大鼠失神经支配骨折修复的实验研究

【作者】 张双明；

【导师】 陈洪波；

【作者基本信息】 湖北中医学院 ， 中医骨伤科学， 2008， 硕士

【摘要】 目的研究强骨胶囊促进大鼠失神经支配骨折愈合的作用机理。方法雄性SD大鼠,10%水合氯醛腹腔麻醉,在距坐骨结节0.5cm处切断坐骨神经,横形截断股骨中段,建立失神经支配骨折动物模型,造模成功后,将大鼠随机分为强骨胶囊大剂量组、强骨胶囊中剂量组、强骨胶囊小剂量组、丹参组和空白对照组,每组30只。强骨胶囊大剂量组,给予强骨胶囊180mg/kg/d灌胃,每日一次;强骨胶囊中剂量组,给予强骨胶囊90mg/kg/d灌胃,每日一次;强骨胶囊小剂量组,给予强骨胶囊45mg/kg/d;丹参组,给予丹参注射液40mg/kg/d,灌胃,每日一次;空白对照组,遭摸后不作处理。各组大鼠分别在术后1周、2周和3周取骨折中心上下各1cm全段股骨标本,测算骨痂体积。骨痂取材切片,常规HE染色,观察骨痂结构。NGF、BMP-2和bFGF免疫组织化学染色,凝胶成像分析系统计算每个视野阳性细胞数。检测血清Ca、P和ALP。结果(1)骨痂体积测算结果:术后1周,各组骨折处均可见增粗。强骨胶囊大剂量组和强骨胶囊中剂量组骨痂体积值大于丹参组(P＜0.05)和空白对照组(P＜0.05),强骨胶囊小剂量组骨痂体积值大于空白对照组(P＜0.05)。术后2周,各组骨痂体积均有不同程度的增大。强骨胶囊大剂量组和强骨胶囊小剂量组骨折断端新生软骨量增多,骨痂外直径增生显著。强骨胶囊大剂量组骨痂体积明显大于丹参组(P＜0.01)、空白对照组(P＜0.01)和强骨胶囊小剂量组(P＜0.01),中剂量组骨痂体积明显大于空白对照组(P＜0.01)和丹参组(P＜0.05),强骨胶囊小剂量组大于丹参组(P＜0.05)和空白对照组(P＜0.05)。术后3周,强骨胶囊大剂量组骨缺损消失,骨痂外直径增粗,骨痂生长更明显,强骨胶囊中剂量组和强骨胶囊小剂量组骨折处有骨性骨痂形成,丹参组和空白对照组骨痂增生不明显。强骨胶囊大剂量组和强骨胶囊中剂量组骨痂体积达到高峰且大于丹参组(P＜0.01)和空白对照组(P＜0.01),强骨胶囊大剂量组也大于强骨胶囊小剂量组(P＜0.05),强骨胶囊小剂量组大于丹参组(P＜0.05)和空白对照组(P＜0.05)。(2)HE染色结果:术后1周,强骨胶囊大剂量组成骨细胞数目多于其余各组,其它各组以血肿和炎性渗出为主要表现,软骨细胞较少。术后2周,强骨胶囊大剂量组可见软骨骨痂和骨性骨痂,软骨细胞团周边有少量编织骨形成。强骨胶囊中剂量组有少量新生骨小梁,骨折断端内外形成骨样组织。其余各组纤维性骨痂生成明显。术后3周,强骨胶囊大剂量组类骨质较多,外骨痂明显增多。强骨胶囊中剂量组骨小梁排列不规则。强骨胶囊小剂量组骨折处新生软骨已接近全部骨化。丹参组新生血管丰富,骨小梁排列紊乱。空白组纤维骨痂消失,代之以软骨骨痂与骨性骨痂。(3)免疫组化染色结果:NGF:术后1周,强骨胶囊大剂量组骨折断端纤维母细胞与少量的软骨细胞NGF阳性颗粒染色呈淡棕黄色,阳性细胞数明显多于中剂量组(P＜0.01)和强骨胶囊小剂量组(P＜0.01),强骨胶囊中剂量组多于空白对照组(P＜0.05)。