本文選題：樹(shù)突狀細(xì)胞 + 口蹄疫病毒　； 參考：《河北醫(yī)科大學(xué)》2008年博士論文

【摘要】： 目的:口蹄疫(foot-and-mouth disease,FMD )是由口蹄疫病毒(foot-and-mouth disease virus, FMDV)引起的牛、羊、豬等偶蹄動(dòng)物的一種急性、高度接觸性、發(fā)熱性傳染病。FMDV也可感染人類(lèi),并表現(xiàn)出與動(dòng)物類(lèi)似的臨床癥狀。因此,被世界衛(wèi)生組織(WHO)把口蹄疫定為人獸共患傳染病。雖然該病已經(jīng)存在了四百多年,但尚不清楚機(jī)體對(duì)FMDV的免疫應(yīng)答機(jī)制。樹(shù)突狀細(xì)胞(dendritic cells,DC)不僅是動(dòng)物機(jī)體的哨位細(xì)胞(sentinel cell),而且還是機(jī)體適應(yīng)性免疫應(yīng)答的啟動(dòng)者,特別是DC還能夠交叉提呈非復(fù)制性抗原(例如滅活的病毒等),故被稱(chēng)為機(jī)體最強(qiáng)大的抗原提呈細(xì)胞,但目前尚不清楚DC是否參與加工、提呈FMDV抗原。由于國(guó)家嚴(yán)格規(guī)定從事口蹄疫活病毒研究必須在生物安全三級(jí)實(shí)驗(yàn)室以上的環(huán)境條件下進(jìn)行,因此,本研究以滅活FMDV疫苗為抗原材料,通過(guò)觀察口服滅活FMDV疫苗后咽部引流淋巴結(jié)內(nèi)樹(shù)突狀細(xì)胞的分布變化,明確DC是否作為FMDV的抗原提呈細(xì)胞參與免疫應(yīng)答,并進(jìn)一步研究DC提呈FMDV抗原的機(jī)制。近年來(lái),FMDV感染模型已經(jīng)在BALB/c小鼠成功復(fù)制,因此,如果試驗(yàn)結(jié)果證實(shí)了DC是滅活FMDV的抗原提呈細(xì)胞,將以BALB/c小鼠為試驗(yàn)對(duì)象,通過(guò)體內(nèi)外試驗(yàn),進(jìn)一步探討小鼠樹(shù)突狀細(xì)胞向CD4+T細(xì)胞和CD8+T細(xì)胞提呈滅活FMDV抗原的機(jī)制。適應(yīng)性免疫包括細(xì)胞免疫和體液免疫兩部分。眾所周知,如果沒(méi)有細(xì)胞免疫應(yīng)答的適當(dāng)啟動(dòng),特別是由CD4+T細(xì)胞介導(dǎo)的免疫應(yīng)答,體液免疫應(yīng)答就不可能得到建立,而且由CD8+T細(xì)胞介導(dǎo)的細(xì)胞免疫也是機(jī)體抵御病毒感染最有效的機(jī)制之一,所以,本研究選用CD4+T細(xì)胞和CD8+T細(xì)胞作為接受DC提呈抗原的靶細(xì)胞。同時(shí),CD4+T細(xì)胞和CD8+T細(xì)胞的活化也可作為反映MHC-II類(lèi)分子和MHC-I類(lèi)分子途徑提呈抗原的指標(biāo)。 方法:FMDV主要經(jīng)消化道、呼吸道和受損傷的皮膚等途徑感染動(dòng)物,并可在咽部大量繁殖,無(wú)論是自然感染動(dòng)物,還是疫苗接種動(dòng)物。因此,咽部可能在機(jī)體抗FMDV感染免疫中發(fā)揮特殊重要作用。根據(jù)黏膜免疫學(xué)理論,咽部引流淋巴結(jié)就是咽部黏膜免疫應(yīng)答的誘導(dǎo)區(qū),而咽部黏膜就是效應(yīng)區(qū)。 1.DC與FMDV關(guān)系的確定:為探明咽部DC是否參與滅活FMDV抗原的加工與提呈,研究者模擬自然感染途徑給BALB/c小鼠口服接種滅活FMDV疫苗,然后,在接種疫苗后不同時(shí)間點(diǎn)摘取咽部引流淋巴結(jié)(用墨汁追蹤法證實(shí)小鼠咽部引流淋巴結(jié)為頸淺淋巴結(jié)),按常規(guī)進(jìn)行固定、脫水、透明和包埋,制備6-8μm厚石蠟組織切片,用兔抗牛S-100多克隆抗體對(duì)淋巴結(jié)進(jìn)行免疫組織化學(xué)染色,觀察頸淺淋巴結(jié)內(nèi)DC的分布,根據(jù)DC在頸淺淋巴結(jié)內(nèi)的數(shù)量變化與分布變化判斷其是否參與FMDV抗原的提呈。 2.口服滅活FMDV疫苗誘導(dǎo)的細(xì)胞免疫應(yīng)答:將36只8周齡BALB/c小鼠隨機(jī)分為6組,每組6只。1組、2組、3組、4組、5組小鼠口服滅活A(yù)sia I-O型雙價(jià)滅活疫苗(100μl/只),并分別于接種疫苗后24h、48h、72h、96h、120h在無(wú)菌條件下收集外周血,制備血清。第6組小鼠口服滅菌PBS(pH7.2)緩沖液(100μl/只)作為對(duì)照。