伪狂犬病病毒感染诱导的小胶质细胞动态学变化的研究

发布时间：2018-02-20 01:25

本文关键词： 伪狂犬病病毒小胶质细胞动态变化细胞增殖极化　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：猪伪狂犬病(Aujeszky's disease)最早发现于美国,在1920到1940年间呈世界性散在发生,并且在之后30年间呈爆发性,其发病率明显增加。目前,猪伪狂犬病在我国仍然是威胁养猪业的重要疾病之一。伪狂犬病病毒(Pseudorabies Virus,PRV)属于疱疹病毒科病毒,为有囊膜的双链DNA病毒。PRV感染动物后可以沿着感染部位附近外周神经复制移行,逐渐进入中枢神经系统(CNS),导致脑组织发生病变。研究伪狂犬病病毒诱导的小胶质细胞(microglia)动态变化及microglia动态变化对疾病发展进程的影响具有重要意义。本实验主要通过建立PRV人工感染小鼠模型,研究伪狂犬病病毒感染诱导的小胶质细胞动态学变化。实验采用PRV-Bartha K61疫苗株皮下人工感染小鼠,使用6×104 TCID50、1×105 TCID50以及2.7×105 TCID50三组剂量作为人工感染剂量。三组剂量均在人工感染后6 d导致小鼠出现死亡,小鼠存活率分别为93%、93%和85%。然而在人工感染后第7 d,小鼠存活率差异显著,6×104 TCID50剂量实验组存活率无变化,而另外两组分别降至67%及14%。各剂量组人工感染后均导致小鼠大脑及脑干出现病变,其病变程度与人工感染剂量呈正相关。1×105 TCID50剂量实验组既能使小鼠保持较高的存活率,又可导致明显的脑组织病变,所以采用其为后续实验人工感染剂量。通过免疫组织化学染色,激光共聚焦及流式细胞术分析PRV感染诱导的microglia动态变化。用增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)抗体和microglia及巨噬细胞特异性蛋白抗体Iba1(ionized calcium binding adapter molecule 1)标记增殖的microglia,用Iba1免疫组化染色方法观察microglia细胞形态。结果显示,PRV感染后大脑组织中出现增殖的microglia,其形态由静息状态(Resting)转化为活化状态(Activated)。应用5-溴脱氧尿嘧啶核苷(5-bromo-deoxy-uridine,Brd U)示踪技术,证明microglia非原位增殖。通过用G蛋白偶合受体激酶1(Gr1)抗体,抗中性粒细胞表面抗原Ly6G抗体及抗单核细胞表面抗原Ly6C抗体,经流式细胞术分析增殖microglia的表型特征,结果显示增殖microglia为CD11b+CD45hi Gr1+Ly6G-Ly6Chi,证明其来源于外周血炎症性单核细胞。通过Semi-quantitative PCR方法对PRV感染后microglia极化状态分析的结果表明,M1型标志性细胞因子诱导型一氧化氮合酶(i NOS)、肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和IL-12的表达量随着感染经过时间递增。M2型细胞因子IL-10和YM1在PRV人工感染后6-7 d表达量上升,说明microglia在PRV感染后向M1及M2方向均有极化,microglia的极化状态与PRV致病性的关系有待进一步研究。
[Abstract]:Aujeszkys disease was first found in the United States and spread worldwide between 1920 and 1940, and the incidence increased significantly in the following 30 years. Pseudorabies virus (Pseudorabies virus) belongs to the herpesviridae virus, which is a double-stranded DNA virus with encapsulated membrane. PRV can replicate and migrate along the peripheral nerve near the infected site. It is important to study the dynamic changes of microglia induced by pseudorabies virus and the effect of dynamic changes of microglia on the development of the disease. The mouse model of PRV infection was established. To study the dynamic changes of microglia induced by pseudorabies virus infection, PRV-Bartha K61 vaccine strain was used to infect mice subcutaneously. The doses of 6 脳 10 ~ 4 TCID 50 脳 10 ~ 5 TCID50 and 2.7 脳 10 ~ 5 TCID50 were used as the artificial infection doses. All the three groups resulted in the death of mice 6 days after artificial infection. The survival rate of mice was 93% and 85%, respectively. However, there was no significant difference in the survival rate of the experimental group at 6 脳 10 ~ 4 TCID50 on the 7th day after artificial infection. However, the other two groups were reduced to 67% and 14.The pathological changes in the brain and brain stem of mice were caused by artificial infection in each dose group. The degree of lesion was positively correlated with the dose of artificial infection. The experimental group could keep the survival rate of mice higher than that of the experimental group at the dose of 1 脳 10 5 TCID50. And it can cause obvious brain lesions, so we use it as a follow-up dose of artificial infection, and we use it by immunohistochemical staining. The dynamic changes of microglia induced by PRV infection were analyzed by confocal laser and flow cytometry. Proliferating cell nuclear antigen-PCNA antibody and microglia and macrophage specific protein antibody Iba1(ionized calcium binding adapter molecule 1 were labeled with proliferating cell nuclear antigen-PCNA. The proliferating microglia was immunized with Iba1. The morphologies of microglia cells were observed by chemical staining. The results showed that the proliferative microglia appeared in the brain tissue after microglia infection, and its morphology was transformed from resting to activated. The 5-bromo-deoxy-uridineuridine (5-bromo-deoxy-uridine) tracing technique was used. It was proved that microglia was not proliferated in situ. The phenotypic characteristics of proliferative microglia were analyzed by flow cytometry by using G protein coupled receptor kinase 1 Gr1 antibody, anti neutrophil surface antigen Ly6G antibody and anti monocyte surface antigen Ly6C antibody. The results showed that the proliferative microglia was CD11b CD45hi Gr1 Ly6G-Ly6ChiChi. it was proved that it originated from peripheral blood inflammatory monocytes. The results of microglia polarization analysis after PRV infection by Semi-quantitative PCR showed that M1-type iconic cytokine inducible nitric oxide synthase (no synthase). The expression of TNF- 伪, IL-6) and IL-12 increased with the time of infection. The expression of M2 cytokines IL-10 and YM1 increased 6-7 days after PRV infection. It is suggested that the relationship between the polarization state of microglia and the pathogenicity of PRV in M1 and M2 directions after PRV infection needs to be further studied.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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