术后2周,强骨胶囊中剂量组和强骨胶囊小剂量组成骨细胞胞浆内有阳性颗粒,强骨胶囊中剂量组阳性颗粒多于丹参组(P＜0.05)和空白对照组(P＜0.05)。强骨胶囊大剂量组阳性细胞数明显多于丹参组(P＜0.01)和空白对照组(P＜0.01)。术后3周,强骨胶囊中剂量组和强骨胶囊小剂量组骨痂成骨细胞染色阳性,强骨胶囊中剂量组和强骨胶囊小剂量组阳性细胞数多于大剂量组(P＜0.05)。强骨胶囊大剂量组成骨细胞轻度着色染色阳性。BMP-2:强骨胶囊大剂量组BMP-2阳性细胞数在术后1、2周时呈上升趋势,第2周时达到高峰,第3周时降低。中剂量组BMP-2阳性细胞数在术后第3周时高于其他组,与空白对照组和丹参组相比差异有显著性(P＜0.01)。bFGF:术后1周,强骨胶囊大剂量组和强骨胶囊中剂量组纤维骨痂中的成纤维细胞和血管内皮细胞,未分化间充质细胞及幼稚的成骨细胞有较均匀的棕色细颗粒状bFGF呈阳性表达,强骨胶囊中剂量组阳性细胞数明显多于强骨胶囊小剂量组(P＜0.05)、对照组(P＜0.05)和丹参组(P＜0.05)。术后2周,强骨胶囊大剂量组和强骨胶囊中剂量组在成骨细胞和幼稚的软骨细胞、新生骨基质和外骨膜成骨细胞bFGF表达阳性,阳性细胞数上升至高峰,明显多于空白对照组(P＜0.01)和丹参组(P＜0.01)。术后3周,强骨胶囊大剂量组阳性细胞数量及阳性反应程度较前明显下降,着色程度较前减弱,与空白对照组和丹参组相比有统计学意义(P＜0.05)。其它各组染色较浅,阳性细胞数明显减少。(4)血清Ca、P和ALP结果:术后1周,各组之间血清Ca浓度无明显差异(P＞0.05);术后2周,强骨胶囊中剂量组血清Ca浓度高于其他组,与丹参组相比(P＜0.01)、与对照组相比(P＜0.05),与大剂量组之间相比差异无显著性(P＞0.05)。术后3周,强骨胶囊小剂量组血清Ca浓度最高,与丹参组相比(P＜0.05)和空白对照组相比(P＜0.05),其他各组比较差异均无显著性(P＞0.05)。术后1周,各组之间血清P浓度无明显差异(P＞0.05);术后2周,强骨胶囊小剂量组血清磷浓度降低较明显,强骨胶囊大剂量组与小剂量组和空白对照组相比(P＜0.05);术后3周,各组血清P浓度均下降,强骨胶囊中剂量组下降最明显,强骨胶囊大剂量组和中剂量组与小剂量相比(P＜0.05)。术后第1、2、3周,除丹参组在第2周下降外,其余各组血清中ALP浓度均升高。术后第1周时,强骨胶囊小剂量组ALP浓度高于空白对照组(P＜0.05),其余各组差异无显著性(P＞0.05)。术后第2周时,强骨胶囊中剂量组血清ALP浓度升高明显,与丹参组相比(P＜0.01)、强骨胶囊小剂量组(P＜0.05)及空白对照组(P＜0.05),但与大剂量组之间的比较差异无显著性(P＞0.05)。大剂量组与丹参组相比(P＜0.05)。术后第3周时,大剂量组的血清ALP升高更明显,大剂量组与丹参组相比(P＜0.01)、强骨胶囊小剂量组相比(P＜0.05)及中剂量组相比(P＞0.05),而其余各组之间的比较差异无显著性(P＞0.05)。结论1.失神经支配骨折的修复与细胞因子的表达有关。2.强骨胶囊能通过诱导NGF、bFGF和BMP-2的表达促进失神经支配骨折的修复。3.强骨胶囊能改善神经元修复过程中的微环境,促进骨痂生成、生长质量,促进失神经支配骨折的愈合速度。更多还原

