学、病理学、超微病理学、分子生物学四个层面观察电针“百会穴”对模型大鼠骨痂的动态变化情况，了解头部持续性的电针刺激对骨折愈合的影响。研究方法取128只健康雄性SD大鼠，随机分为：正常组32只（A组）、胫骨骨折模型组32只（B组）、胫骨骨折模型并头部电针“百会穴”组32只（C组）、中度脑外伤并胫骨骨折模型组32只（D组）。各组实验动物予以相应的干预后，分别于7d、14d、21d、28d分批处死。肉眼观察骨痂的形状、大小、颜色等形态学改变；拍片后，采用Perkins骨痂体积测量公式计算骨痂体积并行统计学比较；留取部分骨痂标本行HE切片观察病理学变化；留取第1周处死大鼠骨折部位软组织行超微病理学电镜观察；留取部分骨痂标本、脑组织标本，免疫组化法检测骨形态发生蛋白-2（bonemorphogenetic protein-2，BMP-2）、转化生长因子-β1（transforming growthfactor beta1，TGF-β1）表达，并在400倍光镜下观察，计算阳性表达细胞的积分光密度（integral optical density,IOD）值；同时应用蛋白质印迹技术(Western blotting)检测脑源性神经营养因子(brain derivedneurotrophic factor, BDNF)蛋白含量，并对数据进行统计学分析。研究结果1建立胫骨骨折、中度脑外伤动物模型，各组给予相应干预，实验结果显示D组骨痂体积比B组、C组大（P<0.05），C组骨痂体积比B组大（P<0.05）。2骨痂组织在400倍光镜下观察结果：B组为经典的骨折愈合表现；C组骨痂生长较B组明显增快；D组骨痂生长速度最快。3第1周骨痂组织在电镜下观察结果：可见成纤维细胞、成软骨细胞、间充质细胞、骨生成细胞及内皮细胞等。D组成纤维细胞及成软骨细胞较其他组更活跃。4骨痂标本BMP-2免疫组化IOD值统计检测结果：B组在第1周、第2周、第3周、第4周表达与A组有显著性差异；C组在第1周、第2周、第3周、第4周表达与A组有显著性差异，与B组比较在第1周、第2周有显著性差异；D组在第1周、第2周、第3周、第4周表达与A组有显著性差异，与B组、C组比较在第1周有显著性差异。5骨痂标本TGF-β1免疫组化IOD值统计检测结果：B组在第1周、第2周、第3周、第4周表达与A组有显著性差异；C组在第1周、第2周、第3周、第4周表达与A组有显著性差异，与B组比较在第2周、第3周、第4周有显著性差异；D组在第1周、第2周、第3周、第4周表达与A组、B组、C组均有显著性差异。6脑组织标本BMP-2免疫组化IOD值统计检测结果：A组、B组、C组之间未见显著性差异；D组在第1周、第2周与A组、B组、C组比较有显著性差异。7脑组织标本TGF-β1免疫组化IOD值统计检测结果：B组与A组比较无显著性差异；C组在第1周、第2周、第3周、第4周表达与A组有显著性差异，与B组比较在第2周、第3周、第4周有显著性差异；D组与A组、B组、C组比较在第1周、第2周、第3周、第4周均有显著性差异。8Western blotting检测骨痂BDNF蛋白含量IOD值统计结果：C组在第1周、第2周与B组比较有显著性差异；D组在第1周、第2周与B组比较有显著性差异。研究结论1持续性电针刺激大鼠“百会穴”可以促进胫骨骨折愈合，不仅可以使骨痂生成体积增大，而且还可以使骨痂生成时间提前。2持续性电针刺激大鼠“百会穴”可以使破骨细胞、成骨细胞、成纤维细胞、软骨细胞增多，进一步促进骨折愈合。3电针刺激“百会穴”可以在骨折修复的各个时期促进骨折愈合，结合免疫组化检验统计结果，早期骨痂内BMP-2含量增高，后期骨痂内TGF-β1含量增高可能是电针刺激“百会穴”引起骨折愈合加速的原因之一。4脑外伤可引起早期骨痂内BMP-2含量增高，后期TGF-β1含量增高；脑组织内早期BMP-2含量增高，后期TGF-β1含量增高，这与电针刺激“百会穴”后BMP-2、TGF-β1的变化类似。两者促进骨折愈合的机理可能有相似之处。5电针“百会穴”可以在前两周内明显提高BDNF在骨痂中的含量,从而加速骨折部位神经修复，这也许是电针促进骨折愈合的原因之一。更多还原

【Abstract】 BackgroundFracture is a common clinical disease. With domestic and foreign scholarin-depth understanding of fracture healing, we have begun explore ways topromote healing from all aspects. We have found the fact in the clinical workand literature, when limb fractures with brain damage, fracture healing isaccelerated and osteotylus gorwn excessivly.But the specific theoretical basisis still not clear. Electroacupuncture, as a traditional treatment of fractures,nonunion means, has got continuous improvement. With the integration ofvarious theoretical system of medicine and the progress of science andtechnology, the mechanism of action studies have aroused widespread concernand attention.PurposeEstablish SD rat tibial fracture models. Observe the change ofosteotylus through morphology, molecular biology, pathology, superfine pathology when using electroacupuncture on “baihui”, to understand thesustainability of the head electroacupuncture effects on fracture healing.Methods128healthy male SD rats were randomly divided into4groups--32normal groups (group A), the tibia fracture model group (group B),32electroacupuncture “baihui” and tibia fracture model group (group C),32moderate brain injury and tibia fracture model group (group D). Groups ofexperimental animals shall be put to death after the intervention, respectivelyin the7d,14d,21d and,28d. Observe the osteotylus change of shape, size,color etc. Under the X-ray film, calculate the osteotylus and statisticalcomparison by Perkins osteotylus volume formula. Slice partial osteotylusand observe pathological changes. Choose the soft tissue from tibia fractureanimal models, observe pathological changes under electron microscope.Using partial brain tissue specimens and osteotylus specimens, detect BMP-2、TGF-β1throught