小鼠播散性巨细胞病毒感染的免疫应答和致病机制研究1小鼠脾巨噬细胞 DNA 感受器 AIM2识别 MCMV DNA 激活其炎性体通路的整体研究2Th17细胞在急性播散性 MCMV 感染致病机制中的作用（可编辑）

小鼠播散性巨细胞病毒感染的免疫应答和致病机制研究1小鼠脾巨噬细胞 DNA 感受器 AIM2识别 MCMV DNA 激活其炎性体通路的整体研究2Th17细胞在急性播散性 MCMV 感染致病机制中的作用（可编辑） 小鼠播散性巨细胞病毒感染的免疫应答和致病机制研 究1小鼠脾巨噬细胞 DNA 感受器 AIM2识别 MCMV DNA 激活 其炎性体通路的整体研究2Th17细胞在急性播散性 MCMV 感 染致病机制中的作用 分 类 号 学号 D200978329学校代码 10487密级博士学位论文 小鼠播散性巨细胞病毒感染的免疫应答和致病机制研究 1)小鼠脾巨噬细胞 DNA 感受器 AIM2 识别 MCMV DNA 激活 其炎性体通路的整体研究 2)Th17 细胞在急性播散性 MCMV 感染致病机制中的作用学位申请人: 李旭芳 学科专业 : 儿科学 指导教师 : 方 峰 教授 答辩日期: 2012 年 5 月 A dissertation submitted to Huazhong University of Science and Technology for the Degree of Doctor of Medicine The Studies on Immune Responses and Pathogenesis of Mice with Disseminated Murine Cytomegalovirus Infection: 1 Recognition of MCMV DNA by AIM2 as DNA sensor and activation of AIM2 inflammasome pathway in spleen macrophages of mice with disseminated MCMV infection in vivo 2 Roles of Th17 cells in the pathogenesis of the acute disseminated MCMV infectionD.Candidate: Li Xufang Major: Pediatrics Supervisor: Fang Feng Huazhong University of Science & Technology Wuhan 430074, P. R. China May, 2012 独创性声明 本人郑重声明,本学位论文是本人在导师指导下进行的研究工作及取得的研 究成 果的总结。尽我所知,除文中已经标明引用的内容外,本论文不包含任何其他 个人或 集体已经发表或撰写过的研究成果。对本文的研究做出贡献的个人和集体, 均已在文 中以明确方式标明。本人完全意识到本人将承担本声明引起的一切法律后 果。 学位论文作者签名: 日期: 年 月 日学位论文版权使用授权 本学位论文作者完全了解学校有关保留、使用学位论文的规定,即:学校有权保 留并向国家有关部门或机构送交论文的复印件和电子版,允许论文被查阅和借阅。本 人授权华中科技大学可以将本学位论文的全部或部分内容编入有关数据库进行检索, 可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。 保密? ,在_____年解密后适用本授权书。 本论文属于 不保密?。 (请在以上方框内打“?” ) 学位论文作者签名: 指导教师签名:日期: 年 月 日 日期: 年 月 日 基金项目 ??教育部博士点基金项目《CMV 激活天然免疫细胞 dsDNA 感受器相 关炎性体通路对抗 CMV免疫的指导作用》,项目编号:2009014211007 目 录 缩略词 表??????????????????????????????????????????????????????????????????? ?????????1 中文摘 要????????????????????????????????????????????????????????????????????????????3 英文摘 要????????????????????????????????????????????????????????????????????????????7 前 言?????????????????????????????????????????????????????????????????????????? 13 一、 小鼠脾巨噬细胞 DNA 感受器 AIM2 识别 MCMV DNA 激活其 炎性体通路的整体研 究?????????????????????????????????????????????????? 20材料和方 法????????????????????????????????????????????????????????????????? 21 结 果???????????????????????????????????????????????????????????????????? 29 讨 论???????????????????????????????????????????????????????????????????? 33 参考文 献???????????????????????????????????????????????????????????????????? 37 二、Th17细胞在急性播散性 MCMV 感染致病机制中的作用 实验一 MCMV 体外诱导 Th17 细胞分化的探索 ?????????????????? 