乌司他丁抑制破骨细胞活化及与基质金属蛋白酶2和9的关系:预防假体骨溶解的潜在价值  

Inhibitory effect of ulinastatin on osteoclast activation and the relationship of ulinastatin to matrix metalloproteinase-2 and matrix metalloproteinase-9:potential of preventing prosthetic osteolysis

作　　者：茹江英[1] 赵建宁[1] 郭亭[1] 俞磊[1] 丁浩[1] 江辉[1] 

机构地区：[1]解放军第二军医大学附属南京临床医学院(解放军南京军区南京总医院),江苏省南京市210002

出　　处：《中国组织工程研究》2014年第35期5633-5639,共7页Chinese Journal of Tissue Engineering Research

基　　金：江苏省基础研究计划(自然科学基金)-面上研究项目(BK2012776)~~

摘　　要：背景:推测尿胰蛋白酶抑制剂可能在假体磨屑诱发的机体炎症反应中,对局部组织在缺血缺氧、长期慢性炎症环境下起保护作用,并可抑制破骨细胞的增殖和活化。目的:探讨尿胰蛋白酶抑制剂在核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)诱导RAW264.7细胞分化、增殖及形成成熟破骨细胞中的作用及与基质金属蛋白酶2和9的关系。方法:采用不同浓度尿胰蛋白酶抑制剂(0,500,5 000 U/mL乌司他丁)处理小鼠单核/巨噬细胞株RAW264.7后24,48,72 h。实验分为4组:空白组(RAW264.7细胞)、RANKL诱导组(0 U/mL乌司他丁)、500 U/mL乌司他丁组和5 000 U/mL乌司他丁组。结果与结论:1MTT法检测结果表明,尿胰蛋白酶抑制剂乌司他丁浓度在0-5 000 U/mL对RAW264.7细胞增殖无明显影响(P>0.05)。2抗酒石酸酸性磷酸酶染色法检测结果表明,与RANKL诱导组相比,乌司他丁组抗酒石酸酸性磷酸酶阳性细胞数量均显著减少(P<0.05),呈时间剂量依赖关系。3免疫组织化学结果显示,与RANKL诱导组比较,乌司他丁组的基质金属蛋白酶9染色阳性细胞百分率均显著降低。4Western blot结果显示,单独RAW264.7细胞仅表达少量基质金属蛋白酶9,加入RANKL 48 h后基质金属蛋白酶9蛋白大量表达,5 000 U/mL乌司他丁组培养72 h后,基质金属蛋白酶9蛋白表达显著减少。5明胶酶谱分析结果显示,与RANKL诱导组比较,5 000 U/mL乌司他丁组基质金属蛋白酶9活性水平均显著降低(P<0.05)。结果表明,尿胰蛋白酶抑制剂对RANKL诱导破骨细胞活化具有一定的抑制作用,且可降低基质金属蛋白酶9的表达水平及活性。BACKGROUND：It is presumed that urinary trypsin inhibitor could have protective effects on local and systemic tissues and could inhibit osteoclast proliferation and activation under long-term chronic inflammation conditions and in ischemic and anoxic environment which was induced by prosthetic wear. OBJECTIVE：To investigate the inhibitory effect of ulinastatin on receptor activator for nuclear factor-κb ligand-induced differentiation, proliferation and osteoclastogenesis of RAW264.7 cells and its effects on matrix metal oproteinase-2, matrix metal oproteinase-9 expression level and activity. METHODS：Mouse monocyte/macrophage cellline RAW264.7 was treated with different concentrations of urinary trypsin inhibitor （0, 500, 5 000 U/mL） for 24, 48 and 72 hours. Experiments were divided into four groups：the blank group （RAW264.7 cells）, receptor activator for nuclear factor-κb ligand-induced group （0 U/mL ulinastatin）, 500 U/mL ulinastatin group and 5 000 U/mL ulinastatin group. RESULTS AND CONCLUSION：（1） MTT results indicated that there was no significant difference on the proliferation of RAW264.7 cells treated with urinary trypsin inhibitor at 0-5 000 U/mL （P〉0.05） （2） Tartrate-resistant acid phosphatase staining results revealed that compared with receptor activator for nuclear factor-κb ligand-induced group, the number of tartrate-resistant acid phosphatase-positive cells was significantly less in the ulinastatin group （P〈0.05）, showing a time-dose dependent manner. （3） Immunohistochemisical results found that compared with receptor activator for nuclear factor-κb ligand-induced group, the percentage of matrix metal oproteinase-9-positive cells was apparently lower in the ulinastatin group. （4） Western blot assay results demonstrated that matrix metal oproteinase-9 expression was low in the RAW264.7 cells alone. At 48 hours after addition of receptor activator for nuclear factor-κb ligand, matrix metal oproteinase-9 protein expression was large. At 72 hours

关 键 词：植入物 人工假体 乌司他丁 骨组织工程 基质金属蛋白酶2 基质金属蛋白酶9 破骨细胞 人工关节 无菌性松动 骨溶解 江苏省自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]