【摘要】： 在病毒免疫過(guò)程中,不僅需要體液免疫,而且需要細(xì)胞免疫。如在新城疫的免疫方面,細(xì)胞免疫與體液免疫協(xié)同發(fā)揮作用。Ghumman(1976)等發(fā)現(xiàn),接種新城疫活苗和滅活苗后2-3天,就能檢測(cè)到淋巴細(xì)胞內(nèi)DNA合成增加。Agrawal(1991)等用白細(xì)胞游走抑制試驗(yàn)來(lái)評(píng)價(jià)接種ND疫苗后細(xì)胞免疫反應(yīng),發(fā)現(xiàn)接種后確實(shí)存在細(xì)胞免疫反應(yīng)。機(jī)體針對(duì)新城疫的細(xì)胞免疫和體液免疫之間究竟是怎么變化的呢?為什么經(jīng)常出現(xiàn)免疫失敗呢?這些問(wèn)題沒(méi)有明確的答案。 酶聯(lián)免疫斑點(diǎn)檢測(cè)技術(shù)(ELISPOT)結(jié)合了細(xì)胞培養(yǎng)技術(shù)與酶聯(lián)免疫吸附技術(shù)(ELISA)的長(zhǎng)處,能夠分析經(jīng)特異抗原活化后分泌細(xì)胞因子(如IFN-γ、TNF-α等)的單個(gè)效應(yīng)細(xì)胞的頻數(shù),具有敏感、特異、易于重復(fù)的優(yōu)點(diǎn)。本研究將ELISPOT技術(shù)應(yīng)用于新城疫細(xì)胞免疫的研究,通過(guò)對(duì)脾淋巴細(xì)胞濃度,培養(yǎng)時(shí)間,抗原刺激量進(jìn)行選擇,建立了檢測(cè)NDV誘導(dǎo)特異性IFN-γ、IL-4的ELISPOT方法。結(jié)果顯示,試驗(yàn)的特異性與培養(yǎng)時(shí)間和刺激抗原密切相關(guān),在一定范圍內(nèi),培養(yǎng)時(shí)間越長(zhǎng)、刺激抗原越多,試驗(yàn)的特異性越低,當(dāng)抗原量為5ug/孔,培養(yǎng)時(shí)間為7h時(shí),試驗(yàn)的特異性最強(qiáng)。ELISPOT斑點(diǎn)數(shù),隨細(xì)胞濃度的增加而增加,當(dāng)小鼠的脾淋巴細(xì)胞濃度為106/孔時(shí),最有利于斑點(diǎn)的計(jì)數(shù),試驗(yàn)誤差最小。用建立的ELISPOT方法對(duì)NDV免疫小鼠的IFN-γ、Il-4水平進(jìn)行檢測(cè)發(fā)現(xiàn),IFN-γ、Il-4與血清抗體的產(chǎn)生存在著一定的時(shí)差關(guān)系,在血清抗體陽(yáng)性檢出的前期,IFN-γ的分泌降至最低水平,Il-4升至最高水平。
[Abstract]:In the process of virus immunization, not only humoral immunity, but also cellular immunity is needed. For example, in the immunity aspect of Newcastle disease, the synergistic effect of cellular immunity and humoral immunity on. Ghumman (1976 was found, 2-3 days after inoculation of live and inactivated Newcastle disease vaccine, The leukocyte migration inhibition test was used to evaluate the cellular immune response after inoculation of ND vaccine. It was found that there was a cellular immune response after inoculation. How on earth does the cellular and humoral immunity of the body change in response to Newcastle disease? Why do you often fail to get immunized? There are no clear answers to these questions. (ELISPOT) combines the advantages of cell culture and enzyme linked immunosorbent assay (ELISA) to analyze cytokines (such as IFN- 緯) secreted by specific antigens. The frequency of single effector cells of TNF- 偽 is sensitive, specific and easy to repeat. In this study, ELISPOT technique was applied to the study of cellular immunity of Newcastle disease (NDV). By selecting the concentration of splenic lymphocytes, culture time and the amount of antigen stimulation, a ELISPOT method for detecting the specific IFN- 緯 and IL-4 induced by NDV was established. The results showed that the specificity of the test was closely related to the culture time and the stimulation antigen. In a certain range, the longer the culture time was, the more the stimulating antigen was, the lower the specificity of the test was. When the amount of antigen was 5ug/ pore, the culture time was 7 h. The specificity of the experiment was the strongest. The number of ELISPOT spots increased with the increase of cell concentration. When the concentration of spleen lymphocytes in mice was 106 / well, the number of spots was the most favorable, and the error of the experiment was the least. The levels of IFN- 緯 and Il-4 in mice immunized with NDV were detected by using the established ELISPOT method. It was found that there was a time-difference relationship between IFN- 緯, Il-4 and the production of serum antibodies. The secretion of IFN- 緯 decreased to the lowest level and Il-4 to the highest level.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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