以血清IFN-γ水平為指標(biāo)(反映機(jī)體的Th1應(yīng)答和CD8+T細(xì)胞應(yīng)答),用ELISA法檢測(cè)小鼠對(duì)滅活FMDV疫苗的細(xì)胞免疫應(yīng)答。 3. BALB/c小鼠單核細(xì)胞源樹(shù)突狀細(xì)胞(monocyte-derived dendritic cells, MoDC)的制備:按常規(guī)方法進(jìn)行,并用免疫細(xì)胞化學(xué)法和流式細(xì)胞術(shù)對(duì)其進(jìn)行鑒定。 4. MoDC對(duì)滅活FMDV抗原的泛素化作用:將滅活FMDV疫苗負(fù)載于MoDC,并在RPMI 1640培養(yǎng)基中進(jìn)行細(xì)胞培養(yǎng),然后在不同時(shí)間點(diǎn)將MoDC裂解,以裂解液為材料進(jìn)行SDS-PAGE,轉(zhuǎn)膜后用western blotting法檢測(cè)MoDC內(nèi)的泛素化蛋白以及泛素化作用出現(xiàn)的時(shí)間。 5.負(fù)載滅活FMDV的MoDC對(duì)淋巴結(jié)T細(xì)胞免疫應(yīng)答的啟動(dòng)作用:將負(fù)載了滅活FMDV的MoDC與淋巴結(jié)CD4+T細(xì)胞和CD8+T細(xì)胞在RPMI 1640培養(yǎng)基中進(jìn)行共培養(yǎng)。用抗CD4抗體或抗CD8抗體分別阻斷CD4+T細(xì)胞或CD8+T細(xì)胞的活化,分別在第9h、12h、24h、36h、48h收集上清液,用ELISA檢測(cè)IFN-γ的含量。 6. MoDC提呈滅活FMDV抗原的機(jī)制:分別用溶酶體抑制劑和蛋白酶體抑制劑處理MoDC,2h后將滅活FMDV負(fù)載于MoDC,隨即與CD4+T細(xì)胞或CD8+T細(xì)胞共培養(yǎng),分別在第9h、12h、24h、48h收集上清液,用ELISA檢測(cè)其IFN-γ的含量。 結(jié)果:1. DC與FMDV關(guān)系的確定:小鼠頸淺淋巴結(jié)內(nèi)DC的數(shù)量在口服FMDV滅活疫苗24h后開(kāi)始增多,散在分布于皮質(zhì)區(qū)內(nèi)。48h后小鼠頸淺淋巴結(jié)內(nèi)DC的數(shù)量明顯增多,并且主要分布于副皮質(zhì)區(qū)。在72h后,咽部DC向頸淺淋巴結(jié)副皮質(zhì)區(qū)的遷移達(dá)到頂峰,依然聚集在副皮質(zhì)區(qū)。至96h,頸淺淋巴結(jié)內(nèi)DC的數(shù)量急劇下降,在皮質(zhì)區(qū)和副皮質(zhì)區(qū)內(nèi)只有少量DC呈現(xiàn)散在分布狀,此即樹(shù)突狀細(xì)胞疲勞(dendritic cell exhaustion)。然而,120h后,頸淺淋巴結(jié)內(nèi)DC的數(shù)量再次增多,且主要分布于皮質(zhì)區(qū)。而PBS對(duì)照組未見(jiàn)類(lèi)似改變。這說(shuō)明DC就是FMDV的抗原提呈細(xì)胞。 2.口服滅活FMDV疫苗誘導(dǎo)的細(xì)胞免疫應(yīng)答:接種疫苗24h后,小鼠血清內(nèi)的IFN-γ含量開(kāi)始升高(10.1568±0.4689),但與PBS對(duì)照組相(10.0512±0.0684)比差異不顯著(P0.05),而48h后,接種疫苗組小鼠血清內(nèi)的IFN-γ含量明顯升高(10.7161±0.2199),與PBS對(duì)照組(10.0748±0.0571)相比,差異極顯著(P0.01)。在接種72h后,接種疫苗組小鼠血清內(nèi)的IFN-γ含量達(dá)到最高峰(11.6784±0.4590),與PBS對(duì)照組(10.1249±0.1498)相比,差異極顯著(P0.01)。96h后,IFN-γ含量開(kāi)始下降(10.9839±0.3633),與PBS對(duì)照組(10.0933±0.0954)相比,差異顯著(P0.05),到120h時(shí),試驗(yàn)組(10.5285±1.3903)與PBS對(duì)照組相(10.0534±0.1543)比差異不顯著(P0.05)。這表明口服滅活FMDV疫苗所誘導(dǎo)的細(xì)胞免疫應(yīng)答與DC向引流淋巴結(jié)內(nèi)的遷移模式相一致。 3. BALB/c小鼠MoDC的制備:免疫細(xì)胞化學(xué)試驗(yàn)和流式細(xì)胞術(shù)檢測(cè)均顯示,本試驗(yàn)所制備的MoDC純度大于99%。 