發(fā)布時間：2018-07-20 16:45

【摘要】：免疫應(yīng)答基本過程包括感應(yīng)階段、反應(yīng)階段和效應(yīng)階段,各階段中涉及到了淋巴細胞、抗原呈遞細胞(Antigen-presenting cell,APC)以及起調(diào)節(jié)作用的一些細胞因子。目前對家禽免疫功能的科學(xué)研究大部分都是通過對淋巴細胞、細胞因子以及抗體表達檢測進行評價,但是對APC的相關(guān)研究卻較少。樹突狀細胞(Dendritic cell,DC)作為已知功能最強的APC,它在機體內(nèi)的發(fā)育情況直接決定了免疫功能強弱。肺臟直接與外界空氣接觸,支氣管相關(guān)淋巴組織(Bronchus associated lymphoid tissue,BALT)等相關(guān)黏膜免疫是抵御病原入侵的第一道防線。探究家禽肺臟DC的發(fā)育對肺臟相關(guān)免疫機制的研究有著重要意義,也是本試驗的設(shè)計目的。本試驗分為兩部分:首先選擇1d、4d、7d、10d、14d、21d、28d共7個日齡組雛雞作為研究對象,流式細胞術(shù)和免疫組化染色法檢測各日齡雛雞肺臟DC數(shù)量變化以及分布情況,然后通過熒光定量PCR方法檢測各日齡組肺臟中CCR6和CCR7趨化因子表達水平,探究雛雞肺臟中DC發(fā)育情況;第二部分試驗選擇1d、4d、7d、14d、21d、28d共6個日齡組,各日齡組雛雞免疫新城疫疫苗(Newcastle disease vaccine,NDV),采用免疫組化雙染法檢測各日齡組免疫后6h、12h、24h、4d和7d時,肺臟中NDV和DC數(shù)量變化與分布情況,從而研究不同日齡雛雞肺臟中DC對抗原免疫反應(yīng)的強度,探究不同時期DC抗原呈遞功能的發(fā)育情況。流式細胞術(shù)與免疫組化試驗結(jié)果發(fā)現(xiàn):1日齡時DC就已出現(xiàn)于雛雞肺臟中并隨日齡增長,細胞比例不斷增加,7-14日齡時DC數(shù)量增長率最高;雛雞1日齡時,DC于三級支氣管氣道內(nèi)壁處出現(xiàn),之后隨肺臟發(fā)育,逐漸散布至肺臟間質(zhì)等非淋巴組織部位。實時熒光定量PCR試驗發(fā)現(xiàn):7-14日齡時雛雞肺臟中CCR6表達量顯著增高,并在14日齡時達到峰值,同時CCR7表達量降低,表明此階段肺臟中DC數(shù)量迅速增長且主要為未成熟樹突狀細胞(Immature dendritic cell,imDC);14日齡后CCR6表達量開始降低,同時CCR7表達量隨日齡不斷上升,表明該日齡后imDC數(shù)量增長率下降,但imDC快速成熟,mDC數(shù)量迅速增加。免疫組化雙染試驗發(fā)現(xiàn):免疫NDV對雛雞肺臟中DC數(shù)量增長有著極顯著促進作用,7日齡組促進效果最為明顯;雛雞免疫后肺臟中DC能夠快速在NDV處聚集并將其吞噬,其中14日齡組DC吞噬效率最高,且DC吞噬后向BALT處聚集,誘導(dǎo)局部免疫應(yīng)答。綜上所述,本試驗從數(shù)量比例、分布、細胞因子表達水平以及抗原攝取能力等多方面探究不同日齡雛雞肺臟中DC發(fā)育以及抗原呈遞功能的變化情況,為進一步研究禽類DC奠定基礎(chǔ),并為研究禽類免疫發(fā)育、適時免疫和疾病防治等提供參考依據(jù)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S831

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