摘要：目的观察免疫应答时胸腺内降钙素基因相关肽(CGRP)能神经纤维的可塑性,为CGRP在胸腺的调节作用提供线索。方法用PPD免疫Balb/c小鼠 ，胸腺切片用免疫组织化学法处理，光镜下观察。结果在正常Balb/c小鼠胸腺被囊、小梁内有较丰富的CGRP能神经纤维分布，胸腺实质中也 有阳性纤维存在。用 PPD免疫28d后，胸腺被囊、小梁内血管周围的CGRP阳性纤维明显增加，这 些纤维直径较粗、膨体少，多数围绕血管形成网状；有的聚集成束直行。胸腺实质内阳性纤维的数量少，免疫后较对照组略有增加，分布不均。此外，胸腺髓质中的一些较大的、三角形或椭圆形的上皮样细胞，亦呈CGRP免疫反应阳性。结论CGRP与胸腺在免疫应答时的结构、机能变化有关。

中图号：R322.8文献标识码：A文章编号：1007－8738(2000)03－0215－04

Plasticity of calcitonin gene related peptidergic nerve fibers in the mouse thymus following PPD immunization

DUAN Xiao-liWANG Bai-renWANG LingJU Gong

（Department of Neuroimmunomodulation, Institute of PLA Neuroscience, Fourth Military Medical University, Xi'an, 710032, Shaanxi Province, China）

Abstract:Aim To elucidate the plasticity of calcitonin gene rel ated peptidergic(CGRP) nerve fibers in mouse thymus and to provide clues for the roles of immunomodulation of CGRP.Methods Balb/c mice were immunized peritoneally by purified protein from tuberculin derivatives(PPD). Sections of the thymic tissues were treated immunohistochemically and oberserved microscopically. Results In normal group, there were relatively abundant CGRP like immunoreactive(LI) fibers in capsule and tubercle. A few positive fibers were seen in parenchyma as well. Twenty eight days after PPD immunization, the perivesicular CGRP LI fibers were significantly increased. These fibers were thick in diameter with few buttons and formed reticular structures surrounding blood vessels or bundles going straightly.The CGRP LI fibers in parenchyma of the thymus were slightly increased in number and distributed unevenly. In addition, a few big triangular or ovoid epithelial like cells in the medulla of thymus were also CGRP LI. Conclusion The present results indicate that CGRP is related to the changes of structure and function of thymus during immune response.  rel ated peptidergic(CGRP) nerve fibers in mouse thymus and to provide clues for the roles of immunomodulation of CGRP.Methods Balb/c mice were immunized peritoneally by purified protein from tuberculin derivatives(PPD). Sections of the thymic tissues were treated immunohistochemically and oberserved microscopically. Results In normal group, there were relatively abundant CGRP like immunoreactive(LI) fibers in capsule and tubercle. A few positive fibers were seen in parenchyma as well. Twenty eight days after PPD immunization, the perivesicular CGRP LI fibers were significantly increased. These fibers were thick in diameter with few buttons and formed reticular structures surrounding blood vessels or bundles going straightly.The CGRP LI fibers in parenchyma of the thymus were slightly increased in number and distributed unevenly. In addition, a few big triangular or ovoid epithelial like cells in the medulla of thymus were also CGRP LI. Conclusion The present results indicate that CGRP is related to the changes of structure and function of thymus during immune response.

Keywords: calcitonin gene related peptide; purified protein derivative; thymus; mouse; immunohistochemistry▲

神经、内分泌和免疫系统间存在着复杂的相互联系。许多证据表明，脑可通过神经内分泌通路和自主神经传出纤维影响免疫器官的活动。胸腺是中枢性淋巴器官，胸腺的免疫功能受神经内分泌系统的调控。这种调控作用的形态学基础之一，是胸腺内有丰富的交感、副交感和传入纤维的支配^〔1〕。许多证据表明，CGRP是一种重要的神经免疫通讯介质^〔2，3〕，胸腺内有丰富的CGRP能神经纤维^〔4〕，但在免疫应激时这些纤维是否有形态学上的变化还未见报道。我们用PPD免疫Balb/c小鼠建立了免疫应答模型，并用免疫组织化学方法，观察了胸腺内CGRP能神经纤维在免疫前后的形态、数量变化，以为进一步探讨CGRP对胸腺功能的调节提供实验依据。

