CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells
CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells
复制标题
DOI：
10.1053/gast.2003.50153
复制
发表时间：
2003-04-01
期刊：
影响因子：
29.4
通讯作者：
Podolsky, DK
中科院分区：
文献类型：
--
作者：
Hisamatsu, T;Suzuki, M;Podolsky, DK
背景和目标：CARD 15/NOD 2基因（一种假定的细胞内模式识别受体）的突变与克罗恩病的风险有关。由于肠上皮细胞对肠道微生物起着屏障作用，我们研究了CARD 15/NOD 2在肠上皮细胞中的表达和功能。研究方法：通过逆转录聚合酶链反应（RT-PCR）评估肠上皮细胞系和原代肠上皮细胞中CARD 15/NOD 2信使RNA（mRNA）的表达。使用SW 480细胞系通过北方印迹法测定细胞因子对CARD 15/NOD 2表达的调节。使用抗CARD 15/NOD 2抗血清通过免疫沉淀和免疫印迹的组合来评估SW 480细胞中的活性CARD 15/NOD 2蛋白。为了鉴定CARD 15/NOD 2在肠上皮细胞中的功能作用，使用用野生型CARD 15/NOD 2或与克罗恩病相关的3020 insC突变体稳定转染的Caco 2细胞进行鼠伤寒沙门氏菌的庆大霉素保护测定。结果CARD 15/NOD 2 mRNA在肠上皮细胞系和原代肠上皮细胞中均有表达。肿瘤坏死因子（TNF α）可上调SW 480细胞CARD 15/NOD 2 mRNA和蛋白表达。存活的内化S.稳定转染CARD 15/NOD 2表达质粒的Caco 2细胞中鼠伤寒沙门氏菌的表达低于未转染的Caco 2细胞或MOCK转染子。相反，与克罗恩病相关的变体的表达不能限制细菌[存活]。结论：CARD 15/NOD 2在肠上皮细胞中表达，并且可能作为对管腔细菌的先天粘膜应答的关键组分作为抗菌因子。这项活动的失败可能会导致克罗恩病的发生。
Background & Aims: Mutations in the CARD15/NOD2 gene, a putative intracellular pattern recognition receptor, have been linked to the risk for Crohn's disease. Because intestinal epithelial cells play a role as the barrier to luminal microorganisms, we investigated the expression and function of CARD15/NOD2 in intestinal epithelial cells. Methods: Expression of CARD15/NOD2 messenger RNA (mRNA) in intestinal epithelial cell lines and primary intestinal epithelial cells was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Regulation of expression of CARD15/NOD2 by cytokines was determined by Northern blot using the SW480 cell line. Active CARD15/ NOD2 protein in SW480 cells was assessed by the combination of immunoprecipitation and immunoblotting using anti-CARD15/NOD2 antisera. To identify the functional role of CARD15/NOD2 in intestinal epithelial cells, gentamicin protection assays of Salmonella typhimurium were performed using Caco2 cells stably transfected with either wild-type CARD15/NOD2 or the 3020insC mutant associated with Crohn's disease. Results. CARD15/NOD2 mRNA was expressed in both intestinal epithelial cell lines and primary intestinal epithelial cells. CARD15/NOD2 mRNA and protein were up-regulated by tumor necrosis factor et (TNFalpha) in SW480 cells. The number of viable internalized S. typhimurium in Caco2 cells stably transfected with CARD15/NOD2 expression plasmid was lower than untransfected Caco2 cells or MOCK transfectant. In contrast, expression of a variant associated with Crohn's disease was unable to constrain bacteria[ survival. Conclusions: CARD15/NOD2 is expressed in intestinal epithelial cells and may serve as a key component of innate mucosal responses to luminal bacteria as an antibacterial factor. Failure in this activity may contribute to the development of Crohn's disease.