CAJ | 학술논문

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-κB p65、P-p65、P-IκBα、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-1β、IL-6、TNFα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-κB p65、P-p65、P-IκBα、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-time PCR结果表明基因敲除小鼠肠道组织中IL-1β、IL-6和TNFα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(x2 =12.421,P=0.001)和有无肝脏及淋巴结转移有关(x2=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-κB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.
목적 초보탐토Claudin-7(Cln-7)기인고제유도소서장도염증급기치암적궤제.방법 Cln-7기인고제조소서화정상대조조소서출생4d후피처사병관찰기장도점막병리변화；채용단백인적기술검측NF-κB p65、P-p65、P-IκBα、린산화조단백H3화배양합매-2재소서장도중적표체정황；채용실시형광정량PCR기술검측소서장도조직중염증인자IL-1β、IL-6、TNFα적기인표체수평；채용면역조화기술검측43례결직장암환자종류표본급15례정상대장점막조직중Cln-7단백적표체,병분석기여림상병리인소지간적관계.결과 Cln-7기인고제소서재출생4d좌우즉출현명현탈수、차정지생장,병재출생후7d좌우사망.Cln-7기인고제소서장도점막출현명현적염증、증식현상；기인고제소서장도조직중NF-κB p65、P-p65、P-IκBα、린산화조단백H3、배양합매-2단백표체비대조조소서균현저승고(P＜0.01)；Real-time PCR결과표명기인고제소서장도조직중IL-1β、IL-6화TNFα적mRNA수평여대조조소서비교차이균구유통계학의의[소장분별위(24.45 ±0.12)비(0.35 ±0.42),t=8.468,P =0.001;(34.26±0.10)비(1.63±0.05),t=8.673,P =0.001;(12.35±0.02)비(1.37 ±0.01),t =4.743,P=0.025.대장분별위(31.25 ±0.15)비(1.56 ±0.02),t=5.436,P =0.005;(21.75 ±0.11)비(1.97 ±0.02),t=4.217,P =0.025;(18.25±0.09)비(1.66±0.01),t=4.217,P=0.025)].면역조화결과현시Cln-7재대장암조직중적표체명현저우정상대장조직,차여대장암분화정도(x2 =12.421,P=0.001)화유무간장급림파결전이유관(x2=9.462,P=0.001;x2=9.875,P=0.001).결론 고제Cln-7기인가통과격활NF-κB등통로유도장도염증,삼여료결직장암적발생화발전과정；Cln-7기인가능시잠재적억암기인.
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-κB p65,P-p65,P-IkBα,P-Histone H3 and Cycloxygenase (COX-2) were detected using Western blot method,and inflammation cytokines such as IL-1β,IL-6,TNFα 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-κB p65,Phospho-p65,Phospho-IκBoα,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-1β,IL-6 and TNFα were up-regulated significantly at mRNA levels in intestines for knock out mouse compared with that in control normal mice using the real-time PCR (small intestine:24.45 ± 0.12 vs.0.35 ± 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±0.02,t=4.217,P=0.025;18.25±0.09vs 1.66±0.01,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 =0.001) and liver metastasis,as well as lymph node metastasis (x2 =9.462,P =0.001 ; x2 =9.875,P =0.001).Conclusions Cln-7 may participate in inflammation and carcinogenesis through NF-κB pathway,and be a potential tumor suppressor gene for colon cancer.