摘要
目的:观察骨质疏松性椎体骨折愈合过程中,局部血管内皮生长因子动态表达,探讨牛骨形态发生蛋白促进SD大鼠骨质疏松性椎体骨折愈合的作用机制。方法:实验于2005-07/2006-07在解放军兰州军区总医院骨研所完成。①牛骨形态发生蛋白的制备:青海小牦牛大腿骨,-4℃条件下,粉碎,按Urist改良方法提取。②选择雌性SPF级8个月龄SD大鼠96只,去除双侧卵巢建立骨质疏松模型。随机分为2组:实验组和对照组,每组48只。③术后3个月行L5椎体开窗建立人工骨折模型,实验组置牛骨形态发生蛋白10mg+纤维蛋白复合体;对照组注入等量无血清培养液。④于手术后3,7,10,14,21,28,56,84d每组麻醉状态处死6只,用苏木精-伊红染色、免疫组织化学及定量聚合酶链反应检测骨折愈合不同时间的标本血管内皮生长因子及血管内皮生长因子mRNA的动态表达,阳性信号为胞浆内棕色颗粒。结果:96只SD大鼠全部进入结果分析。①骨折愈合不同时间组织学观察:实验组术后7d纤维骨痂形成,21d新生骨组织与旧骨组织出现融合,56～84d软骨性骨痂已逐渐被骨性骨痂取代,骨折部位髓腔再通,出现成熟板层骨,骨折基本愈合。对照组术后7d未见典型纤维性骨痂;21d新生骨组织大都游离与孤立存在,术后28d骨小梁仅有少部分连结成网状,56～84d部分骨缺损存在,不见成熟板层骨,大部分髓腔不通,骨折愈合延迟。②骨折不同时间血管内皮生长因子免疫组织化学结果:实验组血管内皮生长因子在7d左右可见有阳性表达的细胞,14d有分泌高峰,对应循环数(CT值)最小,两组比值最大。而对照组未见明显分泌高峰。③实时荧光定量聚合酶链反应检测结果:对照组在造模后1,2,4,6,8周时,椎体内的血管内皮生长因子mRNA的表达量明显低于实验组(P<0.01)。结论:牛骨形态发生蛋白可促进大鼠骨质疏松性椎体骨折部位血管内皮生长因子的动态表达,可在受体骨折部位局部促进骨折生长。
AIM： To study the biological function of local vascular endothelial growth factor （VEGF） and the mechanism of bovine bone morphogenetic protein to accelerate osteoporotic vertebral fracture healing in SD rats.
METHODS： The experiment was completed in the Institute of Orthopaedics, General Hospital of Lanzhou Military Medical University of Chinese PLA during July 2005 and July 2006. ①The preparation of bovine bone morphogenetic protein： The small bovine femur were crushed under -4 ℃ and extracted with the method of Urist. ② Ninety-six 8-month-old female Sprague-Dawley （SD） rats of SPF grade were adopted to remove ovary and establish the osteoporosis models, then were randomized into two groups of 48 in each. ③Three months postoperation, artificial fracture models were made by Ls vertebral fenestration. The bovine bone morphogenetic protein group 10 mg＋fibdn compound （experimental group） and the equal serum-free medium （control group） were injected, respectively.④Six mice of each group were put to death under anesthesia postoperatively of 3, 7, 10, 14, 21, 28, 56, 84 days. The callus of each rat was examined by hematoxylin-eosin （HE）, immunohistochemistry to detect the expression of VEGF and in real time polymerase chain reaction （PCR） to study the expression of VEGF mRNA.
RESULTS： Ninety-six SD rats were all analyzed in the final results. ①Histology： In experimental group, fibrous callus were formed in 7 days postoperatively, regenerating bone and bone were merged in 21 days, but in 56-84 days, cartilaginous callus was replaced by osseous callus, marrow was repast in fracture part, and the bone was mature; But in control group, no fibrous callus presented postoperatively at 7 days, regenerating bone were more isolated or dissociated at 21 days and reticulate structure existed at 28 days, the defect bone weren＇t healed at 56-84 days, the fracture healing were put off. ②Immunohistochemistry： VEGF could be seen positive 7 days post-transplantation, and there had the highest quantities and CT was lowest at 14 days. However there were not obvious peak in controls.③Real-time PCR： The expression of VEGF mRNA in control group was markedly lower than that in experimental group at weeks 1, 2, 4, 6, 8 postoperatively （P〈 0.01）.
CONCLUSION： Bovine bone morphogenetic protein can enhance the dynamic expression of VEGF during the bone fracture healing of osteoporosis rat. VEGF play very important roles in accelerating fracture healing of osteoporosis rats.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第14期2695-2698,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