40 材料和方 法????????????????????????????????????????????????????????????????? 40 结 果???????????????????????????????????????????????????????????????????? 46 讨 论???????????????????????????????????????????????????????????????????? 50 参考文 献???????????????????????????????????????????????????????????????????? 51 实验二 Th17 细胞在急性播散性 MCMV 感染致病机制中的作用 ??? 54 材料和方 法????????????????????????????????????????????????????????????????? 54 结 果???????????????????????????????????????????????????????????????????? 61 讨 论???????????????????????????????????????????????????????????????????? 69 参考文 献???????????????????????????????????????????????????????????????????? 72 全文小 结?????????????????????????????????????????????????????????????????????????? 74 综 述?????????????????????????????????????????????????????????????????????????? 75 附 录?????????????????????????????????????????????????????????????????????????? 87 致 谢?????????????????????????????????????????????????????????????????????????? 88 华 中 科 技 大 学 博 士 学 位 论 文 缩略词表 英文缩写 英文全名 中文名 AIM2 absent in melanoma 2 黑色素瘤缺失基因 2 ASC apoptosis-associated speck-like protein 包含 CARD结构域的 containing a caspase recruitment CARD 凋亡相关点样蛋白 domain caspase-1 cysteine-aspartic protease-1 半胱氨酸天冬氨酸蛋白 酶 1 CIA collagen-induced arthritis 胶原诱导的关节炎 CMV cytomegalovirus 巨细胞病毒 DAI DNA-dependent activator of IRF DNA 依赖性 IRF 激活因 子 dsDNA double stranded DNA双链 DNA dsRNA double stranded RNA 双链 RNA EAE Experimental autoimmune encephalomyelitis 实验诱导的自身免疫性 脑脊髓炎 ELISA enzyme linked immunosorbent assay 酶联免疫吸附试验 GAPDHGlyseraldehyde-3- phosphate dehydrogenase 3-磷酸甘油醛脱氢酶 HBV hepatitis B virus 乙型肝炎病毒 HCMV human cytomegalovirus 人巨细胞病毒 HCV hepatitis C virus 丙型肝炎病毒 HIV human immunodeficiency virus 人免疫缺陷病毒 HSV herpes simplex virus 单纯疱疹病毒 IFN interferon干扰素 IRF interferon regulation factor干扰素调节因子 IL-1 βinterleukin-1 β 白细胞介素 1β IL-17interleukin-17 白细胞介素 17 1华 中 科 技 大 学 博 士 学 位 论 文 IL-18interleukin-18 白细胞介素 18 IL-21interleukin-21 白细胞介素 21 IL-23interleukin-23 白细胞介素 23 MCMV murine cytomegalovirus 小鼠巨细胞病毒 MyD88 myeloid differentiation factor 88 髓系分化蛋白 88 MOImultiplicity of infection感染复数 MEFmouse embryonic fibroblast 小鼠胚胎成纤维细胞 NF- κB nuclear factor-kappa B 核因子 κB NK natural killer cell 自然杀伤细胞 NLR Nucleotide-binding oligomerization NOD样受体 domain-like receptor NALP3 NACHT, LRR and PYD domain-containing 包含 NACHT、LRR 和 protein 3 PYD结构域的蛋白 3 PAMP pathogen-associated molecular pattern 病原相关的分子模式 PBS phosphate buffered saline 磷酸盐缓冲液 pro-caspase-1 pro- cysteine-aspartic protease-1 半胱氨酸蛋白酶 1 前体 pro-IL-1 β pro-interleukin-1 β 白细胞介素 1β 前体 pro-IL-18 pro-interleukin-18 白细胞介素 18前体 PFU plaque formation unit 空斑形成单位 PMAPhorbol-12-myristate-13-acetate 佛波酯 Th1 T helper 1 cell 辅助性 T细胞 1型 Th2 T helper 2 cell 辅助性 T细胞 2型 Th17 