CAJ | 학술논문

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(0，500，5000 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5000 U/mL乌司他丁组。结果与结论：①M TT法检测结果表明，尿胰蛋白酶抑制剂乌司他丁浓度在0-5000 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。④W estern 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，5000 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，5000 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。
배경：추측뇨이단백매억제제가능재가체마설유발적궤체염증반응중，대국부조직재결혈결양、장기만성염증배경하기보호작용，병가억제파골세포적증식화활화。목적：탐토뇨이단백매억제제재핵인자κB 수체활화인자배체(receptor activator for nuclear factor-κB ligand，RANKL)유도RAW264.7세포분화、증식급형성성숙파골세포중적작용급여기질금속단백매2화9적관계。방법：채용불동농도뇨이단백매억제제(0，500，5000 U/mL오사타정)처리소서단핵/거서세포주RAW264.7후24，48，72 h。실험분위4조：공백조(RAW264.7세포)、RANKL유도조(0 U/mL오사타정)、500 U/mL오사타정조화5000 U/mL오사타정조。결과여결론：①M TT법검측결과표명，뇨이단백매억제제오사타정농도재0-5000 U/mL대RAW264.7세포증식무명현영향(P>0.05)。②항주석산산성린산매염색법검측결과표명，여RANKL유도조상비，오사타정조항주석산산성린산매양성세포수량균현저감소(P<0.05)，정시간제량의뢰관계。③면역조직화학결과현시，여 RANKL 유도조비교，오사타정조적기질금속단백매9염색양성세포백분솔균현저강저。④W estern blot결과현시，단독RAW264.7세포부표체소량기질금속단백매9，가입RANKL 48 h후기질금속단백매9단백대량표체，5000 U/mL오사타정조배양72 h후,기질금속단백매9단백표체현저감소。⑤명효매보분석결과현시，여RANKL유도조비교，5000 U/mL오사타정조기질금속단백매9활성수평균현저강저(P<0.05)。결과표명，뇨이단백매억제제대 RANKL 유도파골세포활화구유일정적억제작용，차가강저기질금속단백매9적표체수평급활성。
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 after culture in the 5 000 U/mL ulinastatin group, matrix metal oproteinase-9 protein expression was evidently reduced. (5) Gelatin zymography results showed that compared with the receptor activator for nuclear factor-κb ligand-induced group, matrix metal oproteinase-9 expression was significantly lower in the 5 000 U/mL ulinastatin group (P<0.05). Results suggested that urinary trypsin inhibitor inhibited receptor activator for nuclear factor-κb ligand-induced osteoclastogenesis and diminished matrix metal oproteinase-9 expression and activity.