4. MoDC對(duì)滅活FMDV抗原的泛素化作用:所有負(fù)載滅活FMDV MoDC中均發(fā)生了FMDV抗原的泛素化現(xiàn)象,而未負(fù)載滅活FMDV的MoDC中泛素化蛋白質(zhì)檢測(cè)則呈現(xiàn)陰性反應(yīng)。在負(fù)載滅活FMDV疫苗0.5h后,MoDC內(nèi)就出現(xiàn)了泛素化FMDV蛋白,分子量大約為75kD;3h后,泛素化蛋白含量稍有增加,但從不同時(shí)間點(diǎn)來(lái)看,泛素化蛋白含量變化趨勢(shì)不明顯。本試驗(yàn)所使用的兩種單克隆抗體—鼠抗多鏈泛素化蛋白質(zhì)單克隆抗體和鼠抗單鏈泛素化蛋白質(zhì)單克隆抗體(克隆號(hào)分別為FK1和FK2)均在western blot試驗(yàn)中呈現(xiàn)陽(yáng)性反應(yīng),說(shuō)明MoDC以?xún)煞N不同的方式對(duì)滅活FMDV蛋白質(zhì)抗原進(jìn)行泛素化。 5. MoDC提呈滅活FMDV抗原的機(jī)制:在滅活FMDV負(fù)載MoDC與CD4+T細(xì)胞共培養(yǎng)9h即有大量IFN-γ產(chǎn)生,而與FMDV負(fù)載MoDC共培養(yǎng)的CD8+T細(xì)胞在受到抗原刺激后24h才大量釋放IFN-γ。用溶酶體抑制劑或蛋白酶體抑制劑分別處理MoDC,2h后再分別與CD4+T細(xì)胞或CD8+T細(xì)胞共培養(yǎng),按前述方法檢測(cè)上清液中IFN-γ的含量,結(jié)果發(fā)現(xiàn),兩種抑制劑均在共培養(yǎng)后9h顯著抑制CD4+T細(xì)胞產(chǎn)生IFN-γ。值得注意的是,溶酶體抑制劑顯著抑制滅活FMDV負(fù)載DC刺激CD8+T細(xì)胞產(chǎn)生IFN-γ的能力,而蛋白酶體抑制劑卻促進(jìn)CD8+T細(xì)胞產(chǎn)生IFN-γ,但是,同時(shí)用兩種抑制劑處理DC,CD8+T細(xì)胞產(chǎn)生IFN-γ的能力又顯著下降。這些復(fù)雜現(xiàn)象提示MoDC主要以交叉提呈抗原的方式激活淋巴結(jié)T細(xì)胞,并以產(chǎn)生IFN-γ為特征。但是,CD8+T細(xì)胞產(chǎn)生IFN-γ的時(shí)間明顯晚于CD4+T細(xì)胞,并且IFN-γ釋放也呈現(xiàn)多樣性。結(jié)論:1.咽部DC是滅活FMDV的抗原提呈細(xì)胞,但在DC捕獲滅活FMDV抗原后的遷移過(guò)程中表現(xiàn)出“疲勞”特征(Dendritic cell exhaustion)。 2.小鼠口服接種滅活FMDV疫苗可以誘導(dǎo)細(xì)胞免疫應(yīng)答,并以產(chǎn)生IFN-γ為特征。 3.滅活FMDV抗原被DC捕獲后發(fā)生了兩種不同的泛素化現(xiàn)象,即多鏈泛素化和單鏈泛素化。但泛素化FMDV蛋白質(zhì)抗原主要被FK2抗體標(biāo)記,而FK1抗體標(biāo)記的較少。由于FK1只特異性地結(jié)合多鏈泛素化蛋白質(zhì),FK2則同時(shí)結(jié)合單鏈泛素化蛋白質(zhì)和多鏈泛素化蛋白質(zhì),并且二者都不與游離的泛素結(jié)合。所以,滅活FMDV蛋白質(zhì)抗原主要在DC內(nèi)被多鏈泛素化。 4.泛素化的FMDV蛋白質(zhì)抗原既可被蛋白酶體降解,也可在溶酶體內(nèi)被加工處理,所產(chǎn)生的抗原肽主要以交叉提呈的方式提呈給CD4+T細(xì)胞或CD8+T細(xì)胞。CD4+T細(xì)胞只能識(shí)別裝載著抗原表位的MHC-II類(lèi)分子,所以,溶酶體抑制劑造成的IFN-γ含量顯著下降清楚地表明,DC能夠以溶酶體-MHC-II類(lèi)分子途徑按常規(guī)提呈FMDV抗原。而蛋白酶體抑制劑對(duì)CD4+T細(xì)胞分泌IFN-γ的強(qiáng)大抑制作用則提示,DC內(nèi)還可能存在以蛋白酶體-MHC-II類(lèi)分子途徑交叉提呈FMDV抗原的機(jī)制。并且CD4+T細(xì)胞被抗原肽激活后主要分化為T(mén)h1細(xì)胞亞類(lèi),分泌高水平的IFN-γ。從上述結(jié)果不難看出,FMDV負(fù)載的DC則主要以“內(nèi)吞小體-溶酶體-MHC-I類(lèi)分子途徑”交叉提呈抗原肽給CD8+T細(xì)胞,從而啟動(dòng)CTL應(yīng)答。與DC遷移疲勞現(xiàn)象一致,CD8+T細(xì)胞產(chǎn)生IFN-γ的能力在被活化后48h顯著下降,也呈現(xiàn)出向抑制的應(yīng)答疲勞狀態(tài)。
