IFNGR在山羊腹腔肠系膜前神经节的表达

doi:10.3864/j.issn.0578-1752.2015.05.14

摘要/Abstract

摘要： 【目的】为了解山羊腹腔肠系膜前神经节（celiac cranial mesenteric ganglion, CCMG）是否具备接受γ-干扰素（interferon-γ, IFN-γ）作用的条件，即是否有IFN-γ受体（interferon-γ receptor, IFNGR）存在。【方法】取健康雌、雄性成年山羊的CCMG，经40 g·L^-1多聚甲醛磷酸缓冲固定液固定4 h后，用自来水冲洗10 h，经梯度酒精脱水、二甲苯透明、石蜡包埋后，制作石蜡切片。石蜡切片分4套：第1套做H.E.染色；第2套经脱蜡复水，抗原热修复后，再进行免疫组化SP法染色；第3套经免疫组化SP法染色后用苏木精复染；第4套用作阴性对照组。染色后在光学显微镜下观察记录切片，使用Motic数码显微镜下拍照后，对图片分析处理，计算CCMG的IFNGR相对表达量。将相对表达量数据用SPSS18.0软件分析处理，并利用单因子方差分析（One-way ANOVA, LSD）进行显著性检验。取健康雌性、雄性成年山羊的CCMG，经充分研磨后，使用总RNA提取试剂盒提取总RNA，再以牛的IFNGR1 cDNA序列为模板设计IFNGR1的引物，克隆山羊IFNGR1 cDNA序列并进行扩增。将扩增产物使用琼脂糖凝胶电泳检测是否有目的条带，并进行DNA双向测序。将测序结果在NCBI上进行比对和确认，并与其他的物种的序列进行了同源性比对。【结果】免疫组化染色显示，在CCMG内IFNGR免疫阳性产物分布广泛，在神经元胞体、卫星细胞及神经纤维中均有不同程度的着色。其中神经元均为IFNGR免疫阳性。几乎所有的神经元胞质为黄褐色，IFNGR呈中等阳性，仅有少数神经元胞质染色弱为淡黄色，IFNGR呈弱阳性；在神经元细胞核中，核质部分明显呈棕褐色，IFNGR为强阳性，而核仁染色相对较弱或无着色，IFNGR为弱阳性或阴性。在非神经元结构中，卫星细胞染色呈棕褐色或棕色，IFNGR为强阳性或中等阳性。神经纤维和血管内皮细胞为黄褐色或淡黄色，IFNGR为中等阳性或弱阳性。图像分析表明与其他非神经元结构成分相比，神经元胞体IFNGR的相对表达量差异性极显著（P＜0.01）。PCR检测结果显示，IFNGR1 PCR产物的泳道在300 bp与400 bp处看到一个清晰的白色条带。测序结果显示，在山羊CCMG中克隆出了376 bp的IFNGR1的部分cDNA序列。通过在NCBI上与其他物种的基因序列同源性比对显示，这段序列与绵羊的同源性最高（98%，XM_004011371.1），其次为牛（97%，NM_001035063.1），野猪（84%，NM_001177907.1），家兔（73%，XR_085137.1），与褐家鼠（66%，NM_053783.1）的同源性最低。【结论】在山羊CCMG中有IFNGR的表达与分布，并且IFNGR主要在交感节后神经元表达与分布。所以CCMG具备对IFN-γ刺激做出反应的条件，CCMG的交感节后神经元可能是 IFN-γ作用的主要靶细胞，提示山羊CCMG可能作为IFN-γ对胃肠道免疫调节途径和自主神经对胃肠道调节的神经途径之间相互协调的关键点。

关键词: &gamma, -干扰素受体；腹腔肠系膜前神经节；免疫组化SP法；PCR；山羊

Abstract: 【Objective】This experiment was conducted to detect the existence of IFNGR in the celiac superior mesenteric ganglion (CCMG) in goats.【Method】The CCMG were taken from male and female goats, respectively. The CCMG were fixed with 40 g·L^-1 paraformaldehyde in phosphate buffer for 4 hours. Fixation was followed by thorough rinsing in water for 10 h, then dehydrated in graded alcohols, embedded in paraffin and cut into thick sections to prepare for immunohistochemical and H.E. staining. The sections were divided into 4 groups. The first group stained with H.E. staining. The second group was deparaffinized with xylene and ethanol, and processed for immunohistochemical SP staining. The third group was stained with immunohistochemical SP staining and hematoxylin counterstain. The last group was used as the negative control group. After staining, the specimens were examined and photographed with Motic microscope with digital camera. The digital images were analytically processed and got the relative expression of IFNGR with image-analysis software. All data were processed by SPSS18.0 software, mono factor analysis of variance was used for Significance test. After CCMG thoroughly grinded, the total RNA was extracted with extract RNA kit, and primers were designed according to IFNGR1 cDNA sequence of bovine. Then, the IFNGR1 cDNA of goat was cloned and amplified by PCR. The amplification was detected whether there was the target band by agarose gel electrophoresis and DNA sequencing. The sequencing results were compared and affirmed in the NCBI, and compared with sequences of other species. 【Result】The results of immumohistochemical staining showed that: IFNGR immunopositivity was widely distributed in CCMG of goat, and different levels of staining existed in neurons, satellite cells and nerve fibers. All the neurons were IFNGR positive. Almost every cytoplasm of neurons were dyed tan, were IFNGR positive, only a few cytoplasmic stained weak were weakly positive. In the nucleus of neurons, the karyoplasms were strongly positive, and the nucleolus were weakly positive or negative. In the non-neuronal structure, the satellite cells and nerve fibers were moderately or weakly positive. The relative expression of IFNGR in neuron was very significantly higher than that in non-neuronal structure (P＜0.01). The PCR detection results showed that there was a clear white target band at 300-400 bp in the line of PCR production. The DNA sequenced analysis showed that the full length of IFNGR1 gene amplified by PCR was 376 bp. Homologous comparison with other species indicated that IFNGR1 gene of goat homology with sheep (98%, XM_004011371.1) was the highest, followed by cattle (97%, NM_001035063.1), Sus scrofa (84%, NM_001177907.1), Oryctolagus cuniculus (73%, XR_085137.1) and Rattus norvegicus (66%, NM_053783.1), in the NCBI.【Conclusion】The results suggested that the IFNGR in the CCMG of goats mainly expressed and located in sympathetic postganglionic neurons which were provided with the conditions for the role of IFN-γ, which implied that the CCMG may act as critical point to coordinate the immune regulation of IFN-γ with neuroregulation of autonomic nerve on gastrointestinal tract.

Key words: interferon-&gamma, receptors, celiac superior mesenteric ganglion, immunohistochemical SP method, PCR, goat

李强，王志豪，金秀芳，徐永平，郭晓，董伟，刘文刚. IFNGR在山羊腹腔肠系膜前神经节的表达[J]. 中国农业科学, 2015, 48(5): 959-965.

LI Qiang, WANG Zhi-hao, JIN Xiu-fang, XU Yong-ping, GUO Xiao, DONG Wei, LIU Wen-gang. Expression of IFNGR in the Celiac Superior Mesenteric Ganglion of Goats[J]. Scientia Agricultura Sinica, 2015, 48(5): 959-965.

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