小鼠巨细胞病毒免疫致病机制的研究

发布时间：2018-06-07 00:39

本文选题：小鼠巨细胞病毒 + IL-17　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分炎性因子IL-17在鼠巨细胞病毒致病机制中的作用 [目的] 1)建立鼠巨细胞病毒(Murine cytomegalovirus,MCMV)播散性感染动物模型,整体水平观察炎性因子IL-17在肝脏及唾液腺组织中的表达情况,探讨其在MCMV致病过程中发挥的作用； 2)应用IL-17抗体中和MCMV播散性感染模型小鼠体内的炎性因子IL-17,进一步研究IL-17在MCMV免疫致病机制中的地位。 [方法] 1.建立MCMV全身播散性感染模型：选择4.5周龄的雌性BALB/c小鼠,随机分为两组：MCMV感染组与模拟感染对照组。在MCMV感染后3天、7天、14天和28天各组随机选取4只小鼠,给予乙醚麻醉,摘除眼球取血后颈椎脱臼处死,分离血清,无菌收取唾液腺及肝脏组织,部分经4%多聚甲醛固定后石蜡包埋,部分组织置于-70℃冰箱保存。 2.病毒滴度测定：取胎鼠胚肺成纤维细胞作为培养细胞,采用标准蚀斑试验检测病毒感染后不同时间点唾液腺及肝脏组织内的感染性病毒滴度。 3.组织内IL-17及IL-17R mRNA的表达：用Trizol法提取唾液腺及肝脏组织总RNA,检测RNA纯度将其逆转录为cDNA；采用RT-PCR法检测IL-17及IL-17R mRNA在唾液腺及肝脏组织中的表达水平。每份样本同时检测目的基因和内参GAPDH基因。 4.组织内IL-17蛋白表达：制备组织石蜡切片,采用免疫组织化学技术检测唾液腺及肝脏组织中IL-17的表达水平与分布特征。 5.组织病理改变：石蜡包埋唾液腺及肝脏组织切片后行苏木紫-伊红染色,评估唾液腺及肝脏组织的病理性损伤。 6.血清ALT水平：用罗氏生化分析仪检测血清ALT水平。 7.IL-17抗体阻断研究：首先建立MCMV全身播散性感染模型,再腹腔注射IL-17抗体以中和体内表达的IL-17(IL-17阻断组),同时设立正常对照组、MCMV感染对照组和同型抗体对照组,每组小鼠4只。在病毒感染后第7天,各组小鼠给予乙醚麻醉,摘除眼球取血后颈椎脱臼处死,分离血清,无菌收取唾液腺及肝脏组织,部分经4%多聚甲醛固定后石蜡包埋,部分唾液腺及肝脏组织置-70℃冰箱保存。用Western-blot法检测唾液腺及肝脏组织中IL-17蛋白表达水平；采用标准蚀斑试验检测组织中MCMV感染性病毒滴度；用RT-PCR法测定组织中IL-17、IL-17R, IFN-y和IL-10基因转录(mRNA)水平；用HE染色法评估肝脏及唾液腺组织的病理性损伤程度；用罗氏生化分析仪检测血清ALT水平。 [结果] 1.组织内感染性病毒滴度：唾液腺组织中病毒滴度明显高于肝脏组织,在病毒感染后第7天明显升高,并在14天达到峰值,随后病毒滴度逐渐减低；而肝脏组织中病毒滴度在感染后第3天升高,第7天病毒滴度明显降低,在感染后第14天病毒滴度已低于标准蚀斑试验法的检测水平,无法形成病毒感染性蚀斑。 2.组织病理性损伤：①唾液腺组织：在MCMV感染后第7天,唾液腺组织内炎性细胞浸润大量出现；在病毒感染后第14天,炎性细胞浸润更加明显,炎性灶逐渐扩大,并与邻近炎性灶相互融合；在病毒感染后28天病理性损伤逐渐减轻,但是仍可见炎性细胞浸润；②肝组织：MCMV感染组小鼠肝组织在病毒感染后第3天可见肝细胞气球样变性,嗜酸性变明显,门管区可见炎性细胞浸润,肝细胞点状坏死明显；感染后第7天病变达顶峰,肝细胞嗜酸性变,点灶状坏死明显,坏死区有少量炎性细胞浸润,门管区可见大量炎性细胞浸润；随后病变逐渐缓慢减轻,炎性灶缩小。 3.血清ALT水平：MCMV感染组小鼠血清ALT水平明显高于正常对照组[(144±11) vs(49.6±5), p0.05]。 4.