Claudin-7基因敲除诱导小鼠肠道炎症及其致癌机制的研究  

A study on intestinal inflammation and carcinogenesis in Claudin-7 knock-out mice

作　　者：丁磊[1] 高宏[1] 陈奕至[2] 黄静[1] 朱煜冰[1] 孔璐[3] 昌红[2] 彭吉润[4] 

机构地区：[1]首都医科大学附属北京世纪坛医院肿瘤外科,北京100038 [2]首都医科大学附属北京世纪坛医院病理科,北京100038 [3]首都医科大学肿瘤研究所生物化学及分子生物学系,北京100038 [4]北京大学人民医院肝胆外科中心,北京100038

出　　处：《中华普通外科杂志》2013年第2期120-124,共5页Chinese Journal of General Surgery

基　　金：国家自然科学基金资助项目（81030049）;首都医科大学基础一临床科研合作基金资助项目（12JLl5）;首都医科大学附属北京世纪坛医院院内基金资助项目（2012-C13）

摘　　要：目的初步探讨Claudin-7（Cln-7）基因敲除诱导小鼠肠道炎症及其致癌的机制。方法Cln-7基因敲除组小鼠和正常对照组小鼠出生4d后被处死并观察其肠道黏膜病理变化；采用蛋白印迹技术检测NF—KBp65、P—p65、P—IKBct、磷酸化组蛋白H3和环氧合酶-2在小鼠肠道中的表达情况；采用实时荧光定量PCR技术检测小鼠肠道组织中炎症因子IL-1p、IL-6、TNFct的基因表达水平；采用免疫组化技术检测43例结直肠癌患者肿瘤标本及15例正常大肠黏膜组织中Cln-7蛋白的表达，并分析其与临床病理因素之间的关系。结果Cln-7基因敲除小鼠在出生4d左右即出现明显脱水、且停止生长，并在出生后7d左右死亡。Cln-7基因敲除小鼠肠道黏膜出现明显的炎症、增殖现象；基因敲除小鼠肠道组织中NF—KBp65、P．p65、P—IKBct、磷酸化组蛋白H3、环氧合酶-2蛋白表达比对照组小鼠均显著升高（P〈0．01）；Real—timePCR结果表明基因敲除小鼠肠道组织中IL-1B、IL-6和TNFct的mRNA水平与对照组小鼠比较差异均具有统计学意义[小肠分别为（24．45±0．12）比（0．35±0．42），t=8．468，P：0．001；（34．26±0．10）比（1．63±0．05），t=8．673，P=0．001；（12．35±0．02）比（1．37±0．01），t=4．743，P=0．025。大肠分别为（31．25±0．15）比（1．56±0．02），t=5．436，P=0．005；（21．75±0．11）比（1．97±0．02），t=4．217，P=0．025；（18．25±0．09）比（1．66±0．01），t=4．217，P=0．025）]。免疫组化结果显示Cln-7在大肠癌组织中的表达明显低于正常大肠组织，且与大肠癌分化程度（x。=12．421，P=0．001）和有无肝脏及淋巴结转移有关（x。=9．462，P=0．001；X2=9．875，P=0．001）。结论敲除Cln-7基因可通过激活NF—KB等通路诱导肠道炎症，参与了结直肠癌的发生和发展过程；Cln-7基因可能是潜在的抑癌基因。Objective To explore the mechanisms of intestinal inflammation and colon carcinogenesis in Cln-7 gene knock-out mice. Methods Cln-7 gene knock out mice and normal control mice were sacrificed 4 days after birth, intestine mucosal pathomorphology changes were observed, and protein NF-KB p65, P-p65, P-IKBct, P-Histone H3 and Cyeloxygenase （COX-2） were detected using Western blot method, and inflammation cytokines such as IL-113, IL-6, TNFo~ were also investigated with real-time PCR. The expressions of Cln-7 protein were detected in colon cancer samples by immunohistochemistry SP method. Results Chronic dehydration and failing to grow was found in Cln-7 knock-out mice, which usually die within 7 days after birth. Severe intestinal defects including attenuated villi, epithelial cell sloughing, and mucosal ulcers could be detected. The protein expression levels of NF-KB p65 ,Phospho-p65, Phospho-IKBoL, Phospho-Histone H3 and COX-2 were all significantly increased （P 〈0. 01 ） in small and large intestines respectively in gene knockout mice compared with control normal mice. IL-1 [~, IL-6 and TNFc~ were up-regulated significantly at mRNA levels in intestines for knock outmouse compared with that in control normal mice using the real-time PCR （small intestine ：24. 45 -＋ 0. 12 vs. O. 35 -＋ 0. 42, t = 8.468,P = 0. 001 ;34. 26 -＋ 0. 10 vs. 1.63 ＋ 0. 05, t = 8. 673, P = 0. 001 ; 12. 35 -＋ 0. 02 vs. 1.37 ＋- 0.01, t = 4. 743, P = 0. 025 ; large intestine ： 31.25 ＋- 0.15 vs. 1.56 ＋-- 0.02, t = 5. 436, P = 0. 005 ; 21.75＋0.11 vs. 1.97-＋O. 02,t=4.217,P=0.025;18.25-＋0.09 vs 1.66-＋O. 01,t=4.217,P=0.025）. The expression of Cln-7 protein was remarkably decreased in colon cancer tissues compared with that in normal colon tissue, and its expression level was closely correlated with differentiation grade（ x2 = 12. 421, P = O. 001 ） and liver metastasis, as well as lymph node metastasis （x2 = 9. 462, P = O. 001 ; x2 = 9. 875, P = 0. 001 ）. Conclusions Cln-7 may participate in inflammation

关 键 词：结直肠肿瘤 小鼠 基因敲除 炎症趋化因子类 组蛋白类 Claudin-7基因 

分 类 号：R730.2[医药卫生—肿瘤]