何　伟¹，白万柱¹，王晓宇¹，宿杨帅¹，张晓宁¹，景向红¹，朱　兵¹

(1中国中医科学院针灸研究所,北京100700)

摘要：研究目的：研究大鼠外耳耳甲部的神经活性物质特征。研究方法：SD大鼠6只，体质量180-200g。将大鼠4%多聚甲醛心脏灌流固定，用剪刀剪下外耳耳甲部组织，4%多聚甲醛后固定2h，25%蔗糖过夜脱水。然后组织进行冰冻切片，并用免疫组化和免疫荧光染色技术，观察大鼠外耳耳甲部组织辣椒素受体1（VR1），降钙素基因相关肽（CGRP），酪氨酸羟化酶（TH）等的表达，并对将放大倍数相同的图片中三种物质阳性表达神经纤维的总长度进行统计。研究结果：大鼠耳甲部的皮肤及皮下组织都存在VR1，TH和CGRP阳性表达。以外耳软骨为界，耳甲腹侧的皮肤及皮下组织三种神经活性物质的表达：与TH， CGRP阳性神经纤维长度比较， VR1阳性神经纤维长度最长（P<0.01）；TH， CGRP表达阳性神经纤维长度比较无统计学意义（P>0.05）。大鼠耳甲背侧皮肤及皮下组织神经活性物质的表达：与CGRP表达阳性神经纤维长度比较，VR1 ，TH表达阳性神经纤维长度更长（P<0.01）；TH与CGRP表达阳性神经纤维长度比较，差异无统计学意义（P>0.05），见图5。大鼠耳甲背侧皮下组织神经束内三种物质表达荧光强度比较：与 TH比较，VR1，CGRP阳性表达荧光强度更强（P均<0.01）；与 CGRP比较，VR1阳性表达荧光强度更强（P<0.01）。结论：大鼠外耳耳甲部存在CGRP， VR1，TH表达阳性神经纤维，三种物质的表达部位和量均有差异，以VR1表达最多。

关键词：耳甲；免疫组化；CGRP；TH；VR1

The characteristics of the neuroactive substances in the auricular concha of the rats

HE Wei1, BAI Wanzhu1, WANG Xiaoyu1, SU Yangshuai1, ZHANG Xiaoning1, JING Xianghong1, ZHU Bing1

（1 Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700）

Abstract: Objective: To investigate the characteristics of the active substances of the nerve fibers innervating the auricular concha in the rats. Methods: Six Sprague Dawley rats, weighing 180-220 g, were introduced. The rats were fixed with 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde via transcardial perfusion. Then the tissues of the auricular concha were collected. Following perfusion–fixation, the collected tissues were post-fixed in 4% paraformaldehyde at 4 °C for 2 hours and cryoprotected in phosphate-buffered 20% sucrose at 4 °C for 24 hours. Following post-fixation, the tissue was embedded in artificial medium (Shandon Cryomatrix™, 120 mL, Thermo Scientific, USA), frozen, and cut into 20μm sections. Then immunofluorescence staining techniques were performed to investigate the expression of calcitonin-gene-related peptide (CGRP), Tyrosine Hydroxylasen (TH), and Vanilloid Receptor Subtype 1 (VR1) ofthe sections. The nucleus was labelled with DAPI. Slides were recorded with confocal imaging system (FV1200, Olympus, Japan) and analyzed by the Olympus Image Processing Software by an investigator. Approximately 4 randomized sections from each ratswere counted for the sum length of positive nerve fibers, and the average fluorescent intensity. All immunohistochemistry for each staining combination was performed at the same time to ensure staining consistency. Results: The immunopositive expression of VR1, TH and CGRP was observed in the cutaneous and subcutaneous tissue of the concha of the rat. As to the cutaneous and subcutaneous tissue ventral to the auricular cartilage: the length of the VR1 positive nerve was longer than the length of the TH or VR1 positive nerve (P<0.01). There were no significant difference between the length of the TH and the VR1 positive nerve (P>0.05). As to the cutaneous and subcutaneous tissue dorsal to the auricular cartilage: the length of the VR1 or theTH positive nerve was longer than that of the CGRP positive nerve respectively (P<0.01). There were no significant difference between the length of the VR1 and the CGRP positive nerve (P>0.05). The average fluorescent intensity of VR1 the CGRP positive nerve was more than that of the TH positive nerve (P<0.01). The average fluorescent intensity of VR1 positive nerve was more than that of the CGRP positive nerve (P<0.01). Conclusion: There were CGRP, VR1 and TH positive nerve fibers in the auricular concha of rats. The distribution and the amount of the CGRP, VR1 and TH positive nerve fibers were different, and the expression of VR1 was the most.

Key words: Auricular acupuncture, Immunohistochemistry, CGRP, TH, VR1