组织内IL-17和IL-17R mRNA表达：①唾液腺组织：MCMV感染组在感染后各时间点的IL-17mRNA表达均高于正常对照组,在MCMV感染后逐渐升高,并在感染后14天明显高于模拟感染对照组,差异具有统计学意义[(0.4103±0.0636) vs(0.245±0.027),p0.05],IL-17在唾液腺组织中的表达量与唾液腺组织的病理性损伤明显相关(R=0.616,p0.05)；IL-17R表达在MCMV感染后呈现逐渐上升趋势,并且在感染后第14天明显高于模拟感染对照组,差异具有统计学意义[(0.345±0.012)vs(0.17±0.023),p0.05],其高表达与唾液腺组织病理性损伤显著相关(R=0.751,p0.05)。②肝脏组织：MCMV感染后,肝组织中IL-17mRNA表达逐渐升高,于感染后7天达峰值[(0.67±0.104)vs(0.287±0.046),p0.05],14天后缓慢下降,其表达量与肝脏组织损伤显著相关(R=0.773,p0.05)；模拟感染组IL-17R在肝脏组织的表达相对稳定,而MCMV感染组IL-17R表达在各时间点均高于模拟感染对照组,感染后呈逐渐上升趋势,在第7天显著升高,差异有统计学意义[(0.7035±0.0901)vs(0.442±0.112),p0.05)],随后表达趋于稳定,其表达量与肝组织损伤明显相关(R=0.712,p0.05)。 5.组织内IL-17蛋白表达：MCMV感染组唾液腺组织中IL-17蛋白表达在感染后逐渐升高并在14天达到高峰,随后逐渐降低。IL-17阳性细胞主要分布在腺管周围及炎性细胞浸润密集部位；感染组肝脏组织在感染后3天IL-17表达量明显高于模拟感染组,第7天阳性细胞数增加,随后降低,细胞主要分布于汇管区及肝小叶炎性细胞密集部位。 6.IL-17抗体阻断实验：①病毒滴度：与感染对照组及同型抗体对照组相比,IL-17抗体阻断组在MCMV感染后第7天肝脏组织病毒滴度显著降低(p0.05),而唾液腺中病毒滴度与MCMV感染对照组及同型抗体对照组比较无明显差异(p0.05)；②组织内IL-17蛋白表达：IL-17抗体阻断组肝脏组织IL-17表达较MCMV感染组及同型抗体对照组比较明显下降(p0.05),而唾液腺组织IL-17表达量与MCMV感染组及同型抗体对照组相比差异无统计学意义,说明腹腔注射IL-17抗体未能有效中和唾液腺组织中IL-17；③组织病理改变：在病毒感染后第7天,IL-17抗体阻断组肝组织病理性损伤程度明显低于MCMV染对照组及同型抗体对照组；而唾液腺组织病理改变与MCMV感染对照组及同型抗体对照组比较无明显差别。④肝脏组织内IL-17、IL-17R、IFN-y和IL-10基因转录(mRNA)水平：与同型抗体对照组及MCMV感染对照组相比,IL-17抗体阻断组的IFN-γ[(0.56±0.06) vs(0.55±0.13)vs(0.96±0.2),p0.05]与IL-10[(0.55±0.073)vs(0.51±0.07)vs(0.903±0.18),p0.05]的表达明显增加；IL-17表达明显降低[(0.77±0.15)vs(0.80±0.14)vs(0.44±0.07),p0.05]；而IL-17R表达无明显差异[(0.81±0.16)vs(0.89±0.38)vs(0.87±0.23),p0.05]；⑤血清ALT水平：与MCMV感染对照组及同型抗体对照组相比较,IL-17抗体阻断组的ALT水平显著降低[(146±15)vs(102±11)vs(37±12),p0.05]。 [结论] 1.MCMV感染组织内IL-17/IL-17R高表达与组织病理性损伤严重程度密切相关；阻断肝组织IL-17表达可有效减轻肝组织病理损伤和促进肝功能恢复； 2.感染早期肝组织内IL-17相关免疫反应有利于肝脏内病毒的清除； 3.腹腔注射IL-17抗体可有效阻断肝组织中IL-17表达,却不能中和唾液腺组织中IL-17表达； 4.