摘要
外周免疫器官起源于中胚层，形成于胎儿晚期，主要包括脾脏、淋巴结和消化道、呼吸道和泌尿生殖道的淋巴小结等，是免疫活性细胞定居、增殖和对抗原刺激发生免疫应答的场所。外周免疫器官中淋巴细胞由中枢免疫器官迁移而来，经抗原刺激而分化增殖，继而产生免疫学功能。外周免疫器官作为产生免疫应答的重要场所，是反映动物机体健康状况的晴雨表，其组织结构与功能随着免疫应答而发生变化。哺乳动物出生后通过口服母乳不断获得母源抗体，而雏鸡出壳后，母源抗体获得相对有限。在出壳初期，雏鸡如何保护自身不受病原体的侵扰，外周免疫器官如何发育、功能是否完善、能否提供及时有效的免疫保护呢?这些已成为目前亟待解决的重要研究课题。
本研究选择9、11、13、15、18、20日龄鸡胚及1、4、7、14、21、35、56日龄雏鸡，每个时期分别采集5只鸡(胚)的脾脏、盲肠及食管与腺胃结合处等部位，并制作冰冻切片。利用HE染色方法观察雏鸡不同生长发育阶段外周免疫器官组织发育过程和结构特征；利用免疫组化法结合计算机图像分析技术研究不同生长发育阶段T、B淋巴细胞及其亚群在外周免疫器官中的出现、迁移、组织定位、增殖等一系列发育过程；利用图像分析软件和数据统计软件分析不同生长阶段外周免疫器官中T、B淋巴细胞及其亚群的比例、动态变化规律及相互关系。
研究结果表明：
1、在组织结构方面，脾脏实质内的白髓与红髓在胚胎18日龄后明显可辨，4日龄雏鸡脾脏中形成明显可辨的动脉周围淋巴鞘和椭球周围淋巴鞘；盲肠基部T、B淋巴细胞在胚胎20日龄时开始出现，即初步形成盲肠扁桃体；食管与腺胃结合处在4日龄时形成明显的隐窝结构，即食管扁桃体初步形成；14日龄时，三种器官中首次出现生发中心；随着日龄的增长，外周免疫器官特征结构不断发育成熟，脾脏在21日龄时达到成熟水平，盲肠扁桃体和食管扁桃体在35日龄时达到成熟水平。
2、在T、B淋巴细胞出现时间方面，脾脏中IgM~+和IgA~+细胞在胚胎15日龄时开始出现，IgG~+细胞、CD3~+和CD8~+T淋巴细胞在胚胎18日龄时出现，CD4~+细胞在胚胎20日龄时出现；盲肠扁桃体中CD3~+、CD8~+和IgM~+细胞在胚胎20日龄时开始出现，而CD4~+、IgG~+和IgA~+细胞均在雏鸡出壳后1日龄时出现；食管扁桃体中CD3~+、CD4~+、CD8~+、IgM~+和IgG~+细胞均在胚胎20日龄时开始出现，而IgA~+细胞则在雏鸡出壳后1日龄时出现。
3、在T、B淋巴细胞数量变化方面，外周免疫器官中T、B淋巴细胞的数量随日龄增长，整体均呈上升趋势。脾脏在21日龄时达到稳定，食管扁桃体和盲肠扁桃体均在35日龄时达到稳定；脾脏中T淋巴细胞亚群以CD8~+细胞为主，B淋巴细胞则以IgG~+细胞为主，并在7日龄后数量超过CD3~+T淋巴细胞；盲肠扁桃体中T淋巴细胞亚群以CD8~+细胞为主，B淋巴细胞以IgM~+细胞为主，并在35日龄后数量超过CD3~+T淋巴细胞；21日龄后，食管扁桃体中B淋巴细胞以IgA~+细胞为主，数量超过CD3~+细胞，CD4~+细胞的数量多于CD8~+细胞。
4、在T、B淋巴细胞组织定位方面，脾脏中CD3~+、CD4~+和CD8~+T淋巴细胞主要分布在动脉周围淋巴鞘中，而IgM~+、IgG~+和IgA~+细胞主要分布在椭球周围淋巴鞘和生发中心中；4～7日龄时，盲肠扁桃体中T淋巴细胞主要分布在粘膜固有层的中下部区域，而B淋巴细胞则主要分布在中上部区域，随后各日龄T、B淋巴细胞均匀地分布在粘膜固有层中；在食管扁桃体的T淋巴细胞聚集物中，CD4~+细胞紧密排列，主要占据中央部位，CD8~+细胞主要散布在外周，同时B淋巴细胞又环绕在整个T淋巴细胞聚集物的外周；B淋巴细胞聚集物主要为生发中心，其中存在大量IgM~+、IgG~+和IgA~+细胞，同时T淋巴细胞又环绕在整个B淋巴细胞聚集物的外周。
5、外周免疫器官的组织结构发育和淋巴细胞发生之间存在密切的关系，淋巴细胞迁入淋巴器官刺激组织结构的发育成熟，同时成熟的组织结构又为淋巴细胞发育成熟并行使功能活动提供场所。外周免疫器官的组织结构和淋巴细胞发育在35日龄均达到成熟水平，这也表明雏鸡外周免疫器官的免疫功能在35日龄时达到成熟。
通过对鸡外周免疫器官的组织结构发育和淋巴细胞的发生进行深入研究，从而了解到雏鸡在胚胎期以及出壳初期的免疫状态，这将为禽类分子免疫学、病理生理学增添了新的内容，并为雏鸡适时免疫、致病机理和疾病防治等研究提供必要的理论基础。
The peripheral immune organs are the place where immune competent cells ecize, proliferate and generate immune response including spleen, lymphoid nodes and lymphatic nodules of alimentary canals, respiratory tracts and genitourinary tracts. They originated from mesoderm and form at the advanced stage of fetus. Lymphocytes in peripheral immune organs immigrate from center immune organ. These lymphocytes can proliferate and generate immunological function stimulated by antigens. The peripheral immune organs are the important place that can generate immune response and their structure and function change alone with the immune response. So it is the barometer which reflects the health status of organism. Postnatal mammals can obtain maternal antibody through breast milk. But the quantity of maternal antibody in chickens out of crust is limited. How the chickens out of crust at early stage can be protected against pathogens, how the peripheral immune organs grow and whether its function is perfect to provide effective protection? All these have become the urgent study topics at present.