T helper 17 cell 辅助性 T细胞 17型 TLR toll-like receptor Toll 样受体 TMEV Theiler’s murine encephalomyelitis virus 泰勒虫属鼠脑脊髓炎 病 毒 2华 中 科 技 大 学 博 士 学 位 论 文 小鼠播散性巨细胞病毒感染的免疫应答和致病机制研究 1)小鼠脾巨噬细胞 DNA 感受器 AIM2 识别 MCMV DNA 激活其炎性体通路的整体研究 2)Th17细胞在急性播散性 MCMV 感染致病机制中的作用 中文摘要【目的】 1 在整体水平观察小鼠 MCMV感染后脾巨噬细胞内 AIM2炎性体各组分蛋白 及其 下游促炎因子 IL-1 β 和 IL-18 的时序性表达变化,证实 AIM2 对 MCMV DNA 的 识别并探讨其炎性体通路在抗 MCMV天然免疫与获得性免疫机制中的作用; 2 在整体水平观察 MCMV 播散性感染急性期脾 Th17 细胞的分化及其细胞 因子 IL-17A在不同脏器内的表达变化, Th17细胞和 IL-17A表达与 MCMV载量 及脾和唾液腺病理改变的相关性,探讨 Th17细胞在急性播散性 MCMV感染致病 机制中的作用。 【方法】 3 1 建立动物模型:45只4.5周龄BALB/c小鼠,腹腔接种(5×10 )PFU MCMV Smith 株,建立播散性感染模型,另45只小鼠模拟感染做为对照。于病毒接种后3 d、7 d、 14 d、28 d(急性期末)和45 d,收集小鼠静脉血和无菌分离脾、唾液腺、肝和肺 组织。为获取足够的脾巨噬细胞和血清样本量,各组每个时间点随机抽取3只小鼠 为1小组,其静脉血予以混合,脾脏各取2/3加以混合富集巨噬细胞,其余组织样 本随机抽取3份,故每个时间点的样本数为3; 2 AIM2炎性体通路蛋白表达:分离小鼠脾脏中巨噬细胞,提取蛋白,用Western blot 法AIM2炎性体通路蛋白包括AIM2、ASC、pro-caspase-1和caspase-1 的表达强 度,设GAPDH为内参蛋白,以各目的蛋白与GAPDH蛋白积分光密度比值(K 值) 进行半定量分析; 3华 中 科 技 大 学 博 士 学 位 论 文 3 血清IL-1 β和IL-18水平:用双抗体夹心ELISA法检测血清中AIM2炎性体通路下游 细胞因子IL-1 β和IL-18水平的变化; 4 优化Th17细胞分化诱导方案:分离3只正常小鼠的脾脏淋巴细胞;分别应用无刺 激、灭活MCMV、PMA/Ionomycin、灭活MCMV+PMA/Ionomycin刺激方法;在脾 + + + 脏淋巴细胞培养刺激不同时间后,采用流式细胞术检测CD3 CD4 IL-17A 细胞比 率;并用双抗体夹心ELISA法检测培养上清中IL-17A蛋白浓度,观察分析不同方 法诱导Th17细胞分化的效应,选择最佳的分化诱导方法; 5 脾Th17细胞比率和病毒特异性IL-17A表达水平:取急性期阶段即感染后3 d、7 d、 14 d和28 d 的脾组织,分离脾脏淋巴细胞。采用灭活MCMV+PMA/Ionomycin 刺激 + + + 方法诱导Th17细胞分化,用流式细胞术检测脾脏淋巴细胞中CD3 CD4 IL-17A 细 胞比率。用双抗体夹心ELISA法检测培养上清中IL-17A浓度时,设立单独 PMA/Ionomycin刺激组作为基础刺激对照,计算灭活MCMV/PMA/Ionomycin刺激 和单独PMA/Ionomycin刺激脾淋巴细胞培养上清中IL-17A浓度之差值来评估病毒 特异性IL-17A表达水平。对比分析两组之间的差异; 6 不同脏器组织中IL-17A蛋白表达:用免疫组织化学染色法检测急性期试验小鼠脾、 唾液腺、肝、肺组织中IL-17A蛋白的表达分布情况,对比分析不同脏器组织的局 部免疫特征; 7 脾和唾液腺病理变化:取脾和唾液腺组织切片行HE染色,半定量评估急性期试验 小鼠脾和唾液腺组织的病理变化; 8 MCMV病毒载量:用空斑试验检测急性期各脏器组织内感染性病毒滴度; 9 相关性分析:对脾细胞培养上清中病毒特异性IL-17A和组织中IL-17A蛋白表达水 平与脾和唾液腺组织病理损伤程度及病毒载量进行相关性分析。 【结果】 1 AIM2炎性体通路蛋白表达:MCMV感染后的脾巨噬细胞提取蛋白中,AIM2 炎性 体通路各组分包括AIM2、 ASC、 pro-caspase-1和caspase-1蛋白表达呈现一致性变化, 均在感染后3 d(感染早期)显著升高达峰值,与模拟感染对照组相比差异有统计 学意义, K值分别为 1.120? 0.243 vs 0.230? 0.046; 1.318?0.333 vs 4华 中 科 技 大 学 博 士 学 位 论 文 0.248?0.090;2.049?0.401 vs 0.390?0.120;1.483?0.420 vs 0.176?0.045,均 P0.01;其后表达均下降,与对照组水平相当; 2 血清IL-1 β和IL-18水平: MCMV感染组血清IL-1 β和IL-18浓度于感染后3 d 明显高 于模拟感染对照组,IL-1 β浓度为(111.050?28.500)vs(55.858?2.983)pg/ml, P0.05;IL-18浓度达(99.911?2.222)vs(57.206?6.228)pg/ml,P0.01; 3 优化Th17细胞分化诱导方法:无刺激组和灭活MCMV刺激组均不能有效诱导 Th17细胞表达。与应用相同方法培养刺激1 d+6 h组及应用PMA/Ionomycin培养刺 激6 h组相比,灭活MCMV+PMA/Ionomycin培养刺激6 h组能够有效诱导Th17细 胞分化 [Th17细胞比率分别为 0.163 ? 0.035 vs 0.083 ? 0.032%,P0.05; 0.163 ? 0.035 vs 0.033 ? 0.015 %, P0.01];同时,脾淋巴细胞培养上清中IL-17A 浓度也明显升高[分别为67.246 4.578 vs 45.317 8.793 pg/ml,P0.05; ? ? 67.246 ? 