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⊙基础研究 BASIC RESEARCH⊙
溃疡性结肠炎组织中NF-κB，COX-2和iNOS表达的意义
张 可，邓长生，朱尤庆，杨院平,张燕敏
张可，邓长生，朱尤庆，杨院平，张燕敏，武汉大学中南医院消化内科 湖北省武汉市 430071
张可，女，1977-03-14生，湖北省黄陂人，汉族.武汉大学中南医院消化内科硕士研究生.
项目负责人 张可，430071，武汉大学中南医院消化内科，湖北省武汉市. carrie_jones@263.net
电话: 027-87330314
收稿日期 2002-01-11 接受日期 2002-03-18
Significance of nuclear factor-κB, cyclooxygenase 2 and inducible nitric oxide synthase expression in human ulcerative colitis tissues
Ke Zhang, Chang-Sheng Deng, You-Qing Zhu, Yuan-Ping Yang, Yan-Min Zhang
Ke Zhang,Chang-Sheng Deng,You-Qing Zhu,Yuan-Ping Yang, Yan-Min Zhang,Department of Gastroenterology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China
Correspondence to:Ke Zhang, Department of Gastroenterology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China. carrie_jones@263.net
Received 2002-01-11 Accepted 2002-03-18
Abstract
AIM:The induction of transcription factor NF-κB modulates the expression of multiple genes in immunal and inflammatory reations Ulcerative colitis (UC) exists abnormal activation of epithelial cells, lymphocytes and macrophages and uncontrolled cytokine production. The authors examined the expression and
distribution of NF-κB, p65, COX-2 and inducible nitric oxide synthase (iNOS), of the latter two having NF-κB binding sites in their promoters, to investigate the significance of them in the pathogenesis of UC and the relationship between them.
METHODS:The expression of NF-κBp65, COX-2 and iNOS was examined in 69 cases of paraffin-embedded tissues by immunohistochemical methods. Of the 69 cases, 39 were from endoscopic biopsies of active UC and 30 were normal controls.
RESULTS:P65,COX-2 and iNOS were expressed in all the UC tissues, mainly in epithelial cells, and also inflammatory cells in lamina propria and vascular endothelial cells in variant degrees. They all were expressed negatively or weakly in controls. Their expression was significantly higher in UC tissues than that
in controls (P
<0.01). The expression of p65 was related to endoscopic and pathologic classification. It was significantly higher in endoscopic class II than that in class I (5.8±2.6 vs 3.6±1.9, P
<0.05) and was significantly higher in pathologic class II and III than that in class I (6.1±2.4, 7.3±2.5 vs 4.0±2.3; P
<0.05, P
<0.01, respectively). The expression of iNOS was related to pathologic classification. It was significantly higher in pathologic class III than that in class I and II (7.8±2.5 vs 4.6±2.3, 5.0±1.6; P
<0.01, P
<0.05, respectively). The expression of COX-2 did not relate to endoscopic or pathologic classification, or disease severity. The expression of p65 significantly correlated with that of COX-2 and iNOS (rs1=0.713,rs1=0.706, respectively; P
<0.01). The expression of COX-2 was significantly correlated with that of iNOS (rs1=0.854, P
<0.01).
CONCLUSION:The induction of NF-κBp65 participates in the occurrance and development of UC. COX-2 and iNOS involves in the process of inflammation and damage, and perhaps iNOS plays a more pivotal role. Regulating mechanisms of COX-2 and iNOS expression may be resemble and both have a direct
relationship with the induction of NF-κB.
Zhang K, Deng CS,Zhu YQ, Yang YP, Zhang YM. Significance of nuclear factor-κB, cyclooxygenase 2 and inducible nitric oxide synthase expression in human ulcerative colitis tissues.Shijie Huaren Xiaohua Zazhi 2002;10(5):575-578
摘要
目的:转录因子NF-κB的诱导，调控着免疫和炎症反应中众多基因的表达. 溃疡性结肠炎(ulcerative colitis,UC)存在上皮细胞、淋巴细胞、巨噬细胞的异常激活及细胞因子的表达失调. 我们检测了UC组织中NF-κBp65，及两种启动子含NF-κB结合位点的蛋白COX-2和iNOS的表达和分布，探讨他们在UC发病机制中的作用,及三者之间的关系.
