Functional analysis of defensins in human innate intestinall immunity.

小肠潘氏细胞通过感知细菌和分泌杀菌肽，特别是α-防御素，促进粘膜的先天免疫。目的：研究Toll样受体S及其相关分子在人小肠隐窝中的表达及功能。回肠黏膜取自手术标本，取自知情同意下回肠正常的结肠癌患者。EDTA解离法分离完整隐窝，进一步胶原酶解离制备隐窝单细胞。在单个隐窝或分离的Paneth细胞上进行RT-PCR实验，以检测α-防御素(HD5、HD6)、TLR1-10、CD14、MD2和MyD88mRNAs。用抗HD5和抗TLR抗体对分离的隐窝进行特异性定位分析。分离的完整隐窝暴露于细菌中，诱导Paneth细胞分泌。免疫印迹法检测分泌物对防御素敏感沙门氏菌和HD5的杀菌活性。在分泌检测系统中使用抗TLR2抗体进行抑制实验。TLR1-8、TLR10、CD14和MyD88mRNAs在单个隐窝和分离的Paneth细胞中均有表达。TLR仅表达于分离的隐窝内的Paneth细胞。TLR2抗体可抑制Paneth细胞对细菌的α-防御素分泌。人类小肠隐窝中的Paneth细胞表达Toll样受体，似乎与细菌感染的体外反应有关。

Small intestinal Paneth cells contribute to mucosal innate immunity by sensing bacteria and secreting microbicidal peptides, particularly alpha-defensins. To investigate the expression and function of Toll-like receptor (TLR)s and related molecules in crypts of human small intestine. Ileal mucosa were obtained from surgical specimens reseated from patients with colon cancer with normal ileum under informed consent. Intact crypts were isolated by EDTA dissociation, and further collagenase dissociation was conducted to prepare crypt single cells. RT-PCR experiments were performed on individual crypts or isolated Paneth cells to assay for alpha-defensins (HD5, HD6), TLR1-10, CD14, MD2 and MyD88 mRNAs. Specific localization was analyzed using anti-HD5 and anti-TLR antibodies in isolated crypts. Isolated intact crypts were exposed to bacteria to induce Paneth cell secretion. Secretions were assayd for bactericidal activity against defensin sensitive S.typhimunum and for HD5 by western blot analysis. Inhibition assay was performed using anti-TLR2 antibody in the secretion assay system. TLR1-8, TLR10, CD14 and MyD88 mRNAs were detected in single isolated crypts and isolated Paneth cells. TLR expression was restricted to Paneth cells in the isolated crypts. Alpha-defensin secretion by Paneth cells in response to bacteria was inhibited by TLR2 antibody pre-treatment. Paneth cells in human small intestinal crypts express Toll-like receptors that appear to be implicated in ex vivo responses to bacterial infection.