【Abstract】 Objective To study the mechanism of Qianggu capsule to promote the recovery of rat’s denervated fracture.Methods Male SD rats,10%Chloral Hydrate abdominal anesthesia,sever the sciatic nerve in the ischial tuberosity 0.5 cm and cut bone defect in the middle of the femur,establish denervated fracture animal model,randomly divided into Qianggu capsule large dose group(LDG),Qianggu capsule medium dose group (MDG),Qianggu capsule small dose group(SDG),Injectio Salviae Miltiorrhiae group (SMG)and control group(CG),each group have 30 rats.LDG was given Qianggu capsule 180mg/kg/d intragastric administration,once a day;MDG was given Qianggu capsule 90mg/kg/d intragastric administration,SDG was given Qianggu capsule 45mg/kg/d intragastric administration,once a day,SMG was given Injectio Salviae Miltiorrhiae 40mg/kg/d,intragastric administration,once a day;CG no disposal.In each group after one week,two weeks and three weeks cut osteotylus 1 cm from the center of the whole femoral fracture,to measure osteotylus volume.To drawe the materials from osteotylus,conventional HE staining and observe structural changes in osteotylus.NGF,BMP-2 and bFGF immunohistochemical staining,gel image analysis system to calculate each vision positive cells.Results(1)Osteotylus volume result:It is thus evident that the osteotylus volume in every group was increased in the first week of postop.,LDG and MDG increased obviously than SMG(P＜0.05)and CG (P＜0.05),SDG more than CG(P＜0.05)..In the second week of postop.,Neogenesis cartilage volume increased obviously in broken ends of fracture in LDG and SDG,external diameter of Osteotylus hyperplasia.osteotylus volume in every group increased in varied degrees.LDG hyperplasia obviously more than SMG(P＜0.01),CG (P＜0.01)and SDG(P＜0.01),MDG increased obviously than SMG(P＜0.05)and CG(P＜0.01),SDG more than SMG(P<0.05)and CG(P＜0.05).In the third week of postop.,LDG bone coloboma disappeared,extemal diameter of osteotylus more thicken,osteotylus grew more obviously,MDG and SDG have porosis in the place of fracture,SMG and CG hypercallosis not obviously.Osteotylus volume in LDG and MDG achieve peak and exceed SMG(P＜0.01)and CG(P＜0.01),LDG exceed SDG (P＜0.05),SDG exceed SMG(P＜0.05)and CG(P＜0.05).(2)HE staining Results: In the first week postop.,the amount of osteoblast in LDG more than other groups,and other groups hematoma and inflammatory exudation increased,cartilage cells less.In the second week postop.,LDG have cartilage osteotylus and bony osteotylus,cartilage cells surrounding have generated manipulus woven bone,MDG have manipulus newborn trabecular bone,osteoid tissue generated inside and outside broken ends of fracture.Other groups fiber osteotylus generate obviously.In the third week of postop.,LDG have many osteoid,osteotylus immature and low intensity,external osteotylus increased obviously and observed a lot of osteoclast and osteoclasts.MDG trabecular bone were irregular.SDG fracture ends nearly all sclerotization.SMG neovascularization richly,trabecular bone disorder,osteoclast relatively less.CG fiber osteotylus disappeared,replaced by cartilage osteotylus and boney osteotylus.(3) Immunohistochemical staining results:NGF:In the first week postop.,Fibroblast and a small amount of cartilage positive particles NGF irnmunohistochemical staining were pale brown in LDG,the number of positive cells was higher than MDG(P＜0.01)and SDG(P＜0.01).SMG more than CG(P＜0.05).In the second week postop.,MDG and SDG positive cell particles increased significantly in cytosol of osteoblast,MDG more than SMG(P＜0.05)and CG(P＜0.05).The number of positive cell in LDG was significantly more than SMG(P＜0.01)and CG(P＜0.01).In the third week postop.,MDG and SDG intraosteotylus osteoblast cells staining positive and the number of positive cells more than LDG(P＜0.05).LDG osteoblasts showed weak positive staining.BMP-2:The number of BMP-2 positive cells in LDG upward trend during 2weeks,in the second week at the peak,lower in the third weeks.BMP-2 positive cells in MDG higher than other groups,compared with CG and MG the difference was significant(P＜0.01).BFGF:In the first week postop.,LDG and MDG fibrous osteotylus fibroblasts and vascular endothelial cells,undifferentiated mesenchymal cells and naive osteoblast nucleus have uniform granular brown positive expression of bFGF,MDG positive cells was higher than the other groups,significantly higher than SDG(P＜0.05),CG(P＜0.05)and SMG(P＜0.05).In the second week postop.,LDG and MDG in osteoblasts and naive chondrocytes,new bone matrix,periosteal bone cells expression of bFGF positive,positive cells increased up to peak,significantly more than CG(P＜0.01)and SMG(P＜0.01).In the third week postop.,the number of positive cells staining and positive responses in LDG decreased obviously compared with the past,the dgree of coloring weakened than the former,compared with CG and SMG have statistical significance(P＜0.05).Other groups were less positive cells and coloring weakened.(4)Serum Ca,P and ALP results:In the first week postop.,serum Ca in every groups there was no significant difference(P＞0.05).In the second week postop.,serum Ca in MDG was higher than other groups,compared with SMG(P＜0.01)and CG(P＜0.05).compared with LDG no significant difference(P＞0.05).In the third week postop.,serum Ca in SDG was the highest,compared with SMG(P＜0.05)and CG(P＜0.05),the other groups showed no significant difference(P＞0.05).In the first week postop.,serum P in every group there was no significant difference(P＞0.05),In the second week postop.,serum P in SDG decreased obviously,LDG compared with SDG there were significant differences(P＜0.05),In the third week postop.,serum P decreased in every group,MDG decreased obviously,LDG and MDG compared with SDG(P＜0.05).Serum ALP increased in every groups during 1,2 and 3 weeks after operation except for SMG in the first two weeks fell.In the first week postop.,ALP in SDG higher than CG(P＜0.05),while the other group no significant difference (P＞0.05),In the second week postop.,serum ALP in MDG increased obviously compared with SMG(P＜0.01),SDG(P＜0.05)and CG(P＜0.05),compared with LDG the difference was no significant(P＞0.05),LDG compared with SMG(P＜0.05).In the third week postop.,serum ALP increased more significantly in LDG,LDG compared with SMG(P＜0.01),SDG(P＜0.05)and MDG(P＞0.05).There were no significant difference between the other group(P＞0.05).Conclusion1.The recovery of denervation fracture have relate to the expression of cytokine.2.Qianggu capsule can promote the recovery of denervation fracture by inducing the expression of NGF,bFGF and BMP-2.3.Qianggu capsule can improve the microenvironment in the process of neurons recovery and promote osteotylus formation,growth quality,and accelerate the healing of denervation fracture.更多还原