4.578 vs 24.474 ? 5.134 pg/ml,P0.01]; 4 脾Th17细胞比率和病毒特异性IL-17A表达水平:MCMV感染后3 d,7 d,14 d, 28 d,小鼠脾淋巴细胞体外经优化方法诱导分化,Th17细胞比率均高于模拟感染 对照组[分别为 0.67?0.13 vs 0.22?0.02%,P0.004;0.82?0.02 vs 0.18? 0.06%, P0.004; 1.14?0.09 vs 0.19?0.04%, P0.000; (0.47?0.11) vs (0.20 ?0.06)%,P0.017]。MCMV感染组脾淋巴细胞培养上清中病毒特异性IL-17A 蛋白浓度在感染后3、7 d虽有升高,但与模拟感染对照组相比无统计学差异 (P0.05);而感染后14 d达峰值,明显高于对照组[81.98?12.37 vs 44.96?8.44 pg/ml, P0.01];至感染后28 d 虽有下降,但仍高于对照组[57.58?8.14 vs 35.10 ?10.41 pg/ml,P0.05]; 5 不同脏器组织中 IL-17A 蛋白表达:MCMV 感染鼠的肝、肺组织中仅于感染 后 3d可见少量 IL-17A+细胞;脾和唾液腺组织中可见明显增多的 IL-17A+细胞,并 于感染后 14 d达峰值,且部分唾液腺分泌管上皮细胞也表达 IL-17A; 5华 中 科 技 大 学 博 士 学 位 论 文 6 脾和唾液腺病理变化:MCMV感染后 14 d脾组织病理损伤程度最重:白髓结构 明显破坏,可见大量吞噬细胞、出血坏死及纤维条索增生;MCMV感染后 14 d 唾液腺组织病理损伤程度也最严重:出现大量炎性浸润灶,组织结构明显破坏; 7 MCMV病毒滴度:肝和脾组织病毒滴度于感染后 3 d升高,其后迅速下降,14 d 检测不到;肺组织病毒滴度低于检测低限,而唾液腺组织病毒滴度呈逐渐增高趋 势,于感染后 14 d达峰值; 8 相关性分析: 脾淋巴细胞培养上清中病毒特异性 IL-17A蛋白浓度与唾液腺组织 病毒滴度呈正相关(r0.74,P0.01);脾和唾液腺组织病理损伤越重,脾细胞 培养上清中病毒特异性 IL-17A 蛋白浓度越高(r0.78, P0.01);唾液腺组织内 的 IL-17A 蛋白表达与 MCMV 滴度变化呈正相关(r0.63,P0.05),并与脾和 唾液腺组织病理损伤程度也呈正相关(r0.68,P0.01)。 【结论】 1 在 MCMV感染早期,小鼠脾巨噬细胞 AIM2感受器能够识别胞内 MCMV DNA; AIM2 炎性体参与 MCMV 感染早期的抗病毒天然免疫应答;同时,通过其下游 细胞因子 IL-1 β 和 IL-18在启动获得性免疫应答过程中发挥重要作用。 2 MCMV感染急性期 IL-17A蛋白表达呈现明显组织差异性分布,其在唾液腺 中高 表达的局部免疫特征可能是 MCMV能够在唾液腺中长期持续复制的关键免 疫因 素之一;Th17细胞可能通过 IL-17A参与 MCMV持续性感染及组织免疫病理 损 伤机制。 【关键词】DNA 感受器;AIM2 炎性体通路;IL-1 β;IL-18;天然免疫;小鼠巨 细胞 病毒;Th17细胞;IL-17A 6华 中 科 技 大 学 博 士 学 位 论 文 The Studies on Immune Responses and Pathogenesis of Mice with Disseminated Murine Cytomegalovirus Infection 1 Recognition of MCMV DNA by AIM2 as DNA sensor and activation of AIM2 inflammasome pathway in spleen macrophages of mice with disseminated MCMV infection 2 Roles of Th17 cells in the pathogenesis of the acute disseminated MCMV infectionAbstract Objectives: 1 To observe the time courses of expression of AIM2 inflammasome in spleen macrophages and its downstream cytokines, IL-1 β and IL-18, in sera from mice with acute disseminated murine cytomegalovirus MCMV infection in order to demonstrate whether AIM2 could recognize MCMV DNA and explore the role of AIM2 inflammasome pathway in innate and acquired immune responses against MCMV in vivo2 To investigate the differential levels of T helper 17 Th17 cells and expression of cytokine, IL-17A, in the different organs during the acute stage of disseminated MCMV infection and to analyze the relationship between expression of IL-17A and MCMV titers or pathological changes in the tissues for further understanding the role of Th17 cells in the pathogenesis of the acute disseminated MCMV infection in vivo Methods: 1 Development of the mouse