鸢尾素促进大鼠胫骨骨折愈合的研究

Effect of irisin on the healing of tibial fracture in rats

目的:探究鸢尾素对大鼠骨折愈合的影响,以及对全身和骨折局部骨代谢的调节作用。方法:建立SD大鼠胫骨骨折模型,随机分为对照组和实验组,对照组骨折局部注射生理盐水,实验组注射1 m L鸢尾素蛋白溶液(100 ng/mL);通过X线、Micro-CT、生物力学和HE染色方法观察骨折早期愈合情况,免疫组织化学染色和ELISA检测全身及骨折局部的骨代谢情况。采用SPSS20.0统计软件进行统计学分析。结果:X线和Micro-CT检测结果显示骨折后第4周实验组新生骨痂明显多于对照组,骨折后第8周实验组骨折线基本消失,而对照组的骨断端影像隐约可见;骨折后第4周和第8周实验组的组织总量(total volume,TV)、骨量(bone volume,BV)、骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)和骨小梁数量(Tb.N)均明显大于对照组(P<0.05)。生物力学分析结果显示实验组骨折后第4周和第8周的最大载荷、强度和能量吸收也明显大于对照组(P<0.05)。HE染色结果显示,与对照组相比,实验组骨折后第4周骨桥形成较多,骨折后第8周形成的板层骨也较厚。免疫组织化学染色结果表明,与对照组相比,实验组更明显地增强了氧化聚明胶(oxypolygelatin,OPG)的表达并抑制了核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)的表达。同时在实验组骨痂区域的骨髓组织中观察到更强的解偶联蛋白1(uncoupling protein 1,UCP1)免疫阳性染色。ELISA检测结果表明,在骨折后第4周和第8周,与对照组相比,实验组大鼠骨碱性磷酸酶(bone alkaline phosphatase,BALP)的血清浓度明显增强,而抗酒石酸酸性磷酸酶5b(tartrate-resistant acid phosphatase 5b,TRAP 5b)的血清浓度明显降低(P<0.05)。结论:鸢尾素能够提高全身和骨折局部的骨代谢活动,增加成骨作用,促进骨折早期愈合。

Objective:To investigate the effect of irisin on fracture healing in rats and the regulating role on bone metabolism of whole body and bone fracture sites. Methods:SD rat tibial fracture models were established and were randomly divided into the control group(local injection of normal saline at the fracture gap) and the experimental group(local injection of 1 mL irisin protein solution,100 ng/mL). X-ray,Micro-CT,biomechanical analysis and HE staining were used to observe the early fracture healing. Immunohistochemical staining and ELISA were used to detect the bone metabolism. SPSS 20.0 software was used to perform the statistical analysis.Results:Results of X-ray and Micro-CT showed new bone calluses in the experimental group were more than those in the control group at the 4 th week after fracture. At the 8 th week after fracture,fracture line in the experimental group was closely to disappear,while fracture line on images of bone fractured end in the control group were seen indistinctly. Total volume(TV),bone volume(BV),bone volume fraction(BVF,BV/TV),trabecular thickness(Tb.Th) and trabecular number(Tb.N) in the experimental group at the 4 th and 8 th week after fracture were also significantly higher than those in the control group(P<0.05). Results of biomechanical analysis indicated that maximum load,stiffness and energy absorption in the experimental group was significantly greater than those in the control group at the 4 th and8 th week after fracture(P<0.05). HE staining results showed that bone bridges in the experimental group were more than those the control group at the 4 th week after fracture,and the lamellar bone in the experimental group was thicker than that in the control group at the 8 th week after fracture. Immunohistochemical staining results showed that the expression of oxypolygelatin(OPG) in the experimental group was significantly enhanced and the expression of receptor activator of nuclear factor-κB ligand(RANKL) in the experimental group was significantly inhibited when compared with those in the control group. At the same time,a stronger uncoupling protein 1(UCP1) immunopositive staining was observed in the bone marrow tissue in the experimental group. ELISA results showed that serum levels of bone alkaline phosphatase(BALP) in the experimental group were significantly increased than that in the control group at the 4 th and 8 th week after fracture,and serum levels of tartrate-resistant acid phosphatase 5 b(TRAP 5 b) were significantly decreased(P<0.05). Conclusion:Irisin can improve bone metabolism of the whole body and bone fracture sites,so as to promote osteogenesis and healing within a short time.