方法:用免疫组化SP法检测39例活动期溃疡性结肠炎内镜活检标本及30例正常对照石蜡包埋组织中NF-κBp65，COX-2和iNOS的表达情况.
结果:UC组p65，COX-2和iNOS均为阳性表达，主要分布于上皮细胞. 固有层炎性细胞及血管内皮细胞也有程度不同的表达.对照组均为阴性或弱阳性表达.三者在UC组的表达与对照组相比，差异均有非常显著性（P
<0.01）. p65的表达与内镜及病理分级有关.内镜II级与I级比较（5.8±2.6 vs 3.6±1.9），差异显著（P
<0.05）.病理II，III级与I级比较（6.1±2.4, 7.3±2.5 vs 4.0±2.3），差异显著（P
<0.05， P
<0.01）. iNOS的表达与病理分级有关，III级与I，II级比较（7.8±2.5 vs 4.6±2.3, 5.0±1.6），差异显著（P
<0.01, P
<0.05）.COX-2的表达在病情轻重，内镜及病理分级间差异无显著性（P
>0.05）. p65与COX-2，iNOS表达显著相关（rs1 分别为0.713, 0.706; P
<0.01），COX-2与iNOS表达显著相关（rs1=0.854, P
<0.01）.
结论:NF-κB的诱导参与UC的发生、发展. COX-2，iNOS也参与UC的炎症及损伤过程，可能iNOS发挥的作用更显著.COX-2，iNOS表达的调控机制可能相似，且与NF-κBp65的诱导有直接关系.
张可，邓长生，朱尤庆，杨院平,张燕敏. 溃疡性结肠炎组织中NF-κB，COX-2和iNOS表达的意义. 世界华人消化杂志 2002;10(5): 575-578
0 引言
NF-κB（nuclear factor-κB）的表达和激活状态的改变，导致炎症反应的持续化，可能在人类溃疡性结肠炎（ulcerative colitis, UC）的发生、发展中起重要作用［1］.在NF-κB家族成员中，p65亚单位是主要的促炎亚单位［2］.UC炎症肠组织中绝大多数二聚体含有一个p65亚单位. COX-2，iNOS是启动子含NF-κB结合位点的两种诱生型酶［3，4］，在大多数正常组织中不表达，而在炎症时高度表达，分别催化PGs，NO的大量合成，在UC的病理过程中发挥着广泛而复杂的作用. 我们检测了活动期UC组织中NF-κBp65，COX-2和iNOS蛋白的表达，探讨他们在UC发病机制中的作用及三者之间的关系.
1 材料和方法
1.1 材料 1998-06/2000-08武汉大学中南医院病理科UC内镜取材石蜡包埋标本39例.均符合1993年太原全国慢性非感染性肠道疾病学术研讨会制定的溃疡性结肠炎诊断标准［5］.男28例，女11例，年龄8～65（平均38）岁.均为活动期，按Truelove-Witts标准进行病情轻重分级［5］：轻度21例，中度11例，重度7例；按Truelove标准进行内镜分级［5］：I级16例，II级20例，III级3例；按Truelove-Richards标准进行病理学分级［5］：I级25例，II级8例，III级6例；按病变累及部位分：直肠炎21例，直乙结肠炎15例，左半结肠炎3例. 30例正常对照取自同期结肠癌切除肠段断端的石蜡包埋标本，距瘤缘约20cm，均经病理学确认为正常组织. 鼠抗人NF-κBp65单克隆抗体，兔抗人COX-2多克隆抗体和兔抗人iNOS多克隆抗体由美国Santa Cruz公司提供. 鼠SP及兔SP试剂盒购自美国Zymed公司.DAB显色剂和EDTA抗原修复液（1mmol/L pH8.0）购自北京中山生物技术公司.
1.2 方法 免疫组化检测步骤如下：组织切片脱蜡至水，30ml/LH2O2浸泡，37℃ 15min，PBS冲洗;置EDTA抗原修复液中，95℃ 10min,自然冷却至室温,PBS冲洗；滴加非免疫性动物血清，37℃ 15min；滴加单克隆抗体NF-κBp65和多克隆抗体COX-2, iNOS,工作浓度依次为1∶250，1∶400，1∶