柯萨奇病毒感染星形胶质细胞及细胞因子变化研究
发布时间：2019-05-27 11:26
【摘要】： 研究目的和意义 柯萨奇病毒(Coxsackievirus)是引起人类感染的最常见的致病原之一,其感染可以导致许多疾病和症状,如呼吸道感染疾病,典型症状为疱疹性咽峡炎,手足口病,严重的心肌炎、心包炎及神经系统疾病。近年来在我国出现了几起暴发流行,以无菌性脑膜脑炎为主要特点。病人的脑脊液中促炎症细胞因子浓度出现异常升高。神经胶质细胞,尤其是小胶质细胞和星形胶质细胞是中枢神经系统(CNS)细胞因子产生的主要来源,但其在Coxsackievirus感染CNS时的功能仍不清楚。因此,本实验研究的主要目的在于探索星形胶质细胞在Coxsackievirus感染后所发生的病理变化及相关免疫反应如细胞因子变化,为明确胶质细胞在感染中的作用、理解CNS感染Coxsackievirus后的发病机制提供必要的实验依据。 运用基因克隆的方法构建趋化因子IP-10(CXCL10)真核表达载体,为进一步研究趋化因子IP-10的生物学功能提供帮助。 材料和方法 新生(1d)的BALB/C小鼠,常规皮肤消毒,断头杀鼠,在无菌条件下迅速剥除硬脑膜,钝性分离脑组织制成混合细胞悬液。培养20-25天,用温和胰酶消化结合定轨摇床振荡的方法分离纯化星形胶质细胞。用Coxsackievirus中具有代表性的病毒Coxsackievirus A16和B3分别感染星形细胞,检测病毒复制、病毒生长曲线、细胞病变、细胞存活率、细胞因子基因转录水平及蛋白分泌变化。运用基因克隆的方法构建IP-10(CXCL10)真核表达载体,转染细胞使之过表达IP-10研究其对细胞生存状态的影响。 结果及结论 1.成功分离并纯化星形胶质细胞和小胶质细胞。采用免疫荧光和流式细胞技术检测纯度在90%以上,方法成熟可重复性高,满足实验要求。 2. Coxsackievirus B3可以感染星形胶质细胞并在其中增殖产生子代病毒。星形胶质细胞对Coxsackievirus A16不敏感,不能在其中有效复制产生子代病毒。 3. Coxsackievirus B3感染星形胶质细胞可引起明显的细胞病变效应(CPE),培养上清中的病毒滴度在24h内达到高峰。Coxsackievirus A16不引起星形细胞CPE出现。 4. Coxsackievirus B3感染可诱导星形胶质细胞的促炎症细胞因子IL-1、IL-6及TNF-α及趋化因子CCL5、IP-10的mRNA水平上调;Coxsackievirus A16同样可以引起星形胶质细胞的促炎症细胞因子IL-1、IL-6及TNF-α及趋化因子CCL5、IP-10的mRNA水平上调,但没有Coxsackievirus B3明显。 5. ELISA定量检测培养上清中的IP-10含量结果表明,与Coxsackievirus A16相比,Coxsackievirus B3感染星形胶质细胞能够诱导产生更多的IP-10,灭活的Coxsackievirus B3也可以诱导星形胶质细胞产生少量IP-10。 6.成功构建IP-10(CXCL10)真核表达载体,转染原代星形胶质细胞,使之表达IP-10。
[Abstract]:Objective and significance of the study (Coxsackievirus) is one of the most common pathogens causing human infection. Its infection can lead to many diseases and symptoms, such as respiratory tract infection, the typical symptom is herpetic pharyngitis. Hand, foot and mouth disease, severe myocarditis, pericarditis and nervous system diseases. In recent years, there have been several outbreaks in China, characterized by aseptic meningeal meningitis. The concentration of pro-inflammatory cytokines in cerebrospinal fluid of the patients increased abnormally. Glial cells, especially microglia and astrocytes, are the main sources of (CNS) cytokines in the central nervous system, but their function in CNS infection with Coxsackievirus is still unclear. Therefore, the main purpose of this study is to explore the pathological changes of astrocytes and related immune responses such as cytokines after Coxsackievirus infection, in order to clarify the role of glial cells in infection. To understand the pathogenesis of CNS infection with Coxsackievirus provides the necessary experimental basis. The eukaryotic expression vector of chemokine IP-10 (CXCL10) was constructed by gene cloning, which provided help for further study of the biological function of chemokine IP-10. Materials and methods BALB/C mice were sterilized by routine skin, killed by decapitation, quickly stripped of dura mater under aseptic condition, and the mixed cell suspension was made by obtuse isolation of brain tissue. The astrocytes were isolated and purified by mild trypsin digestion combined with orbit shaking bed oscillations for 20 to 25 days. Star cells were infected with representative viruses Coxsackievirus A16 and B3 in Coxsackievirus. Virus replication, virus growth curve, cytopathic effect, cell survival rate, cytokine gene transcription level and protein secretion were detected. The eukaryotic expression vector IP-10 (CXCL10) was constructed by gene cloning and overexpressed in the cells to study the effect of IP-10 on the survival status of the cells. Results and conclusion 1. Astrocytes and microglia were successfully isolated and purified. Immunofluorescence and flow cytometry were used to detect the purity of more than 90%. The method was mature and repeatable and met the requirements of the experiment. 2. Coxsackievirus B3 can infect astrocytes and proliferate in them to produce offspring virus. Astrocytes are not sensitive to Coxsackievirus A 16 and can not effectively replicate and produce progeny viruses. 