唾液腺内IL-17受体低表达与IL-17和IL-17受体表达高峰延迟等局部免疫特征可能与唾液腺内病毒逃避免疫清除机制有关； 5.阻断IL-17表达后,肝组织IFN-y及IL-10表达增加,提示三者可相互调节而发挥不同功能。IFN-γ及IL-10高表达可加速肝组织内病毒的清除和减轻炎症损伤。第二部分MCMV启动Caspase-1信号通路对适应性免疫应答的影响 [目的] 1.建立MCMV播散型感染动物模型,在整体水平观察Caspase-1信号通路启动情况,初步探讨其在MCMV致病过程中的作用； 2.研究Caspase-1信号通路对适应性免疫应答Th17细胞的影响 [方法] 1.建立MCMV全身播散型感染动物模型：32只4.5周龄雌性BALB/c、鼠被随机分为两组：MCMV病毒感染组与模拟感染对照组。在MCMV感染后3天、7天、14天和28天各组随机选取4只小鼠乙醚麻醉摘除眼球取血后颈椎脱臼处死,分离血清,无菌收取脾脏组织,部分经4%多聚甲醛固定后石蜡包埋,部分组织置于-70℃冰箱保存； 2.标准蚀斑试实验检测脾脏组织中感染性病毒滴度； 3.Western blot检测脾脏组织中Caspase-1表达强度； 4.免疫组化法检测脾组织中IL-1β和IL-18表达状况； 5.流式细胞技术检测脾脏组织中Th17细胞数量； 6.HE染色法评估脾脏的病理性损伤程度。 [结果] 1.MCMV感染后,脾脏组织中病毒滴度在感染后第3天已经开始升高,第7天病毒滴度逐渐降低,第14天时用标准空斑实验已经检测不到病毒； 2.与模拟感染对照组比较,MCMV感染组在感染后第3天脾组织中Caspase-1[(1.483±0.420) vs (0.176±0.045),p0.05]表达明显升高后逐渐下降； 3.脾脏组织中IL-1p和IL-18表达呈进行性升高,于感染后7天达峰值,14天减少,28天基本接近正常； 4.巨细胞病毒感染后,脾脏的Thl7细胞逐渐增多,并在感染后第14天达高峰[(1.14±0.09) vs (0.19±0.04),p0.05],明显高于模拟感染对照组； 5.脾脏组织病理评估：MCMV感染后第3天,脾脏组织病理损害不明显,第7天时白髓淋巴细胞明显增生,并出现较多中性粒细胞与单核细胞；第14天病变逐渐加重,可见大量巨噬细胞、出血坏死及纤维条索增生；第28天时病理损伤明显减轻。 [结论] 巨细胞病毒感染可使脾脏Caspase-1表达增加,其下游促炎因子IL-1β和IL-18合成和释放增多,进而促进Th17细胞适应性免疫应答反应,在清除病毒的同时也造成了组织免疫病理性损伤。
[Abstract]:Part 1 the role of inflammatory factor IL-17 in the pathogenesis of murine cytomegalovirus
[Objective]
1) to establish an animal model of Murine cytomegalovirus (MCMV) disseminated infection, and to observe the expression of inflammatory factor IL-17 in the liver and salivary glands on the whole level, and to explore its role in the pathogenesis of MCMV.
2) use IL-17 antibody to neutralize the inflammatory factor IL-17 in MCMV mice with disseminated infection, and further study the role of IL-17 in the pathogenesis of MCMV.
