IL-1β改变血凝块结构性质对骨愈合的影响及机制研究

Delayed bone healing (DBH) and bony non-union (BNU) caused by large segmental bone defects remain a challenge for orthopedic surgeons. Evidence now shows that prolonged local inflammation and changes to hematoma structures significantly delay bone healing. However, there are few if any studies that have considered the effect of interleukin-1 beta (IL-1β) on hematoma architectures and subsequent bone healing. In earlier published studies, we examined how IL-1β affected hematoma structures in normal bone healing and DBH. Following up from these findings, in this project we will characterize qualitative differences in hematomas formed in normal bone healing versus BNUs using a segmental bone defect model in rats and IL-1β-related gene and protein expressions will be, in particular, thoroughly evaluated. De novo bone formation, in the presence and absence of IL-1β, will be assessed in DBH and BNU models using radiographic analysis and histological staining. Moreover, the effects of IL-1β neutralizing antibodies, on fibrin polymerization, morphology, rigidity, and clot fibrinolysis in platelet-poor plasma / blood clots will be evaluated by turbidity, compression, and clot lysis assays. Finally, the mechanisms governing the influence of IL-1β on the structure of an ex vivo fibrin clot will be explored. The results from this study may provide new insights into how to accelerate early bone regeneration by modulating the in-situ blood clots.

大节段骨缺损并发的骨延迟愈合和骨不连是临床骨科长期面对的难题之一。大量文献表明骨缺损内部的炎症反应时间过长和血凝块的纤维结构改变均是导致骨延迟愈合和骨不连出现的主要因素，但促炎症性因子IL-1β直接改变血凝块纤维结构对骨缺损愈合的影响却未见报道。申请人前期发现骨延迟愈合和正常骨愈合模型中血凝块的纤维结构存在差异，且IL-1β在前者中显著性高表达。本项目拟通过构建节段性骨缺损正常骨愈合模型和骨不连模型，研究两者中血凝块的纤维结构差异和IL-1β级联炎症反应因子的重要相关基因、蛋白表达差异；通过影像学和组织学染色来证实过表达或抑止IL-1β对骨延迟愈合和骨不连的直接影响；通过混度实验、压力实验和纤维溶解实验等检测不同浓度的IL-1β抗体作用下，贫血小板血浆/全血凝块中纤维结构的变化；最后阐明IL-1β调节纤维结构改变的分子机制，为今后调节血凝块的纤维结构，促进早期骨愈合提供理论依据。

大节段骨缺损并发的骨延迟愈合是临床骨科长期面对的难题之一。大量文献表明骨缺损内部的炎症反应时间过长和血凝块的纤维结构改变均是导致骨延迟愈合和骨不连出现的主要因素，但促炎症性因子IL-1β直接改变血凝块纤维结构对骨缺损愈合的影响却未见报道。本项目主要研究血凝块在骨延迟愈合模型中的基础性结构差异和IL-1β级联炎症反应因子的重要相关基因、蛋白表达情况，发现了血凝块中IL-1β因子蛋白含量在骨延迟愈合模型中达到745.40 ± 99.19 pg/mL，而正常骨愈合模型中IL-1β因子的蛋白含量仅为57.46 ± 4.72 pg/mL (P < 0.01)。并且，通过体外混度实验证实了高浓度500 pg/mL IL-1β作用下延迟时间为57.8 s±24.6，Vmax值为0.6764±0.0456，Tmax值为0.0588±0.0023均显著低于空白对照组（P < 0.01）。从形态学方面，扫描电镜观察到高浓度500 pg/mL IL-1β较空白对照组，纤维直径明显变细（75.47±14.33 nm vs 175.17±38.37 nm），而纤维密度明显增加（20.1 ± 1.84 根/4 μm2 vs 12.4±2.94根/4 μm2）（P <0.01）；荧光电镜下，我们也得到了相似的结论。通过HPLC分析，通过纤维蛋白原转化为纤维蛋白肽A和B（FpA和FpB）的速率来测定IL-1β抑制凝血酶的催化能力。计算曲线下的面积，与对照组相比，IL-1β处理后生成的FpA或FpB量没有显著差异，这表明IL-1β对凝血酶活性的影响有限。通过阐明IL-1β调节纤维结构改变的分子机制，为今后调节血凝块的纤维结构，促进早期骨愈合提供有力的理论依据。