低强度脉冲超声对兔桡骨骨折愈合及小窝蛋白-1基因表达的影响

Effects of low-intensity pulsed ultrasound on fracture healing and caveolin-1 gene expression in the radius defects of rabbits

目的探讨低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)对长管状骨骨折愈合的作用,测定LIPUS作用下兔桡骨骨折部位小窝蛋白-1(caveolin-1)局部表达情况。方法在24只新西兰白兔双侧桡骨中下1/3处制作宽为3 mm的骨缺损模型,按随机数字表法平均分为4组(n=6)。右侧桡骨骨折处给予强度为30 mW/cm^2的LIPUS刺激,每天1次,每次20 min,左侧桡骨骨折处给予切断电源的假刺激。各组实验动物分别于术后第7、14、21、28天,采用X线片、HE染色评价骨折愈合情况,免疫组化染色检测小窝蛋白-1水平与定位,实时荧光定量PCR检测小窝蛋白-1、软骨和骨特异基因Col2a1、Col10a1及骨钙蛋白(osteocalcin)的mRNA水平。结果自第14天起,实验侧X线评分和矿化骨痂面积均显著大于对照侧,二者随治疗时间的延长而逐渐增高;HE染色提示实验侧软骨细胞分化、凋亡,形成原始骨小梁和骨小梁的融合等过程均早于对照侧。免疫组化染色显示术后第7、14天,随着软骨细胞的增殖、分化,小窝蛋白-1水平逐渐增加,实验侧显著大于对照侧;第21、28天,迁移至软骨基质表面的间充质细胞开始向成骨细胞分化,小窝蛋白-1水平降低,实验侧显著低于对照侧。实时荧光定量PCR结果显示:实验侧小窝蛋白-1mRNA水平在第7天显著高于对照侧,在第21天却显著低于对照侧;实验侧Col2a1,Col10a1及骨钙蛋白mRNA水平在第7、14天显著高于对照侧,在第21天却显著低于对照侧,在第28天实验侧Col2a1和骨钙蛋白mRNA水平显著低于对照侧。结论 LIPUS可通过促进软骨内成骨加速长管状骨骨折愈合,推测可能与先上调软骨细胞,随后下调间充质细胞小窝蛋白-1的表达有关。

Objective T o explore the effect of low-intensity pulsed ultrasound(LIPUS)on long bone fracture healing and to examine caveolin-1 gene expression in the radius defects of rabbits.Methods A total of 24 New Zealand rabbits with 3-mm bone defects at lower 1/3 in both radii were randomly assigned to 4 groups(n=6).Daily LIPUS treatment was performed to the right fracture sites at a intensity of 30 mW/cm2 for 20 minutes?while the left sites received sham treatment with power off.To assess the effects of LIPUS on bone defects>X-ray imaging and hematoxylin-eosin staining were applied 7,1 4,2 1,2 8 days after the surgery.Additionally,the immunohistochemical staining was used to determine the subcllular localization of caveolin-1 and semi-quantify the caveolin-1 level,qPCR was performed to detect the mRNA level of caveolin-1 jgene Col2a1and CoZlOa1,and osteocalcin.Results On day 14,the radiological score of the right radii and mineralized callus area were significantly higher than that of the left ones,both of them were elevated with time flied.Histological examination suggested that the differentiation and apoptosis of chondrocytes along with the formation and bridging of the bone trabeculas appeared earlier in the right radius defects.The immunohistochemical staining showed that on day 7 and 1 4,the level of caveolin-1 increased with the proliferation and differentiation of condrocytes,and was significantly higher in callus tissues on the right sites.On day 21 and 2 8,the mesenchymal stem cells migrated to the surface of cartilage matrix started to differentiate into osteoblasts,the level of caveolin-1 decreased,and was significantly lower on the right sites.The result of qPCR indicated that compared with the left sites the caveolin-1 gene expression on the right sites was significantly higher on day 7,while significantly lower on day 21.The mRNA expression levels of Col2a1,Col10a1and osteocalcin on the right sites were significantly higher on day 7 and 1 4,but they were significantly lower on day 21 and 28,except for CoZlOal on day 28.Conclusions Advancing endochondral ossification is considered to be a crucial mechanism during long bone fracture healing promoted by LIPUS.The caveolin-1 gene expression first increased in the chondrocytes then decreased in the mesenchymal stem cells during the process.