[Abstract]:Objective: foot-and-mouth disease (FMD) is an acute, highly contagious, infectious disease of cattle, sheep, pigs and other hoofed animals caused by foot-and-mouth disease virus (foot-and-mouth disease virus, FMDV), and the febrile infectious disease.FMDV can also infect humans and show similar clinical symptoms. Therefore, the WHO (WHO) has a foot-and-mouth disease. Although it has been in existence for more than 400 years, it is not clear that the body's immune response to FMDV is not clear. Dendritic cells (DC) is not only the post cell (sentinel cell) of animal body (sentinel cell), but also the promoter of the adaptive immune response to the organism, especially the DC can also be non replicating. Sex antigen, such as inactivated virus, is known as the most powerful antigen presenting cell of the body, but it is not clear whether DC is involved in processing and presenting FMDV antigen. Because the state strictly stipulates that the study of foot-and-mouth disease virus must be carried out under the environmental conditions above the biological safety level 3 Laboratory, therefore, this study is to inactivate the FMDV epidemic. The vaccine was an antigen material. By observing the distribution of dendritic cells in the pharynx drainage lymph node after oral inactivation of FMDV vaccine, it is clear whether DC is an antigen presenting cell of FMDV and participates in the immune response, and the mechanism of FMDV antigen presentation by DC has been further studied. In recent years, the FMDV infection model has been successfully replicated in BALB/c mice. Therefore, if the test has been done, if the test has been done, the test of the FMDV infection model has been successfully replicated. The test results confirmed that DC is an antigen presenting cell for inactivating FMDV. It will take BALB/c mice as the test object. Through the experiment in vitro and in vivo, the mechanism of inactivating FMDV antigen by mouse dendritic cells to CD4+T and CD8+T cells is further explored. Adaptive immunity includes two parts of cell