model: Fory-five BALB/c mice aged of 4.5-week-old 3 were inoculated intraperitoneally with salivary gland homogenates containing 5×10 PFU of MCMV Smith strain to establish the model with the disseminated MCMV 7华 中 科 技 大 学 博 士 学 位 论 文 infection, another 45 mice injected with normal salivary gland homogenates served as mock-infected controls. On the 3d, 7d, 14d, 28d the end of acute infection and 45d of post infection PI, nine mice were sacrificed and their venous blood, spleens, salivary glands, livers and lungs were obtained, respectively. In order to get enough volume of sera and number of macrophages from spleen, every 3 mice as one subgroup were randomly selected and their blood samples and the two to third of spleens in size were mixed up as one sample, and the rest spleens and other tissues were randomly selected from 3 mice, so that there were 3 samples at each time point 2 Expression of AIM2 inflammasome proteins: Macrophages were isolated from spleens and their proteins were extracted, then the expression of AIM2 inflammasome proteins including AIM2, ASC, pro-caspase-1 and caspase-1 were detected by using Western blot assay. GAPDH protein served as the internal control. The expressing levels of each protein were semi-quantitatively analyzed by the ratio K value of the integral density values of the desired and internal bands3 Levels of IL-1 β and IL-18 in sera: The levels of IL-1 β and IL-18, downstream cytokines of AIM2 inflammasome pathway, in sera were measured by double antibody sandwich enzyme-linked immunosorbent assay ELISA4 The optimizing method for induction of Th17 cell differentiation: Spleen lymphocytes were isolated from 3 normal mice and then cultured in the four different conditions, including no stimulus, heat-inactivated MCMV, PMA/Ionomycin and heat-inactivated MCMV/PMA/Ionomycin. After preset culture time intervals, cells were harvested for + + + detection of the percentages of CD3 CD4 IL-17A cells by using flow cytometryMoreover, levels of IL-17A in the culture supernatants were measured by ELISA. The induction effect of each group was analyzed for determining the optimizing method for induction of Th17 differentiation5 Expression of Th17 cells and MCMV-specific IL-17A protein from spleens: On the 3d, 7d, 14d, and 28d PI acute stage of infection, isolated spleen lymphocytes were 8华 中 科 技 大 学 博 士 学 位 论 文 stimulated with heat-inactive MCMV/PMA/Ionomycin for 6h and then the + + + percentages of CD3 CD4 IL-17A cells in spleen lymphocytes were determined by flow cytometry. For measurement of viral specific IL-17A