Practical techniques for detection of Toll-like receptor-4 in the human intestine.
Practical techniques for detection of Toll-like receptor-4 in the human intestine.
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检测人肠道中 Toll 样受体 4 的实用技术。
DOI：
10.1007/978-1-59745-541-1_21
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Fukata,Masayuki
中科院分区：
文献类型：
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作者：
Ungaro,Ryan;Abreu,MariaT;Fukata,Masayuki
人类肠道在各种各样的管腔微生物的存在下进化。为了维持肠道内环境的稳定，必须严格调节对这些微生物的粘膜免疫应答。肠道需要能够对病原生物作出反应，同时保持对正常肠道植物群的耐受性。Toll样受体（TLR）在这种微妙的平衡中发挥着重要作用。TLR是跨膜非催化受体蛋白，其通过识别微生物的结构保守的分子模式来诱导对微生物的先天性和适应性免疫应答的激活。肠道上皮细胞TLRs的表达通常被下调以维持对肠道微生物的免疫耐受，但许多实验方法很难检测到肠粘膜中TLRs的低表达，这是人类肠道TLR研究面临的挑战之一。定量方法如PCR在其检测组织样品中特定细胞类型的TLR表达的能力方面受到限制，这在研究TLR信号传导对病理状况的贡献时可能是重要的。在这方面，免疫组织化学（IHC）是有利的，因为人们可以可视化给定组织样品的正常和病理部分内的靶蛋白的分布和定位。我们通过免疫荧光（IF）和IHC方法发现，人类结直肠癌的一个子集过表达TLR4。TLR4在癌组织中的定位通常是不规则的，这使得IHC成为检查这些变化的合适方法。我们将描述我们目前的技术来检测TLR4在石蜡包埋的人大肠切片。建立一个实用的IHC技术，可以提供实验室之间的一致结果将显着提高TLRs在人类肠道健康和疾病中的作用的理解。
The human intestine has evolved in the presence of a diverse array of luminal microorganisms. In order to maintain intestinal homeostasis, mucosal immune responses to theses microorganisms must be tightly regulated. The intestine needs to be able to respond to pathogenic organisms while at the same time maintain tolerance to normal commensal flora. Toll-like receptors (TLRs) play an important role in this delicate balance. TLRs are transmembrane noncatalytic receptor proteins that induce activation of innate and adaptive immune responses to microorganisms by recognizing structurally conserved molecular patterns of microbes. Expression of TLRs by intestinal epithelial cell is normally down-regulated to maintain immune tolerance to the luminal microorganisms.One of the challenges of TLR research in the human intestine is that it is difficult for many experimental methods to detect very low expression of TLRs within the intestinal mucosa. Quantitative methods such as PCR are limited in their ability to detect TLR expression by specific cell types within a tissue sample, which can be important when studying the contribution of TLR signaling to pathological conditions. In this regard, immunohistochemistry (IHC) is advantageous in that one can visualize the distribution and localization of target proteins within both normal and pathologic parts of a given tissue sample. We found that a subset of human colorectal cancers over-express TLR4 by means of immunofluorescence (IF) and IHC methods. Localization of TLR4 within cancer tissue often appears to be patchy, making IHC an appropriate way to examine these changes. We will describe our current techniques to detect TLR4 in paraffin-embedded human large intestine sections. Establishing a practical IHC technique that may provide consistent results between laboratories will significantly enhance understanding of the role of TLRs in human intestinal health and disease.