Examination of in vivo gelatinolytic activity in rheumatoid arthritis synovial tissue using newly developed in situ zymography and image analyzer.
Examination of in vivo gelatinolytic activity in rheumatoid arthritis synovial tissue using newly developed in situ zymography and image analyzer.
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发表时间：
2009-07
影响因子：
3.7
通讯作者：
W. Yoshida;M. Uzuki;J. Nishida;T. Shimamura;T. Sawai
中科院分区：
文献类型：
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作者：
W. Yoshida;M. Uzuki;J. Nishida;T. Shimamura;T. Sawai
目的应用新发展的原位酶谱(ISZ)方法和病理图像分析仪检测类风湿关节炎(RA)滑膜的体内明胶溶解活性，并探讨这一活性与RA若干特征的关系。方法收集类风湿性关节炎(RA)患者术中滑膜标本8例，并以8例骨性关节炎(OA)患者作为对照。对14例滑膜标本进行Larsen分级比较，其中III级4例，IV级5例，V级5例。术后即刻用OCT复合体冷冻。将冷冻切片应用于新开发的用于ISZ的明胶涂层FIZ膜(日本富士胶片公司)，并在37℃下孵育6小时。使用图像分析仪(图像处理器用于分析病理；IPAP)测量两个指标作为体内明胶溶解活性的指标：明胶溶解面积的光密度(ODG)和明胶溶解面积比率(RGA)。此外，我们还研究了这些指标与下列变量的关系：X线改变(Larsen分级)、临床资料(C反应蛋白浓度)、滑膜组织学评分(改良的Rooney评分)以及基质金属蛋白酶(MMPs)-2、MMP9、TIMP-1和TIMP-2的表达(用免疫组织化学方法评估)。结果RA滑膜的RGA明显高于OA，ODG明显低于OA，提示RA具有较高的明胶溶解活性。Larsen分级为IV级和V级的滑膜ODG显著低于III级，但RGA在不同分级之间无显著差异。明胶溶解活性(ODG或RGA)与CRP和改良的Rooney‘s组织学评分均无显著相关性。ISZ结果表明，明胶溶解区域主要分布在基质内，少量散布在基质中。免疫组织化学结果显示，明胶溶解区域有MMP2、MMP9、TIMP 1和TIMP 2的表达。结论在体内，RA患者滑膜的明胶溶解活性强于OA患者。结果还表明，在Larsen分级为IV或V级的患者中，RA滑膜细胞的明胶溶解活性比在III级患者中更强，尽管明胶溶解面积相似。由光密度和明胶溶解面积表示的明胶溶解活性在不同地区，甚至在同一标本中也不同，这表明蛋白酶及其抑制物的产生是不平衡的。我们认为，该酶谱方法有助于阐明RA滑膜的生物酶活性。
OBJECTIVE The aim of this study was to examine in vivo gelatinolytic activity of rheumatoid arthritis (RA) synovium using a newly developed in situ zymography (ISZ) method and pathological image analyzer, and to evaluate the relationship between this activity and several features on RA. METHODS A total of 8 samples of synovium were obtained from RA patients during surgery, and 8 samples from osteoarthritis (OA) patients were examined as controls. Furthermore, total 14 samples of syovium were obtained for comparison among radiographical classifications as Larsen grade (4 cases of grade III, 5 cases of grade IV and 5 cases of grade V). These specimens were frozen with OCT compound immediately after surgery. Frozen sections were applied to a newly developed gelatin-coated FIZ film (Fuji Film Co.Tokyo.Japan) designed for use ISZ, and incubated at 37 degrees C for 6 hours. Using an image analyzer (image processor for analytical pathology; IPAP), two variables were measured as indicators of in vivo gelatynolytic activity: optical density of gelatinolyzed area (ODG), and ratio of gelatinolyzed area (RGA). Also, we investigated the relationship between these indicators and the following variables: radiographic changes (Larsen grades), clinical data (C-reactive protein concentration), histological score of synovial tissue (modified Rooney's score), and expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 (assessed by immunohistochemistry). RESULTS RA synovium had significantly higher RGA and lower ODG than OA, indicating higher gelatinolytic activity in RA. Synovium from cases with Larsen grade IV or V had significantly lower ODG than cases with grade III, but there was no significant difference in RGA between grades. There was no significant correlation between gelatinolytic activity (ODG or RGA) and either CRP or modified Rooney's Histological Score. The results of ISZ indicate that the gelatinolyzed areas were mainly localized in the lining area, with a small amount scattered throughout the stroma. The results of immunohistochemistry indicate that MMP-2, MMP-9, TIMP-1 and TIMP-2 were expressed in areas of gelatinolysis. CONCLUSIONS The present results indicate that in vivo gelatinolytic activity of synovium is stronger in RA than in OA. They also indicate that gelatinolytic activity of RA synovial cells is stronger in cases with Larsen grade IV or V than in cases with grade III, although the gelatinolyzed area is similar. Gelatinolytic activity, as indicated by optical density and the gelatinolyzed area, differed between regions, even within the same specimen, suggesting an imbalance between production of proteinases and their inhibitors. We believe that the present zymography method can contribute to the elucidation of biological enzymatic activity of RA synovium.