第三型硫酸乙醯肝素酶對小鼠骨癒合的影響

Abstract

骨癒合向來是熱門的研究議題，範圍涵蓋臨床上功能與巨觀的解剖構造的恢復以及細胞分子層面的變化。硫酸乙醯醣蛋白（Heparin sulfate proteoglycans, HSPGs）是普遍存在於生物體細胞表面和細胞外基質中的醣蛋白，參與許多生長因子的訊號傳遞。第三型硫酸乙醯肝素酶（heparinase III）是一種可以切割HSPGs上硫酸乙醯肝素（heparin sulfate, HS）的酵素，對於HSPG和其所調節的分子訊號有間接的影響。本實驗的目的為評估heparinase III作用於HS後對小鼠骨缺損癒合的影響。本實驗中小鼠為實驗動物模式，在其遠端股骨製造一直徑1 mm之圓柱狀骨缺損，利用連續給藥膠囊及導管分別將heparinase III和heparinase III緩衝液給到實驗組和控制組的骨缺損處，並在術後第1、4、7、14、21天犧牲小鼠，以微電腦斷層、組織學檢查、定量聚合酶鏈式反應、和免疫化學染色的方式評估癒合情況。實驗組和控制組間最顯著的差別在術後第1天的蝕骨細胞acid phosphatase化學染色結果以及術後第4天蝕骨細胞的Cd68基因表現量。Heparinase III處理的組別在上述的兩種評估中皆表現出顯著較高的蝕骨細胞數量與分佈。由本實驗的結果可以得知heparinase III不會影響骨癒合最終的形態結果，但在早期蝕骨細胞的聚集和骨重吸收的作用中應扮演著重要的角色。

The healing of bone tissuehas always been a popular researchtopic. Besides restorationof clinical functionand returning to a macroscopic normal anatomical structure, changes at the microscopic cellular and molecular level should also be investigated. Heparin sulfate proteoglycans(HSPGs) are found on cell surfaceand in extracellularmatrix, where they participatein many signaling pathways of different growth factors. It is postulated thatmodulating HSPGs may influencethe processof bone healing indirectly.Heparinase III derived from microorganismiscapable of cleavingthe heparansulfate (HS) chain on HSPGs. This study aims to evaluate the effectof heparinase III in bone healing in mouse model. Mice with a distal femur bone defect were used as the animal model. Mice were randomly assigned to either the control group receiving heparinase buffer or the experiment group receiving heparinase III in heparinase buffer. The mice were sacrificed at1, 4, 7, 14, 21 days post-injury, and micro-CT, histological exam, qPCR, and immunohistochemistrywere performedto evaluate bone healing. Results showedthat the main difference betweenthe two groups are acid phosphatase histochemistry results at 1 day post-injury and Cd68 qPCR results at 4 days post-injury, with heparinase III group showing significant higher osteoclastnumbers and distribution. These results indicate that heparinase III may not affectthe final outcomeof bone healing, but certainly play a role in the early stage of osteoclastrecruitmentand bone resorption.