本文關(guān)鍵詞： 偽狂犬病病毒 小膠質(zhì)細(xì)胞 動(dòng)態(tài)變化 細(xì)胞增殖 極化　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬偽狂犬病(Aujeszky's disease)最早發(fā)現(xiàn)于美國(guó),在1920到1940年間呈世界性散在發(fā)生,并且在之后30年間呈爆發(fā)性,其發(fā)病率明顯增加。目前,豬偽狂犬病在我國(guó)仍然是威脅養(yǎng)豬業(yè)的重要疾病之一。偽狂犬病病毒(Pseudorabies Virus,PRV)屬于皰疹病毒科病毒,為有囊膜的雙鏈DNA病毒。PRV感染動(dòng)物后可以沿著感染部位附近外周神經(jīng)復(fù)制移行,逐漸進(jìn)入中樞神經(jīng)系統(tǒng)(CNS),導(dǎo)致腦組織發(fā)生病變。研究偽狂犬病病毒誘導(dǎo)的小膠質(zhì)細(xì)胞(microglia)動(dòng)態(tài)變化及microglia動(dòng)態(tài)變化對(duì)疾病發(fā)展進(jìn)程的影響具有重要意義。本實(shí)驗(yàn)主要通過建立PRV人工感染小鼠模型,研究偽狂犬病病毒感染誘導(dǎo)的小膠質(zhì)細(xì)胞動(dòng)態(tài)學(xué)變化。實(shí)驗(yàn)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 TCID50三組劑量作為人工感染劑量。三組劑量均在人工感染后6 d導(dǎo)致小鼠出現(xiàn)死亡,小鼠存活率分別為93%、93%和85%。然而在人工感染后第7 d,小鼠存活率差異顯著,6×104 TCID50劑量實(shí)驗(yàn)組存活率無變化,而另外兩組分別降至67%及14%。各劑量組人工感染后均導(dǎo)致小鼠大腦及腦干出現(xiàn)病變,其病變程度與人工感染劑量呈正相關(guān)。1×105 TCID50劑量實(shí)驗(yàn)組既能使小鼠保持較高的存活率,又可導(dǎo)致明顯的腦組織病變,所以采用其為后續(xù)實(shí)驗(yàn)人工感染劑量。通過免疫組織化學(xué)染色,激光共聚焦及流式細(xì)胞術(shù)分析PRV感染誘導(dǎo)的microglia動(dòng)態(tài)變化。用增殖細(xì)胞核抗原(proliferating cell nuclear antigen,PCNA)抗體和microglia及巨噬細(xì)胞特異性蛋白抗體Iba1(ionized calcium binding adapter molecule 1)標(biāo)記增殖的microglia,用Iba1免疫組化染色方法觀察microglia細(xì)胞形態(tài)。結(jié)果顯示,PRV感染后大腦組織中出現(xiàn)增殖的microglia,其形態(tài)由靜息狀態(tài)(Resting)轉(zhuǎn)化為活化狀態(tài)(Activated)。應(yīng)用5-溴脫氧尿嘧啶核苷(5-bromo-deoxy-uridine,Brd U)示蹤技術(shù),證明microglia非原位增殖。通過用G蛋白偶合受體激酶1(Gr1)抗體,抗中性粒細(xì)胞表面抗原Ly6G抗體及抗單核細(xì)胞表面抗原Ly6C抗體,經(jīng)流式細(xì)胞術(shù)分析增殖microglia的表型特征,結(jié)果顯示增殖microglia為CD11b+CD45hi Gr1+Ly6G-Ly6Chi,證明其來源于外周血炎癥性單核細(xì)胞。通過Semi-quantitative PCR方法對(duì)PRV感染后microglia極化狀態(tài)分析的結(jié)果表明,M1型標(biāo)志性細(xì)胞因子誘導(dǎo)型一氧化氮合酶(i NOS)、腫瘤壞死因子α(TNF-α)、白介素6(IL-6)和IL-12的表達(dá)量隨著感染經(jīng)過時(shí)間遞增。M2型細(xì)胞因子IL-10和YM1在PRV人工感染后6-7 d表達(dá)量上升,說明microglia在PRV感染后向M1及M2方向均有極化,microglia的極化狀態(tài)與PRV致病性的關(guān)系有待進(jìn)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- 偽, 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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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