苏氏接骨胶囊对胫骨干骨折大鼠模型促早期愈合作用及BMP-2表达的影响

Effect of Su's bone capsule on early healing and BMP-2 expression in rats with tibial shaft fracture

关键词:  苏氏接骨胶囊  骨折愈合  Micro-CT  BMP-2

Key words:  Su's bone capsule  fracture healing  Micro-CT  BMP-2

摘要:

[目的]探讨苏氏接骨胶囊（简称Su）对大鼠胫骨干骨折早期愈合的促进作用及骨形态发生蛋白-2（BMP-2）表达的影响。[方法]36只6周龄雄性SD大鼠按照随机数字表分为假干预组、Su剂量140 mg/（kg·d）组（Su-140组）和Su剂量280 mg/（kg·d）组（Su-280组）。分组建立胫骨干闭合骨折髓内钉固定模型，术后第2天开始给予相应的药物干预。造模后10 d，过度麻醉心脏取血并处死各组大鼠。对大鼠患肢胫骨进行取材后，通过X线对骨折愈合情况进行初步检查；通过Micro-CT技术计算骨痂的骨体积分数（BV/TV）、骨小梁厚度（Tb.Th）、骨小梁数量（Tb.N），并对骨痂进行组织形态学观察；通过免疫组化技术观察骨痂中BMP-2的表达，实时荧光聚合酶链反应（Real Time-PCR）定量检测血清BMP-2的含量。[结果]骨折造模术后10 d，假干预组、Su-140组及Su-280组3组大鼠的骨折断端均可见骨痂形成，骨折端愈合尚稳定，部分标本骨折断端稍有微动。经X线观察Su-280组骨折端愈合情况好于Su-140组及假干预组，Su-140组优于假干预组。Micro-CT检测结果提示，Su-140组和Su-280组的BV/TV值均高于假干预组，差异有统计学意义（P<0.05或P<0.01）。Tb.Th和Tb.N 2个参数，Su-140组与假干预组间比较无统计学意义（P>0.05），Su-280组与假干预组间比较有统计学差异（P<0.01），Su-140组与Su-280组间比较有统计学差异（P<0.01或P<0.05）。免疫组化结果显示，Su-280组骨痂内有大面积的BMP-2表达区域，Su-140的表达区域较少，假干预组更少；RealTime-PCR结果显示，3组间BMP-2的含量差异存在显著性（P<0.05），其中Su-280组与Su-140组和假干预组差异均有显著性（P<0.05），Su-140组和假干预组间差异无显著性（P>0.05）。[结论]苏氏接骨胶囊能促进大鼠胫骨干骨折的早期愈合及BMP-2的表达，且随药物浓度的增加，骨折愈合越快，其机制可能为Su促进BMP-2的表达，进而诱导成骨细胞及胞外基质的分化增殖，促进骨形成及骨愈合。

Abstract:

[Objective] To investigate the effect of Su's bone capsule (Su) on early healing of tibial shaft fracture and the expression of BMP-2 in rats.[Methods] Thirty-six 6-week-old male SD rats were randomly divided into sham intervention group (sham group), Su group 140 mg/(kg·d) dose (Su-140 group) and Su dose 280 mg/(kg·d) dose (Su-280 group). The intramedullary nailing model of closed tibial fracture was established and the corresponding drug intervention were done since the second day after operation. Ten days after modeling, the rats were anesthetized and sacrificed. After the tibia of the affected limb in rats, the healing of the fracture was examined by naked eyes and X-rays. The bone volume/total volume (BV/TV), trabecular thickness (Tb.Th) and trabecular bone (Tb.N) of bone callus were calculated by Micro-CT technique, and the histomorphology of callus was observed. The expression of BMP-2 in callus was observed by immuno-histochemical technique, and the contents of serum BMP-2 was detected by real time PCR.[Results] On the 10 day after the fracture, the callus formation was observed in the fracture of three groups of rats in sham group, Su-140 group and Su-280 group. The most of fracture end was stable, and few was slightly loose. By X-ray observation, the healing result in group Su-280 was better than in Su-140 group and sham group, and that in group Su-140 was better than in sham group. The results of Micro-CT test indicated that the BV/TV values in Su-140 group and Su-280 group were higher than those in sham group, and the difference was statistically significant (P<0.05, P<0.01) respectively. The two parameters of Tb.Th and Tb.N were not statistically significant between sham group and Su-140 group (P>0.05), but there was statistical difference between Su-280 group and sham group (P<0.01) and between Su-140 group and Su-280 group (P<0.01, P<0.05). By immunohistochemistry, it showed that there were more expression of BMP-2 in the Su-280 group and the less expression in Su-140 group and sham; the results of real time PCR showed that there were significant differences in the expression of BMP-2 in three groups (P<0.05), and the same between the Su-280 group and sham group, Su-140 group and sham group (P<0.05, P<0.05), but there was no significant differences between the Su-140 group and sham group (P>0.05).[Conclusion] Su can promote the expression of BMP-2 and early healing of tibial shaft fracture in rats, and with the increase in drug concentration, the faster fracture healing. The mechanism may be the BMP-2 stimulated by Su promote the differentiation and proliferation of osteoblasts and extracellular matrix and bone healing.