levels in culture supernatants by ELISA, isolated spleen lymphocytes were stimulated simultaneously with heat-inactive MCMV/PMA/Ionomycin and PMA/Ionomycin, respectively, and the difference of IL-17A levels between those with above two stimulated methods was considered as MCMV-specific IL-17A level 6 Expression of IL-17A protein in organs: Immunohistochemical stains were employed to detect the expression of IL-17A in the tissues of spleen, salivary gland, liver and lung from experimental mice during acute stage of infection, for analyzing the difference of IL-17A expression in the different organs7 Pathological changes of spleen and salivary gland: The pathological changes of spleen and salivary gland during the acute infection were assessed by histological examination and a semi-quantitative evaluation method 8 Infective MCMV loads: Virus titers of tissue homogenates were determined using a standard plaque assay9 Correlation analysis was performed between the levels of MCMV-specific IL-17A in spleen cell culture supernatants or expression of IL-17A in organs and the pathological changes of spleen and salivary gland or virus titersResults: 1 Expression of AIM2 inflammasome proteins: Expressing levels of all AIM2 inflammasome proteins AIM2, ASC, pro-caspase-1 and caspase-1 in spleen macrophages were significantly increased and peaked on day 3 after MCMV infection, with obviously higher K values of AIM2, ASC, pro-caspase-1 and caspase-1 in MCMV-infected mice than those in mock-infected mice, 1.120?0.243 versus 0.230 ?0.046, 1.318?0.333 versus 0.248?0.090, 2.049?0.401 versus 0.390?0.120, and 1.483?0.420 versus 0.176?0.045, respectively P0.01. Afterwards, the expressing 9华 中 科 技 大 学 博 士 学 位 论 文 levels of the four proteins in MCMV-infected mice rapidly dropped to the control levels2 Levels of IL-1 β and IL-18 in sera: Serum levels of IL-1 β and IL-18 in MCMV-infected mice were also significantly higher than those in mock-infected controls 111.050?28.500 versus 55.858?2.983 pg/ml, P0.05, and 99.911?2.222 versus 57.206?6.228 pg/ml, P0.01, respectively on day 3 after infection 3 Optimizing method for Th17 differentiation: Percentages of Th17 cells in spleen lymphocytes stimulated with heat-inactivated MCMV/PMA/Ionomycin for 6 hours were significantly higher than those with the same stimulus for 1 day and 6 hours and those with PMA/Ionomycin for 6 hours 0.163 ? 0.035 versus 0.083 ? 0.032%, P0.05, and 0.163 ? 0.035 versus 0.033 ? 0.015 %, P0.01, respectively, and levels of IL-17A in spleen lymphocytes culture supernatants showed the same findings 67.246 ? 