A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon
A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon
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DOI：
10.3389/fnana.2019.00037
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发表时间：
2019-04-16
影响因子：
2.9
通讯作者：
Kimura, Hiroshi
中科院分区：
文献类型：
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作者：
Bellier, Jean-Pierre;Yuan, Pu-Qing;Kimura, Hiroshi
胆碱乙酰转移酶(ChAT)是合成乙酰胆碱(ACh)的酶，具有外显子跳跃剪接变异体，在外周神经系统(PNS)中优先表达，因此被称为外周ChAT(外周ChAT)。用预先制备的兔抗大鼠pChAT抗血清(RpChAT)进行免疫组织化学(IHC)研究各种动物的外周胆碱能结构。本研究旨在开发一种针对预测的人pChAT(HpChAT)蛋白的特异性抗血清。针对hpChAT蛋白独特的14个氨基酸序列，成功地制备了一种新的小鼠抗血清。我们用该抗血清(这里称为hpChAT抗血清)对人结肠提取液进行Western印迹，显示只有一条47 kDa的条带，与推测的hpChAT蛋白的大小相匹配。免疫组织化学染色显示，该抗血清在人结肠的许多神经细胞和神经纤维中呈强染色，而在脑内未见，这种染色模式与用rpChAT抗血清在各种动物的结肠中的染色模式似乎是一致的。在抗体吸收试验中，用预先吸附抗原肽的抗血清可完全阻断人结肠的hpChAT免疫反应。此外，人结肠的双重免疫荧光显示，抗hpChAT染色的结构也被抗rpChAT染色，反之亦然。HpChAT抗血清可用于鉴定细胞类型，如壁内神经丛中的Dogiel型细胞以及结肠肌和粘膜的纤维神经支配。本研究结果证明了hpChAT抗血清作为人类结肠免疫组织化学研究的一种新工具的特异性和可靠性，为定位其他人类三叉神经节组织中的胆碱能神经支配开辟了道路。
Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.