immunity and humoral immunity. The appropriate start of the epidemic, especially the immune response mediated by CD4+T cells, can not be established in the humoral immune response, and the cellular immunity mediated by CD8+T cells is also one of the most effective mechanisms for resisting the virus infection. Therefore, this study selects CD4+T cells and CD8+T cells as the target cells for receiving DC antigen presenting antigen. At the same time, the activation of CD4+T cells and CD8+T cells can also serve as indicators of antigen presentation of MHC-II molecules and MHC-I molecular pathways.
Methods: FMDV mainly infect animals through the digestive tract, respiratory tract and damaged skin, and can proliferate in the pharynx, whether it is a natural infection or a vaccine. Therefore, the pharynx may play a special role in the immunity of the body against FMDV infection. According to the theory of mucosal immunology, the pharynx drainage lymph node is the pharynx. Mucosal immune response is induced, while pharyngeal mucosa is the effector area.
The determination of the relationship between 1.DC and FMDV: to determine whether the pharyngeal DC was involved in the processing and presentation of the inactivated FMDV antigen, the researchers simulated the natural infection route to the BALB/c mice by oral inactivation of the FMDV vaccine. Then, the pharynx drainage lymph nodes were extracted at different time points after the inoculation (with the ink tracing method confirmed that the throat drainage lymph nodes of the mice were superficial to the neck. " The 6-8 m thick paraffin tissue sections were prepared by routine fixation, dehydration, transparency and embedding. The lymph nodes were stained with Rabbit anti bovine S-100 polyclonal antibody to observe the distribution of DC in the superficial cervical lymph nodes. According to the changes and distribution changes of the number of DC in the superficial cervical lymph nodes, they were judged to be involved in the presentation of the FMDV antigen.
