本文選題：Toll樣受體 + 牛　； 參考：《揚州大學》2009年碩士論文

【摘要】： 單核細胞和巨噬細胞在抵抗和防御病原體感染中發(fā)揮了重要作用,尤其是在細胞內(nèi)的病原體防御中發(fā)揮了巨大作用。根據(jù)組織內(nèi)環(huán)境以及它們自身所處的不同分化階段,它們都表現(xiàn)出不同的形態(tài)、機能和代謝作用。這些細胞可以通過細胞表面表達的TLR來識別和結(jié)合PAMP引發(fā)信號傳導(dǎo)通路從而激發(fā)宿主的防御系統(tǒng)。至今為止,已經(jīng)發(fā)現(xiàn)了TLR1-TLR10的部分基因序列,然而對牛外周血單核細胞及其巨噬細胞中Toll樣受體的表達形式還不清除。因此本試驗采取單核細胞以及由來巨噬細胞進行研究,主要研究牛外周血中此類細胞Toll樣受體的表達形式,及其在LPS刺激條件下細胞因子的表達,從而得知該細胞在宿主免疫中發(fā)揮的重要作用。 試驗一牛外周血單核細胞和巨噬細胞分離培養(yǎng)和純化方法 本試驗采用流式細胞分離儀和磁性細胞分離儀分離和鑒定了外周血中單核細胞及其由來的巨噬細胞,通過使用Repcell細胞培養(yǎng)板培養(yǎng)巨噬細胞,改進了Taflon Bag培養(yǎng)巨噬細胞的方法,且單核細胞和巨噬細胞的分離百分率分別為97.9%和73.9%。從而簡化的了試驗步驟,為以后的試驗奠定了堅實的基礎(chǔ)。 試驗二牛外周血單核細胞和巨噬細胞Toll樣受體mRNA水平的表達形式 通過RT-PCR和NIH光密度成像系統(tǒng)分析得到單核細胞和巨噬細胞TLRs mRNA的表達形式,并測得在LPS刺激時,TLRs家族中不同TLR對LPS的反應(yīng)能力。研究發(fā)現(xiàn),巨噬細胞在LPS刺激24h后,IL-6和TNF-αmRNA的表達量顯著升高(P0.05),表達量分別為2.02±0.48和1.66±0.39。與此同時IL-8 mRNA的表達量相對提高,即2.14±0.52。TLR1、TLR3、TLR5、TLR8和TLR10的轉(zhuǎn)錄產(chǎn)物顯著下降(P0.05),然而TLR2、TLR4、TLR6和TLR7則保持穩(wěn)定。另外單核細胞的Toll樣受體1-10以及細胞因子的表達量在LPS刺激前后保持穩(wěn)定,其中TLR1、TLR2、TLR3、TLR4、TLR7、TLR8和TLR10的表達量高于1.0, IL-8和IL-6的表達量大于TNF-α,它們在LPS刺激后的表達量分別為1.38±0.41、1.47±0.17和1.21±0.51,與單核細胞相似的是TLR9不管是在正常培養(yǎng)條件下還是LPS刺激條件下都沒有表達。試驗結(jié)果表明,巨噬細胞在受到病原刺激后通過提高IL-6和TNF-αmRNA的表達量從而提高了天然免疫的作用。該研究為探討巨噬細胞在天然免疫中的作用奠定了基礎(chǔ)。 第三章牛外周血單核細胞和巨噬細胞中Toll樣受體蛋白質(zhì)水平的表達形式 本試驗通過使用熒光免疫染色、流式細胞分離儀和共聚焦電鏡,分析得知TLR2和TLR4在單核細胞和巨噬細胞表面的分布形式以及蛋白水平的表達量,并研究了在LPS刺激條件下,TLR2和TLR4的變化情況。試驗結(jié)果表明,單核細胞和巨噬細胞在受到LPS刺激24h后,TLR2和TLR4蛋白水平表達量相對提高,分別為34.1%、47.1%和22.4%、10.1%,TLR2和TLR4蛋白水平的表達量與mRNA水平的表達形式一致,從而充分證實了TLR2和TLR4在單核細胞和巨噬細胞抵抗病原物質(zhì)感染時發(fā)揮了重要作用。
[Abstract]:Monocytes and macrophages play an important role in resisting and defending pathogen infection, especially in intracellular pathogen defense. They all exhibit different morphological, functional and metabolic functions according to their internal environment and their own differentiation stages. These cells can recognize and bind PAMP triggered signal transduction pathway through TLR expressed on the cell surface to stimulate the host's defense system. Up to now, some gene sequences of TLR1-TLR10 have been found, but the expression of Toll-like receptor in bovine peripheral blood monocytes and macrophages has not been eliminated. Therefore, monocytes and macrophages were used to study the expression of Toll-like receptors and cytokines in bovine peripheral blood. Therefore, it is known that this cell plays an important role in host immunity. Isolation, culture and purification of bovine peripheral blood monocytes and macrophages the monocytes and their origin in peripheral blood were isolated and identified by flow cytometry and magnetic cell isolator. By using Repcell cell culture plate to culture macrophages, the method of Taflon bag culture was improved, and the isolation rates of monocytes and macrophages were 97.9% and 73.9%, respectively. Thus the test procedure is simplified and a solid foundation is laid for the later test. The expression of Toll-like receptor mRNA in bovine peripheral blood monocytes and macrophages was analyzed by RT-PCR and NIH optical density imaging system. The ability of different TLR to respond to LPS in the TLRs family was measured when stimulated by LPS. It was found that the expressions of IL-6 and TNF- 偽 mRNA in macrophages were significantly increased after 24 hours of LPS stimulation (P0.05), which were 2.02 鹵0.48 and 1.66 鹵0.39, respectively. At the same time, the expression of IL-8 mRNA increased relatively, that is, the transcription products of TLR3TLR5, TLR5, TLR8 and TLR10 decreased significantly (P0.05), but TLR2TLR4TLR6 and TLR7 remained stable. In addition, the expression of Toll-like receptor 1-10 and cytokines in monocytes remained stable before and after LPS stimulation. The expression levels of TLR1, TLR2, TLR4, TLR7TLR8 and TLR10 were higher than 1.0, IL-8 and IL-6 were higher than that of TNF- 偽, and the expression of TLR9 was 1.38 鹵0.41 鹵0.17 and 1.21 鹵0.51respectively after LPS stimulation. TLR9 was not expressed in normal culture or LPS stimulated condition. The results showed that macrophages enhanced the innate immunity by increasing the expression of IL-6 and TNF- 偽 mRNA after pathogenetic stimulation. This study provides a basis for exploring the role of macrophages in innate immunity. The expression of Toll-like receptor protein in bovine peripheral blood monocytes and macrophages. The distribution of TLR2 and TLR4 on the surface of monocytes and macrophages and the expression of TLR2 and TLR4 on the surface of monocytes and macrophages were analyzed. The changes of TLR2 and TLR4 in LPS stimulated cells were studied. The results showed that the expression of TLR2 and TLR4 protein in monocytes and macrophages increased relatively after 24 hours of LPS stimulation, which were 34. 1% and 22. 4% respectively. The expressions of TLR2 and TLR4 in monocytes and macrophages were consistent with the expression of mRNA. Thus, TLR2 and TLR4 play an important role in monocyte and macrophage resistance to pathogen infection.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

【引證文獻】

相關(guān)博士學位論文 前1條

1 劉利;牛外周血單個核細胞的免疫研究[D];中國農(nóng)業(yè)科學院;2012年

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本文編號：2075861