immunohistochemical method. Observe it undermicroscope (400times), calculate the integral optical density (IDO) of positiveexpression cells and detect the brain derived neurotrophic factor proteinthrough Western blotting, compare statistical differences.Results1Give corresponding intervention to tibial fracture models、electroacupuncture treatment models、 brain injury animal models. Theexperimental results show that group D’osteotylus is larger than group B andC(P <0.05).2Osteotylus tissue is observed under the microscope of400times: groupB shows the classical fracture healing; Group C’s osteotylus grows obviouslyfaster than group B; Group D’s osteotylus grows the fastest. 3Osteotylus tissue of the first week under the electron microscope: we cansee the fibroblasts, chondroblasts, mesenchymal cells, Osteogenesis cells andendothelial cells, etc. The fibroblasts and cmesenchymal cells of group D aremore active than other groups.4Specimens of osteotylus’s BMP-2immunohistochemical: the detectiveresults of group B in week1, week2, week3, week4are significantlydifferent from group A; The results of Group C in week1, week2, week3andweek4are significantly different from group A. The results of group c inweek1, week2are significantly different from group B; The results of GroupD in week1, week2, week3and week4are significantly different from groupA, and are obviously different from group B and group C in week1.5Specimens of osteotylus’s TGF-β1immunohistochemistry: The resultsof group B in week1, week2, week3, week4are significantly different fromgroup A; Group C in week1, week2, week3and week4are significantlydifferent from group A and are significantly different from group B in week1,week2; The results of Group D in week1, week2, week3and week4aresignificantly difference from group A, group B and group C.6Specimens of Brain tissue’s BMP-2immunohistochemistry: The resultsof group A and group B has no significant difference from group C; the resultsof Group D in week1, week2are significantly different from group A, groupB and group C.7Specimens of Brain tissue’s TGF-β1immunohistochemistry: Theresults of group A and group B has no significant difference; The results ofGroup C in week1, week2, week3and week4are significantly differentfrom group A; and are significantly different from group B in week2,3,4.The results of Group D have significant difference from group A, group B andgroup C in week1, week2, week3and week4.8Western blotting detection of Osteotylus of BDNF protein: The result of group C has significant difference from group B in week1, week2; the resultsof Group D have significant difference from group B in week1, week2.Conclusions:1Electroacupuncture “baihui” can promote healing of rats tibial fracture，not only on the volume of the osteotylus,but also on the time of recovering.2Electroacupuncture “baihui” can increase osteoclasts、osteogenesiscells、 fibroblasts and chondroblasts. This will promote the fracture healing.3Electroacupuncture “baihui” can promote all the stage of fracture healing.Form the result of the immunohistochemical, we can find the increasing ofOsteotylus of BMP-2in the early and TGF-β1in the late, which is maybeone of the reason of fracture healing acceleration.4Brain injury can cause the increasing of Osteotylus of BMP-2in theearly and TGF-β1in the late, which is similar to the electroacupuncture“baihui” of fracture healing acceleration.5Electroacupuncture “baihui” increases BDNF in Osteotylus in the early2weeks, which may be one of the reasons for fracture healing acceleration.更多还原