2. the cellular immune response induced by oral inactivated FMDV vaccine: 36 8 week old BALB/c mice were randomly divided into 6 groups, each group of 6.1 groups, 2 groups, 3 groups, 4 groups, and 5 groups of mice inactivated Asia I-O bivalent inactivated vaccine (100 mu l/), and after inoculation, 24h, 48h, 72h, 96h, 120h were collected under aseptic conditions to prepare serum and sixth mice. The oral sterilization PBS (pH7.2) buffer solution (100 mu l/) was used as control. The serum IFN- gamma level was used as an index (reflecting the body's Th1 response and CD8+T cell response), and the cellular immune response of mice to inactivated FMDV vaccine was detected by ELISA method.
3. BALB/c mouse monocyte derived dendritic cells (monocyte-derived dendritic cells, MoDC) were prepared by routine methods and identified by immunocytochemistry and flow cytometry.
4. MoDC to inactivate FMDV antigen ubiquitination: inactivated FMDV vaccine loaded with MoDC, and cell culture in the RPMI 1640 medium, and then cracking MoDC at different time points, using lysate as the material for SDS-PAGE, Western blotting method for the detection of ubiquitination protein in MoDC and the time of ubiquitination in MoDC.
5. load inactivated FMDV MoDC on the immune response of T cells in lymph nodes: co culture MoDC with inactivated FMDV and lymph node CD4+T cells and CD8+T cells in RPMI 1640 medium. The activation of CD4+T cells or CD8+T cells was blocked with anti CD4 or anti CD8 antibodies. The content of IFN- gamma was detected by ELISA.
6. MoDC proposed the mechanism of inactivating FMDV antigen: MoDC was treated with lysosome inhibitor and proteasome inhibitor respectively. After 2h, the inactivated FMDV was loaded on MoDC, then CD4+T cells or CD8+T cells were co cultured, and the supernatant was collected in 9h, 12h, 24h, 48h, respectively.
Results: the determination of the relationship between 1. DC and FMDV: the number of DC in the superficial cervical lymph nodes in mice began to increase after the oral FMDV inactivated vaccine 24h. The number of DC in the superficial cervical lymph nodes scattered in the cortical region was significantly increased and mainly distributed in the paracortical area. After 72h, the migration of the pharynx from the pharynx to the superficial lymph node paracortical area reached the peak. The number of DC in the superficial cervical lymph nodes decreased dramatically in the superficial cervical lymph nodes, and only a small amount of DC in the cortical and paracitical areas showed scattered distribution in the cortex and paracitical areas, that is, dendritic cell fatigue (dendritic cell exhaustion). However, the number of DC in the superficial cervical lymph nodes increased again after 120h, and was mainly distributed in the cortical area. The PBS control group was mainly distributed in the cortical region. No similar changes were observed. This indicates that DC is the antigen presenting cell of FMDV.
2. the cellular immune response induced by oral inactivated FMDV vaccine: after vaccination 24h, the content of IFN- gamma in the serum of mice began to increase (10.1568 + 0.4689), but (10.0512 + 0.0684) compared with the PBS control group (P0.05), but after 48h, the content of IFN- gamma in the serum of the vaccinated mice increased significantly (10.7161 + 0.2199), and the control group of PBS (10.) (10.) The difference was very significant (P0.01). After inoculation of 72h, the content of IFN- gamma in the serum of vaccinated mice reached the highest peak (11.6784 + 0.4590). Compared with the PBS control group (10.1249 + 0.1498), the difference was very significant (P0.01).96h, the content of IFN- gamma decreased (10.9839 + 0.3633), and the difference was significant compared with that of the PBS control group (10.0933 + 0.0954). (P0.05), at 120h, the test group (10.5285 + 1.3903) and the PBS control group (10.0534 + 0.1543) had no significant difference (P0.05). This showed that the cellular immune response induced by the oral inactivated FMDV vaccine was in accordance with the migration patterns of DC in the drainage lymph nodes.
3. the preparation of MoDC in BALB/c mice: immunocytochemistry and flow cytometry showed that the purity of MoDC prepared in this experiment was greater than 99%.
