Functional analysis of defensins in human innate intestinall immunity.

小肠潘氏细胞通过感应细菌和分泌杀微生物肽，特别是α-防御素，促进粘膜先天免疫。目的探讨Toll样受体（TLR）及其相关分子在人小肠隐窝中的表达及功能。回肠粘膜取自在知情同意的情况下从具有正常回肠的结肠癌患者重新切除的手术标本。通过EDTA解离分离完整的隐窝，并进行进一步的胶原酶解离以制备隐窝单细胞。对单个隐窝或分离的潘氏细胞进行RT-PCR实验以测定α-防御素（HD 5、HD 6）、TLR 1 -10、CD 14、MD 2和MyD 88 mRNA。在分离的隐窝中使用抗HD 5和抗TLR抗体分析特异性定位。将分离的完整隐窝暴露于细菌以诱导潘氏细胞分泌。通过蛋白质印迹分析测定分泌物对防御素敏感的鼠伤寒沙门氏菌的杀菌活性和对HD 5的杀菌活性。在分泌测定系统中使用抗TLR 2抗体进行抑制测定。在单个分离的隐窝和分离的Paneth细胞中检测到TLR 1 -8、TLR 10、CD 14和MyD 88 mRNA。TLR表达仅限于孤立的隐窝中的潘氏细胞。TLR 2抗体预处理可抑制Paneth细胞分泌α-防御素。人小肠隐窝中的潘氏细胞表达Toll样受体，其似乎与对细菌感染的离体反应有关。

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.