[method]
1. the model of MCMV systemic disseminated infection was established: female BALB/c mice of 4.5 weeks of age were selected and randomly divided into two groups: MCMV infection group and simulated infection control group. 4 mice were randomly selected for 3 days, 7 days, 14 days and 28 days after MCMV infection, given ether anesthesia, removal of cervical dislocations after exucleation of blood, separation of serum and asepy collection of salivary glands. And liver tissue, partially paraffin embedded after 4% paraformaldehyde, and partially stored in -70 C refrigerator.
2. virus titer measurement: fetal rat embryo lung fibroblasts were taken as culture cells, and the standard plaque test was used to detect the titer of infected virus in salivary glands and liver tissues at different time points after virus infection.
3. expression of IL-17 and IL-17R mRNA in the tissues: the total RNA of salivary glands and liver tissues was extracted by Trizol, and the purity of RNA was detected by reverse transcription to cDNA, and the expression level of IL-17 and IL-17R mRNA in salivary glands and liver tissues was detected by RT-PCR. The target gene and the internal reference gene were detected at the same time.
The expression of IL-17 protein in 4. tissues: tissue paraffin section was prepared, and the expression and distribution of IL-17 in salivary glands and liver tissues were detected by immunohistochemistry.
5. histopathological changes: paraffin embedded salivary glands and liver tissue sections were stained with hematoxylin eosin staining to assess pathological damage of salivary glands and liver tissues.
6. serum ALT level: serum ALT level was detected by Roche biochemical analyzer.
7.IL-17 antibody blocking study: first to establish a MCMV systemic disseminated infection model, and then intraperitoneally injected with IL-17 antibody to neutralize the IL-17 (IL-17 blockage group) expressed in the body, and set up a normal control group. The control group of MCMV infection and the same type antibody control group, 4 mice in each group. After seventh days of the virus infection, each group of mice is given ether anesthesia and extirpate After the ball was taken from the blood, the dislocated cervical vertebra was executed, the serum was separated, the salivary glands and liver tissues were collected asepsis, part of the paraffin was embedded after 4% polyformaldehyde. Some salivary glands and liver tissues were stored at -70 C fridge. The expression of IL-17 protein in the salivary glands and liver tissues was detected by Western-blot; the standard plaque test was used to detect the MCMV in the tissue. The titer of infectious virus, IL-17, IL-17R, IFN-y and IL-10 gene transcription (mRNA) were measured by RT-PCR, and the degree of pathological damage in the liver and salivary glands was evaluated by HE staining, and the serum ALT level was detected by Roche biochemical analyzer.
[results]
1. the titer of infective virus in the tissue: the virus titer in the salivary gland was significantly higher than that in the liver, and the virus titer increased obviously on the seventh day after the virus infection, and reached the peak at the 14 day, then the virus titer gradually decreased, while the virus titer in the liver tissue increased at third days after infection, and the virus titer decreased significantly on the seventh day, and the virus was the fourteenth day after infection. Titer is lower than the standard plaque test method, and can not form virus infected plaque.
2. tissue pathological injury: (1) salivary gland tissue: inflammatory cell infiltration in salivary glands appeared in the salivary gland on the seventh day after MCMV infection; inflammatory cells infiltrated more obviously on the fourteenth day after virus infection, inflammatory foci gradually expanded and fused with adjacent inflammatory foci; 28 days after the virus infection, the rational damage of the disease gradually lessen, but still Inflammatory cell infiltration was seen in the liver tissue: liver tissue in the liver tissue of the MCMV infection group showed that the liver cells were balloon like degeneration, eosinophilic degeneration was obvious on the third day after virus infection, inflammatory cells infiltrated in the portal area, and the necrosis of the liver cells was obvious. The pathological changes reached the peak at the seventh day after infection, the liver cells were eosinophilic and the focal necrosis was obvious. Necrotic areas were obvious. Necrotic areas were in the necrotic area. A small number of inflammatory cells infiltrated and a large number of inflammatory cells infiltrated in the portal area. Then the lesions gradually slowed down and the inflammatory foci were narrowed.