1材料和方法

1.1动物分组和免疫过程3～4wk龄的雄性Balb/c小鼠8只，分为实验组与对照组(各4只)，饲养在自然光线、恒温和定期消毒的环境中，喂以小鼠专用块食和清水。实验组动物接受2次免疫注射，抗原为PPD(中国药品生物制品鉴定所产品)。初次免疫时，将抗原与福氏完全佐剂(Sigma)混合，腹腔内注射，每只动物300μL，内含PPD30μg。存活21d后，实验组再接受第2次免疫注射，将抗原溶于灭菌生理盐水，每只动物腹腔内注射500μL，含抗原30μg。7d后将动物处死。此时间点为再次免疫应答时抗体形成的高峰期^〔5〕。对照组动物在与实验组2个相同的时间点接受相同量的灭菌生理盐水腹腔注射。

1.2组织处理和免疫组织化学所有动物以戊巴比妥钠深麻(60mg/kg，i.p.)，开胸剪开右心房，用注射器经升主动脉灌流生理盐水10mL，继而灌注4℃的含40g/L多聚甲醛的0.1mol/L磷酸缓冲液(PB)50mL。取出胸腺,浸入相同固定液中固定4h，转入含200g/L蔗糖的4℃PB中，直至沉底。将胸腺两叶分开，取一叶的中间1/3段沿矢状面方向做冰冻切片，片厚15μm，隔5取1，贴于明胶处理的载玻片上，置20℃保存。反应时先用3mL/LH2O2(用800mL/L甲醇配制)封闭切片30min，以消除内源性过氧化物酶。结束后用0.01mol/LPBS洗3次(10min/次)。继之用含3mL/LTritonX100，10g/LBSA和30mL/L免血清的0.01mol/L钾磷酸盐缓冲液生理盐水(KPBS，pH7.6)孵育1h，以封闭非特异性结合反应。然后依次加入兔抗CGRP抗体(Chemicon，1∶3000室温24h)，生物素化的羊抗兔IgG(Sigma，1∶500室温12h)及ABC复合物(1∶500，室温4h)。以上每一步骤间均用0.01mol/LPBS(pH7.6)洗涤，10min×3。最后在含有0.5g/LDAB，5g/L硫酸镍铵和0.1mL/LH2O2的0.1mol/L醋酸缓冲液(pH6.2)中呈色10～15min，真至反应的颜色适度为止。切片在室温下晾干，部分切片用中性红复染，梯度酒精脱水、二甲苯透明，中性树胶封片后，在OlympusBH60显微镜下观察并照相，对每只动物所有的胸腺切片均进行观察。在20倍物镜下，从每张切片的左上角开始，每隔1个视野取1个做分析，观察有无CGRP阳性纤维并记录，计算出含阳性纤维视野的数。

2结果

经免疫细胞化学法反应的胸腺切片背景着色较浅，阳性结构清晰，CGRP样免疫反应阳性产物呈蓝紫色或黑色。用中性红复染后，胸腺的组织学结构显示较清楚。空白对照实验未发现阳性染色。

对照组小鼠胸腺内CGRP样免疫反应阳性的纤维主要分布于被膜和小梁内，绝大部分围绕在血管周围。在较大血管的纵断面上，可见阳性纤

图1正常和PPD免疫小鼠胸腺内CGRP样免疫阳性结构

Fig 1 CGRP like immunoreactive structures in the thymus of normal and PPD immunized mice(Immunocytochemical staining ， × 120)

Notes:Microphotographes A and B showing respectively CGRP like immunoreactive fibers in the cortex of the normal and PPD immunized rat thymus. Microphotographes C and D showing CGRP like immunoreactive fibers in the medulla of the normal and PPD immunized rat thymus.Microphotograph E showing CGRP like immunoreactive fibers in the cortex of the thymus in normal rat.Microphotograph F showing the perivasicular CGRP like immunoreactive fibers which form network in the trabecula of thymus in the PPD immunized rat.

维相互交织，在横断面上形成数层环状。也有一些CGRP阳性纤维在被膜和小梁中呈独立地走行。在胸腺实质内阳性纤维的数量很少,在20倍物镜下从每只动物随机选取20个视野，平均约每4个视野可见到有阳性纤维存在。一些阳性纤维从被膜走向皮质，分布于被膜下区，也有少数纤维走行于皮质深层的密集的淋巴细胞之间。皮、髓质交界处和髓质内也有少量CGRP阳性纤维分布。另外，胸腺髓质中一些较大的、三角形或椭圆形细胞亦呈CGRP样免疫反应阳性。

PPD再次免疫后7d，胸腺被囊、小梁内血管周围的CGRP阳性纤维数量明显增加，围绕血管形成复杂的纤维网，网中的纤维密度明显增多，也有的纤维聚集成束直行。阳性纤维的直径较<