In this study, chicken embryos of 9, 11, 13, 15, 18, 20 days and chickens of 1, 4, 7, 14, 21, 35, 56 days were choosed. Spleen, appendix and the conjunction of esophago and stomachus glandularis of 5 chickens in every stage are collected and made frozen sections. Then observe the development process and structural feature of peripheral immune organs in chickens of different development stage by HE staining method. The emergence, immigration, tissue allocation and proliferation of T, B lymphocytes and their subpopulation of different development stage in peripheral immune organs were studied by Immunohistochemistry combine with computer image analysis technique. The proportion, kinesis variation rule and interrelationship of T, B lymphocytes and subpopulation of different development stage in peripheral immune organs were analyzed by image analysis and data statistics software.
The results of study show:
1. White pulp and red pulp in parenchyma of spleen of embryo can be obviously discerned after 18 days. Periarterial lymphoid sheath and ellipsoid periarterial lymphoid sheath also can be obviously discerned in spleen of 4 days chicken. T, B lymphocytes in appendix basement of embryo emerge after 20 days. It is the initial shape of cecal tonsil. Crypt structure of conjunction of esophago and stomachus glandularis form obviously at 4 days. It is the initial shape of esophago tonsil. The germinal center firstly emerges in these three organs at 14 days. With the increase of day age, the characteristic structure peripheral immune organs gradually develop mature. Spleen achieve mature at 21 days and cecal tonsil at 35 days.
2. IgM~+ and IgA~+ cells in spleen of embryo emerge at 15 days. IgG~+ cell, CD3~+ and CD8~+T lymphocytes of embryo emerge at 20 days. CD3~+, CD8~+ and IgM~+ cells in cecal tonsil of embryo emerge at 20 days. However CD4~+, IgG~+ and IgA~+ cells all emerge in 1 day age chicken out of crust. CD3~+, CD4~+, CD8~+, IgM~+ and IgG~+ cells in esophago tonsil of embryo all emerge at 20 days. However IgA~+ cells emerge in 1 day age chicken out of crust.
3. The amount of T, B lymphocytes in peripheral immune organs increase follow with the increase of day age, and hold an upgrade tendency. The amount of T, B lymphocytes in spleen achieved stabilization at 21 days, and in tonsil of esophago and appendix at 35 days. CD8~+ cell is the main T lymphocyte subset in spleen, and B lymphocyte mainly is IgG~+ cell, moreover the amount of these B lymphocytes could exceed CD3~+ T lymphocyte subset after 7 days. CD8~+ cell is the main T lymphocyte subset in tonsil of appendix, and B lymphocyte is IgM~+ cell, and the amount could exceed CD3~+ T lymphocytes after 35 days. After 21 days, B lymphocytes in esophago tonsil are the main IgA~+ cells and the amount exceeds CD3~+ lymphocytes. The amount of CD4~+ lymphocytes is more than CD8+ lymphocytes.
4. CD3~+、CD4~+ and CD8~+ T lymphocytes in spleen mainly distribute in periarterial lymphoid sheath. However IgM~+、IgG~+ and IgA~+ cells mainly distribute in ellipsoid periarterial lymphoid sheath and germinal center. T lymphocytes in appendix tonsil mainly distribute in middle and inferior part of mucous and the B lymphocytes mainly in middle and mucous between 4~7 days. Whereafter T, B lymphocytes equably distribute in mucous. CD4~+ cells arrange tightly and mainly occupy the central part in aggregates of T lymphocytes in esophago tonsil and CD8~+ lymphocytes mainly distribute in periphery. Meanwhile B lymphocytes encircle the periphery of aggregates of T lymphocytes. The aggregates of B lymphocytes is mainly the germinal center with lots of IgM~+、IgG~+ and IgA~+ cells. Meanwihle T lymphocytes encircle the periphery of aggregates of B lymphocytes.
5. There is an intimate relationship between the development of tissue structure of peripheral immune organs and lymphcytopoiesis. The maturation of tissue structure is stimulated by the immigration of lymphocytes and the mature tissue structure provides place where lymphocytes grow mature and functionate. Tissue structure and the development of lymphocytes in peripheral immune organs achieve mature at 35 days. This also indicated the immune function of chicken peripheral immune organs achieve mature at 35days.
The tests carry out a deepgoing study on development of tissue structure and lymphocytic generation of chicken peripheral immune organs. Thus investigate the immune status of chicken in embryo and at early stage out of crust. All these increase new content for molecular immunology and pathophysiology of fowl and provide necessary theory foundation for research in suitable immunization, pathogenic mechanism and prevention and cure of disease of chicken.
引文
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