3. serum ALT level: the serum ALT level of MCMV infected group was significantly higher than that of the normal control group [(144 + 11) vs (49.6 + 5), p0.05].
4. the expression of IL-17 and IL-17R mRNA in the tissues: (1) the salivary gland tissue: the expression of IL-17mRNA in the MCMV infection group was higher than that of the normal control group after infection, and increased gradually after MCMV infection, and was significantly higher than that of the simulated infection control group after infection. The difference was statistically significant [(0.4103 + 0.0636) vs (0.245 + 0.027), p0.05], IL-17 The expression of the salivary gland was significantly correlated with the pathological damage of salivary gland tissue (R=0.616, P0.05). The expression of IL-17R showed a gradual upward trend after MCMV infection, and was significantly higher than that of the simulated infection control group on fourteenth days after infection. The difference was statistically significant [(0.345 + 0.012) vs (0.17 + 0.023), p0.05], and its high expression and saliva The pathological changes of the adeno tissue were significantly correlated (R=0.751, P0.05). After MCMV infection, the expression of IL-17mRNA in the liver tissues increased gradually. The peak value of the liver tissue was 7 (0.67 + 0.104) vs (0.287 + 0.046) after infection, p0.05], decreased slowly after 14 days, and its expression was significantly related to the liver tissue injury (R=0.773, P0.05), and IL-17R in the liver of the simulated infection group was in the liver. The expression of the dirty tissue was relatively stable, and the expression of IL-17R in the MCMV infection group was higher than that of the simulated infection control group at all time points. After infection, the expression increased gradually, and the difference was statistically significant [(0.7035 + 0.0901) vs (0.442 + 0.112), P0.05) at seventh days, and then the expression tended to be stable, and its expression was significantly related to the injury of liver tissue (R=0.71 2, P0.05).
The expression of IL-17 protein in 5. tissues: the expression of IL-17 protein in salivary glands of MCMV infection group increased gradually after infection and reached the peak in 14 days, and then gradually reduced.IL-17 positive cells mainly distributed around the glands and inflammatory cells, and the expression of IL-17 in the liver tissue in the infected group was significantly higher than that of the simulation in 3 days after infection. In the dyed group, the number of positive cells on the seventh day increased, and then decreased.
6.IL-17 antibody blocking experiment: (1) virus titer: compared with the infection control group and the same type antibody control group, the liver tissue virus titer of the IL-17 antibody blockage group decreased significantly (P0.05) at seventh days after MCMV infection, but there was no significant difference between the virus titer in the salivary gland and the MCMV infection control group and the same type antibody control group (P0.05); and (2) the I in the tissue I. The expression of L-17 protein: the expression of IL-17 in the liver tissue of the IL-17 antibody blockage group was significantly lower than that in the MCMV infection group and the same type antibody control group (P0.05), but the expression of IL-17 in salivary gland tissue was not significantly different from that of the MCMV infection group and the same type antibody control group, indicating that the peritoneal injection of IL-17 antibody did not effectively neutralize the IL- in the salivary glands. 17; (3) histopathological changes: in the seventh day after the virus infection, the degree of pathological damage of liver tissue in the IL-17 antibody blockage group was significantly lower than that of the MCMV control group and the same type antibody control group, while the pathological changes of salivary glands were not significantly different from those of the control group of MCMV infection and the same type antibody control group. (4) IL-17, IL-17R, IFN-y and IL in the liver tissue. -10 gene transcription (mRNA) level: compared with the same type antibody control group and the MCMV infection control group, the IFN- gamma (0.56 + 0.06) vs (0.55 + 0.13) vs (0.96 + 0.2), p0.05] and IL-10[(0.55 + 0.073) vs (0.51 + 0.07) vs (0.903 + 0.18), the expression of p0.05] was significantly increased. + 0.07), p0.05], and no significant difference in IL-17R expression [(0.81 + 0.16) vs (0.89 + 0.38) vs (0.87 + 0.23), p0.05]; serum ALT level: compared with MCMV infection control group and Homo antibody control group, the ALT level of IL-17 antibody blockage group was significantly lower [(146 + 15) vs (102 + 11) vs (37 + 12), p0.05].