4.578 versus 45.317 ? 8.793 pg/ml, P0.05, and 67.246 ? 4.578 versus 24.474 ? 5.134 pg/ml, P0.01, respectively. But no expression of Th17 cells and IL-17A was observed in spleen lymphocytes stimulated with no stimulus or heat-inactive MCMV4 Percentages of Th17 cells and expression of IL-17A in spleen lymphocytes: On the 3d, 7d, 14d and 28d PI, percentages of Th17 in spleen lymphocytes stimulated with optimizing induction method were all increased in MCMV-infected mice compared with mock-infected mice 0.67?0.13 versus 0.22?0.02%, P0.004, 0.82?0.02 versus 0.18?0.06%, P0.004, 1.14?0.09 versus 0.19?0.04%, P0.000, and 0.47? 0.11 versus 0.20?0.06%, P0.017, respectively. Levels of MCMV-specific IL-17A were markedly higher on the 14d and 28d after infection in MCMV-infected mice than those in mock-infected mice 81.98?12.37 versus 44.96?8.44 pg/ml, P0.01, and 57.58?8.14 versus 35.10?10.41 pg/ml, P0.05, respectively, but no significance was observed on the 3d and 7d (P0.05). Both of percentages of Th17 cells and levels of MCMV-specific IL-17A peaked on day 14 after MCMV infection 10华 中 科 技 大 学 博 士 学 位 论 文 5 Expression of IL-17A protein in organs: Expression of IL-17A showed a obvous organ disparity in MCMV-infected mice. IL-17A+ cells significantly accumulated in spleen and salivary gland of MCMV infected mice. The number of IL-17A+ cells reached the peak on day 14 and some positive staining could be observed in epithelial cells of secretory duct in salivary gland. Only fewer IL-17A+ staining was found in livers and lungs on day 3 PI6 Pathology of spleen and salivary gland: Histological evaluation showed that the most severe damages could be observed both in spleen and salivary gland on day 14 after infection.White pulp structures were extensively destructed and large numbers of phagocytic cells, dead cells and debris could be seen in spleens, and severe inflammatory infiltrates and loss of normal tissue architecture in salivary gland7 MCMV titers: Viral titers gradually increased and peaked on day 14 in salivary gland and remained higher on day 28. Viral titers peaked on day 3 in liver and spleen and then quickly diminished and virus was not detected on day 14. Virus was undetectable in lung throughout the experiments8 Correlation analysis: Levels of MCMV-specific IL-17A in spleen lymphocytes culture supernatants showed positive correlation with MCMV titers in salivary gland r0.74, P0.01 and injury degrees of spleen and salivary gland r0.78, P0.01. Expression of IL-17A also displayed positive correlation with MCMV titers in salivary gland r0.63,