跨皮电刺激和外源性rhG-CSF对大鼠胫骨骨折部位Ang-1与bF-GF表达及血管再生的影响

Effects of Electrical Stimulation and Exogenous rhG-CSF on the Expressions of Ang-1,bFGF and Angiogenesis in Tibia Fracture Site in Rats

目的:探讨电刺激及外源性重组人粒细胞集落刺激因子(rhG-CSF),对大鼠骨折愈合过程中血管再生因子及血管形成的影响。方法:3月龄SD大鼠80只,随机分为对照组、电刺激组、rhG-CSF组和电刺激+rhG-CSF组,每组20只,制备大鼠胫骨闭合性骨折模型,rhG-CSF组和电刺激+rhG-CSF组分别在骨折后3 h皮下注射rhG-CSF,连续注射5天,电刺激组和电刺激+rhG-CSF组分别在骨折后第5天进行跨皮电刺激大鼠小腿,2周取材刺激9天,4、6、8周取材刺激15天,各组分别在2、4、6、8周取材,每次5只,制成脱钙骨切片;用免疫组织化学染色法对骨折部位促血管再生因子Ang-1、bFGF和血管新生因子CD31标记后统计分析,用血管墨汁灌注法对大鼠骨折部位血管形成情况进行观察。结果:免疫组化结果显示,各干预手段均可促进骨折部位Ang-1、bFGF、CD31的表达量,电刺激+rhG-CSF组与电刺激组、rhG-CSF组相比阳性表达均显著升高。墨汁灌注染色结果显示,各干预手段均可促进大鼠骨折部位血管、血管网的形成,加速骨折后的血管重建,尤其以电刺激+rhG-CSF动员组效果更为显著。结论:采用电刺激及外源性rhG-CSF动员均可促进骨折愈合过程中血管再生因子的表达,加速血运重建,且联合作用优于单一因素,为加速骨折愈合提供基础理论依据。

Objective： Investigate the effects of electrical stimulation and exogenous recombinant human granulocyte colony-stimulating factor （rhG-CSF） on the angiogenesis factor responsible for vascular remodeling during fracture healing in rats. Methods： 80 three-month-old SD rats were randomly assigned to 1 ） the control group, 2） electrical stimulation group, 3） rhG-CSF group, and 4） electrical stimulation ＋ rhG-CSF group. The rhG-CSF group and electrical stimulation ＋ rhG-CSF group were treated with 5 days continuous subcutaneous rhG-CSF injection 3 hours after fracture. The electrical stimulation group and electrical stimulation ＋ rhG-CSF group were treated with electrical stimulation 5 days after fracture. The 2, 4, 6, 8 week groups were stimulated for 9, 15, 15, and 15 days, respectively. Each group was sacrificed at 2, 4, 6, and 8 weeks with 5 rats per group. Bone slices were decalcified, then immunohistochem- ical staining was performed to examine the effects on the expressions of the angiogenesis factor Ang-1, bFGF and angiogenic factor CD31 at the fracture site. Ink perfusion was then performed to visualize vasculature formation at the fracture site. Results： The results of immunohistochemistry indicated that all the in- tervention can promote the expression of Ang-1, bFGF and CD31 at the fracture site. Compare to electrical stimulation group and rhG-CSF group, electrical stimulation ＋ rhG-CSF group can significantly induce the expression of these three factors. Ink perfusion staining showed that the intervention could promote vascular network formation and accelerate revascularization at the fracture site. In particular, electrical stimulation combined with rhG-CSF mobilization ef- fect was even more significant. Conclusions： Electrical stimulation and exogenous rhG-CSF mobilization could promote fracture healing angiogenesis factor which lead to accelerated revascularization. Fracture healing can be accelerated to a further extend by combining these interventions. This result provides theoretical basis for methods that accelerated fracture healing.