雏鸡肺脏中树突状细胞的发育及其抗原呈递功能的研究

发布时间：2018-07-20 16:45

【摘要】：免疫应答基本过程包括感应阶段、反应阶段和效应阶段,各阶段中涉及到了淋巴细胞、抗原呈递细胞(Antigen-presenting cell,APC)以及起调节作用的一些细胞因子。目前对家禽免疫功能的科学研究大部分都是通过对淋巴细胞、细胞因子以及抗体表达检测进行评价,但是对APC的相关研究却较少。树突状细胞(Dendritic cell,DC)作为已知功能最强的APC,它在机体内的发育情况直接决定了免疫功能强弱。肺脏直接与外界空气接触,支气管相关淋巴组织(Bronchus associated lymphoid tissue,BALT)等相关黏膜免疫是抵御病原入侵的第一道防线。探究家禽肺脏DC的发育对肺脏相关免疫机制的研究有着重要意义,也是本试验的设计目的。本试验分为两部分:首先选择1d、4d、7d、10d、14d、21d、28d共7个日龄组雏鸡作为研究对象,流式细胞术和免疫组化染色法检测各日龄雏鸡肺脏DC数量变化以及分布情况,然后通过荧光定量PCR方法检测各日龄组肺脏中CCR6和CCR7趋化因子表达水平,探究雏鸡肺脏中DC发育情况;第二部分试验选择1d、4d、7d、14d、21d、28d共6个日龄组,各日龄组雏鸡免疫新城疫疫苗(Newcastle disease vaccine,NDV),采用免疫组化双染法检测各日龄组免疫后6h、12h、24h、4d和7d时,肺脏中NDV和DC数量变化与分布情况,从而研究不同日龄雏鸡肺脏中DC对抗原免疫反应的强度,探究不同时期DC抗原呈递功能的发育情况。流式细胞术与免疫组化试验结果发现:1日龄时DC就已出现于雏鸡肺脏中并随日龄增长,细胞比例不断增加,7-14日龄时DC数量增长率最高;雏鸡1日龄时,DC于三级支气管气道内壁处出现,之后随肺脏发育,逐渐散布至肺脏间质等非淋巴组织部位。实时荧光定量PCR试验发现:7-14日龄时雏鸡肺脏中CCR6表达量显著增高,并在14日龄时达到峰值,同时CCR7表达量降低,表明此阶段肺脏中DC数量迅速增长且主要为未成熟树突状细胞(Immature dendritic cell,imDC);14日龄后CCR6表达量开始降低,同时CCR7表达量随日龄不断上升,表明该日龄后imDC数量增长率下降,但imDC快速成熟,mDC数量迅速增加。免疫组化双染试验发现:免疫NDV对雏鸡肺脏中DC数量增长有着极显著促进作用,7日龄组促进效果最为明显;雏鸡免疫后肺脏中DC能够快速在NDV处聚集并将其吞噬,其中14日龄组DC吞噬效率最高,且DC吞噬后向BALT处聚集,诱导局部免疫应答。综上所述,本试验从数量比例、分布、细胞因子表达水平以及抗原摄取能力等多方面探究不同日龄雏鸡肺脏中DC发育以及抗原呈递功能的变化情况,为进一步研究禽类DC奠定基础,并为研究禽类免疫发育、适时免疫和疾病防治等提供参考依据。
[Abstract]:The basic process of immune response includes induction stage, reaction stage and effect stage, which involves lymphocytes, Antigen-presenting cells and some cytokines that play a role in regulation. At present, most of the scientific studies on the immune function of poultry are based on the detection of lymphocytes, cytokines and antibodies, but there are few studies on APC. As the strongest known APCs, dendritic cells (DCs) directly determine the immune function of APCs. Lung is in direct contact with the outside air, and bronchus associated lymphoid tissue (Balt) is the first line of defense against pathogen invasion. It is important to explore the development of DC in poultry lung for the study of the immune mechanism related to the lung, and it is also the purpose of this study. The experiment was divided into two parts: firstly, seven day-old chicks were selected for 1 day, 4 days, 7 days, 10 days, 14 days, 21 days and 28 days. Flow cytometry and immunohistochemical staining were used to detect the changes and distribution of DC in the lungs of different day old chicks. Then the expression levels of CCR6 and CCR7 chemokines in lung of different age groups were detected by fluorescence quantitative PCR to investigate the development of DC in the lungs of chicks. Newcastle disease vaccine (NDV) was immunized in chickens of different days of age. The changes and distribution of NDV and DC in lung were detected by immunohistochemical double staining at 4 and 7 days after 6 hours and 12 hours after immunization. The immunoreactivity of DC to antigen was studied in the lungs of chickens of different days, and the development of antigen presenting function in different period was studied. The results of flow cytometry and immunohistochemistry showed that DC appeared in the lungs of chicks at 1 day of age and increased with the age of 7 to 14 days, and the number of DC increased the highest at 7 to 14 days of age. At 1 day of age, DC appeared at the inner wall of the third stage bronchial airway, and then spread to the nonlymphoid tissue such as the interstitium of lung with the development of lung. Real-time fluorescence quantitative PCR assay showed that the expression of CCR6 in lung of chicks was significantly increased at the age of 7 to 14 days, and reached its peak at 14 days of age, while the expression of CCR7 decreased. The results showed that the number of DC in lung increased rapidly and the expression of CCR6 began to decrease after 14 days of age, and the expression of CCR7 increased with the age of day, indicating that the growth rate of IMDC decreased after the age of 14 days. But the number of IMDC matured rapidly increased rapidly. Immunohistochemical double staining assay showed that immunized NDV could significantly promote the growth of DC in the lungs of chicks, and the DC in the lungs of chicks could rapidly gather and phagocystify NDV after immunization. The phagocytosis efficiency of DC was the highest in 14 day old group, and DC phagocytosis gathered to Balt to induce local immune response. To sum up, the changes of DC development and antigen presenting function in lung of different day-old chicks were studied from the aspects of quantity ratio, distribution, expression level of cytokines and ability of antigen uptake. It provides a basis for the further study of avian DC, and provides a reference for the study of poultry immune development, timely immunization and disease prevention and control.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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