4. MoDC's ubiquitination of inactivated FMDV antigen: the ubiquitination of FMDV antigen occurred in all load inactivated FMDV MoDC, while the ubiquitinated protein detection in MoDC without inactivated FMDV showed negative reaction. The FMDV protein appeared in MoDC after FMDV vaccine 0.5h, and the molecular weight was about 75kD. The content of protein-protein was slightly increased, but the change trend of ubiquitinated protein content was not obvious at different time points. The two monoclonal antibodies used in this experiment - mouse anti - Multi Chain ubiquitinated protein monoclonal antibody and mouse anti - single chain ubiquitinated protein monoclonal antibody (clone number FK1 and FK2, respectively) were presented in the Western blot test The positive reaction indicates that MoDC is ubiquitin to inactivate FMDV protein antigen in two different ways.
5. MoDC presented a mechanism for inactivating FMDV antigen: a large number of IFN- gamma produced by the co culture of 9h in the inactivated FMDV load MoDC and CD4+T cells, while the CD8+T cells co cultured with FMDV loaded MoDC only released IFN- gamma in large quantities after being stimulated by the antigen. 8+T cells were co cultured and detected the content of IFN- gamma in the supernatant according to the previous methods. The results showed that the two inhibitors all inhibited the production of IFN- gamma in CD4+T cells after co culture. It is worth noting that the lysosome inhibitor significantly inhibited the ability of the inactivated FMDV load DC to stimulate CD8+T cells to produce IFN- gamma, while the proteasome inhibitor promoted CD8+T to be fine. IFN- gamma is produced by the cell, but the ability to produce IFN- gamma by two inhibitors and the production of IFN- gamma in CD8+T cells is significantly decreased. These complex phenomena suggest that MoDC mainly activates the lymphoid T cells with a cross presenting antigen, and is characterized by the production of IFN- gamma. However, the time of producing IFN- gamma in CD8+T cells is significantly later than CD4+T cells and IFN- gamma release. It also showed diversity. Conclusion: 1. DC of the pharynx is an antigen presenting cell for inactivating FMDV, but it shows "fatigue" (Dendritic cell exhaustion) during the migration of the inactivated FMDV antigen by DC.
2. mice inoculated with inactivated FMDV vaccine can induce cellular immune response and produce IFN- gamma.
3. inactivated FMDV antigen was captured by DC after two different ubiquitination, namely, Multi Chain ubiquitination and single chain ubiquitination. But ubiquitination FMDV protein antigen is mainly marked by FK2 antibody, while FK1 antibody is less marked. Because FK1 only specifically combines multi chain ubiquitination protein, FK2 combines single chain ubiquitination protein and multi chain simultaneously. Ubiquitin protein, and the two do not bind to free ubiquitin, so the inactivated FMDV protein antigen is mainly ubiquitin ubiquitin in DC.
4. the ubiquitinative FMDV protein antigen can be degraded by proteasome and can be processed in lysase. The antigenic peptide produced is presented to CD4+T cells or CD8+T cells in the way of cross extraction..CD4+T cells can only identify the MHC-II molecules carrying the antigen epitopes, so the content of IFN- gamma caused by lysosome inhibitors is significant. The decrease clearly indicates that DC can present FMDV antigen by the lysosome -MHC-II molecular pathway, and the strong inhibitory effect of proteasome inhibitor on the secretion of IFN- gamma by CD4+T cells suggests that there may be a mechanism for the cross presentation of FMDV antigen in the molecular pathway of proteasome -MHC-II. And CD4+T cells are activated by antigenic peptides. The main differentiation is Th1 cell subclass and secrete high level of IFN- gamma. From the above results, it is not difficult to see that the DC loaded by FMDV is mainly used as an antigen peptide of "endocytic body and lysosome -MHC-I molecular pathway" to present an antigen peptide to CD8+T cells, thus starting the CTL response. The ability of CD8+T cells to produce IFN- gamma is consistent with DC migration fatigue. The ability of CD8+T cells to produce IFN- gamma is 48h after being activated. Significant decline also showed a suppressed fatigue response.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392

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