鹿瓜多肽注射液促进骨折愈合过程中血管内皮生长因子表达的机制

Mechanism of cervus and cucumis polypeptide injection in improving the expression of vascular endothelial growth factor in the fracture healing

目的:通过观察鹿瓜多肽注射液经肌肉注射后对兔骨折愈合过程中血管内皮生长因子表达的影响,探讨其作用机制。方法:实验于2005-09/2006-03在武汉大学人民医院骨科、病理学教研室完成。①80只新西兰大白兔制成桡骨中段骨折模型,术后随机分为鹿瓜多肽低剂量组、鹿瓜多肽高剂量组、骨肽对照组和空白对照组,每组各20只,分别予以0.1mL/kg鹿瓜多肽注射液(哈尔滨誉衡药业有限公司,主要成分为梅花鹿的骨骼和葫芦科植物甜瓜的干燥成熟种子提取的天然多肽类物质)、0.3mL/kg鹿瓜多肽注射液、骨肽注射液(蚌埠市宏叶生化制药厂,含有多肽类骨代谢因子、有机钙、无机钙、无机盐、微量元素、氨基酸等)0.2mL/kg和不注射任何药液。②术后1,2,3,4周分批处死动物取材行免疫组织化学染色及图象分析测定血管内皮生长因子表达的强度。免疫组织化学染色片显微镜下放大200倍,每个标本取6张切片,随机取5个视野,计算免疫组织化学染色的平均吸光度值和积分吸光度值进行半定量分析。结果:80只兔全部进入结果分析。①血管内皮生长因子免疫组织化学结果:术后1周,鹿瓜多肽高剂量组和低剂量组纤维性骨痂中细胞外间质、成骨细胞、骨折端骨细胞、成软骨细胞和各种炎性细胞均呈阳性染色,骨肽对照组和空白对照组有少量阳性细胞,阳性程度弱。术后2周,鹿瓜多肽高剂量组和低剂量组软骨骨痂及新生骨小梁中成骨细胞、成软骨细胞、成纤维细胞和新生的血管内皮细胞均呈强阳性染色,骨肽对照组和空白对照组阳性程度较弱。术后3,4周,各组呈强阳性表达,各组间着色差别不明显。②图像分析结果:在骨折愈合过程中,鹿瓜多肽高剂量组和低剂量组血管内皮生长因子的表达量一直强于骨肽对照组和空白对照组(第2周平均积分吸光度依次为5.33±0.22,4.71±0.21,3.80±0.25,3.66±0.17,P<0.05或0.01);鹿瓜多肽高剂量组和低剂量组血管内皮生长因子的表达高峰提前出现,约出现在第2周左右。结论:鹿瓜多肽注射液能有效促进骨折愈合过程中血管内皮生长因子的合成分泌,是其促进骨折愈合的重要机制。

ATM： To evaluate the effect of cervus and cucumis polypeptide injection on the expression level of vascular endothelial growth factor （VEGF） during fracture healing in rabbits, so as to probe into the action mechanism of cervus and cucumis polypeptide injection on fracture healing. METHODS： The experiment was conducted in the Department of Orthopaedics and Department of Pathology, Renmin Hospital of Wuhan University from September 2005 to March 2006. ①Eighty New Zealand rabbits were divided into four equal groups randomly after the double radial bone fracture model was established by operation, which were subjected to intramuscular injections of cervus and cucumis pelypeptide injection （0.2 mL/kg, 0.1 mL/kg, product of Harbin Yuheng Pharmaceutical Co.,Ltd, consisting of natural polypeptides extracted from sika skeleton and dry mature seeds of muskmelon）, bone peptide injection （Bengbu Hongye Biochemical Pharmaceutical Factory, including polypeptide factors of bone metabolism, organic and inorganic calcium, inorganic salt, microelement, amigo acid and so on） or nothing, respectively. ②Rabbits were sacrificed at the 1^st, 2^nd, 3^r or 4^th weeks postoperatively in batches for extracting the samples which were used to detect the expression level of VEGF by immunohistochemistry and photograph analysis. Under the microscope magnifying 200, 6 sections of each sample in 5 random sights were selected to conduct the semiquantitative analysis of the mean absorbance （Am） and integral absorbance （Ai）. RESULTS： Totally 80 rabbits were involved in the result analysis.① Immunohistochemistry results of VEGF： In the 1^st week postoperatively, VEGF expressed in the extracellular mesenchymal, osteoblast, osteocyte bone fracture, chondroblast and various inflammatory cells of fibrous bony callus in high dose group and low dose group, and expressed seldom and mildly in bone peptide group and control group; In the 24 week postoperatively, VEGF expressed strongly in osteoblast, chondroblast and fibroblast of cartilage bony callus and newborn born trabecula as well as newborn vascular endothelial cells in high dose group and low dose group, while still mildly expressed in bone peptide group and control group. During the third and fourth week postoperatively, the expression of VEGF was strongly in all the groups, but the pigmenting was similar between groups. ②Photograph analysis： The expressiDns of VEGF in high dose group and low dose group were higher than those in bone peptide group and control group during the whole process of fracture healing （Am at the 24 week： 5.33±0.22, 4.71±0.21, 3.80±0.25, 3.66±0.17, P 〈 0.05 or 0.01）, and the peak value appeared at the 24 week. CONCLUSION： Cervus and cucumis polypeptide injection can actually promote the production and exudation of VEGF through the process of fracture healing, which is the important action mechanism of fracture healing.