[Conclusion]
The high expression of IL-17/IL-17R in 1.MCMV infected tissues is closely related to the severity of histopathological injury, and blocking the expression of IL-17 in liver tissue can effectively reduce the pathological damage of liver tissue and promote the recovery of liver function.
2. IL-17 associated immune response in the early stage of liver infection is beneficial to the elimination of virus in the liver.
3. intraperitoneal injection of IL-17 antibody can effectively block the expression of IL-17 in liver tissue, but can not neutralize the IL-17 expression in salivary gland tissue.
4. the low expression of IL-17 receptor in salivary glands and the peak delay of IL-17 and IL-17 receptor expression may be related to the mechanism of virus evasion in salivary glands.
5. the expression of IFN-y and IL-10 in liver tissues increased after blocking the expression of IL-17, suggesting that the three groups could adjust each other and exert different functions of.IFN- gamma and IL-10 to accelerate the clearance of virus in the liver tissue and reduce the inflammatory damage. The effect of Caspase-1 signaling pathway on the adaptive immune response in the second part of MCMV
[Objective]
1. establish MCMV disseminated infection animal model, observe the Caspase-1 signaling pathway at the overall level, and explore its role in the pathogenesis of MCMV.
2. to study the effect of Caspase-1 signaling pathway on adaptive immune response to Th17 cells.
[method]
1. the animal model of MCMV systemic disseminated infection was established: 32 4.5 weeks old female BALB/c, and the rats were randomly divided into two groups: MCMV virus infection group and simulated infection control group. After 3 days of MCMV infection, 7 days, 14 days and 28 days, 4 mice were randomly selected to remove the neck dislocated of the eyeball after exucleation of the eyeball, separate the serum and collect the spleen asepsis group. The parts were woven with paraffin fixed and paraffin embedded, and some of them were stored in -70 C refrigerator. 4%.
2. standard plaque test was used to detect infectious virus titer in spleen tissue.
3.Western blot was used to detect the expression intensity of Caspase-1 in spleen tissue.
4. immunohistochemical staining was used to detect the expression of IL-1 and IL-18 in spleen tissue.
5. the number of Th17 cells in spleen tissue was detected by flow cytometry.
6.HE staining was used to evaluate the degree of pathological injury of the spleen.
[results]
After 1.MCMV infection, the virus titer in the spleen tissues began to rise on the third day after infection, and the titer of the virus gradually decreased on the seventh day. The virus was not detected in the standard air spot test at fourteenth days.
2. compared with the simulated infection control group, the Caspase-1[(1.483 + 0.420) vs (0.176 + 0.045) and the p0.05] expression decreased gradually in the spleen tissue of the MCMV infection group after third days of infection.
3. the expression of IL-1p and IL-18 in spleen tissue progressively increased, and the peak value in 7 Tianda after infection, decreased in 14 days, and basically close to normal in 28 days.
After 4. cytomegalovirus infection, the Thl7 cells of the spleen increased gradually, and the peak of fourteenth Tianda after infection [(1.14 + 0.09) vs (0.19 + 0.04), p0.05], was significantly higher than that of the simulated infection control group.
5. the histopathological evaluation of spleen: third days after MCMV infection, the pathological damage of the spleen was not obvious. At the time of seventh days, the white myeloid lymphocyte proliferation was obvious, and more neutrophils and mononuclear cells appeared, and the pathological changes gradually aggravated on the fourteenth day, and a large number of macrophages, hemorrhage and necrosis and fibrous stripe hyperplasia were seen, and the pathological damage was obviously reduced at twenty-eighth days.
[Conclusion]
Cytomegalovirus infection can increase the expression of Caspase-1 in the spleen, increase the synthesis and release of IL-1 beta and IL-18 in the downstream, and then promote the adaptive immune response of Th17 cells, and cause pathological pathological damage of the tissues at the same time.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.1

【参考文献】