3. Coxsackievirus B3 infection with astrocytes could induce obvious cytopathic effect. The virus titer in the culture medium of (CPE), reached the peak within 24 hours. Coxsackievirus A16 did not cause the appearance of CPE in astrocytes. 4. Coxsackievirus B3 infection could increase the mRNA level of pro-inflammatory cytokines IL-1,IL-6 and TNF- 伪 and chemokine CCL5,IP-10 in astrocytes. Coxsackievirus A16 could also up-regulate the mRNA levels of pro-inflammatory cytokines IL-1,IL-6 and TNF- 伪 and chemokine CCL5,IP-10 in astrocytes, but not as much as Coxsackievirus B3. 5. ELISA quantitative detection of IP-10 content in culture medium showed that Coxsackievirus B3 could induce more IP-10, inactivated Coxsackievirus B3 and produce a small amount of IP-10. compared with Coxsackievirus A16. 6. The eukaryotic expression vector IP-10 (CXCL10) was successfully constructed and transformed into primary astrocytes to express IP-10..
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
本文编号：2486106
[Abstract]:Objective and significance of the study (Coxsackievirus) is one of the most common pathogens causing human infection. Its infection can lead to many diseases and symptoms, such as respiratory tract infection, the typical symptom is herpetic pharyngitis. Hand, foot and mouth disease, severe myocarditis, pericarditis and nervous system diseases. In recent years, there have been several outbreaks in China, characterized by aseptic meningeal meningitis. The concentration of pro-inflammatory cytokines in cerebrospinal fluid of the patients increased abnormally. Glial cells, especially microglia and astrocytes, are the main sources of (CNS) cytokines in the central nervous system, but their function in CNS infection with Coxsackievirus is still unclear. Therefore, the main purpose of this study is to explore the pathological changes of astrocytes and related immune responses such as cytokines after Coxsackievirus infection, in order to clarify the role of glial cells in infection. To understand the pathogenesis of CNS infection with Coxsackievirus provides the necessary experimental basis. The eukaryotic expression vector of chemokine IP-10 (CXCL10) was constructed by gene cloning, which provided help for further study of the biological function of chemokine IP-10. Materials and methods BALB/C mice were sterilized by routine skin, killed by decapitation, quickly stripped of dura mater under aseptic condition, and the mixed cell suspension was made by obtuse isolation of brain tissue. The astrocytes were isolated and purified by mild trypsin digestion combined with orbit shaking bed oscillations for 20 to 25 days. Star cells were infected with representative viruses Coxsackievirus A16 and B3 in Coxsackievirus. Virus replication, virus growth curve, cytopathic effect, cell survival rate, cytokine gene transcription level and protein secretion were detected. The eukaryotic expression vector IP-10 (CXCL10) was constructed by gene cloning and overexpressed in the cells to study the effect of IP-10 on the survival status of the cells. Results and conclusion 1. Astrocytes and microglia were successfully isolated and purified. Immunofluorescence and flow cytometry were used to detect the purity of more than 90%. The method was mature and repeatable and met the requirements of the experiment. 2. Coxsackievirus B3 can infect astrocytes and proliferate in them to produce offspring virus. Astrocytes are not sensitive to Coxsackievirus A 16 and can not effectively replicate and produce progeny viruses. 3. Coxsackievirus B3 infection with astrocytes could induce obvious cytopathic effect. The virus titer in the culture medium of (CPE), reached the peak within 24 hours. Coxsackievirus A16 did not cause the appearance of CPE in astrocytes. 4. Coxsackievirus B3 infection could increase the mRNA level of pro-inflammatory cytokines IL-1,IL-6 and TNF- 伪 and chemokine CCL5,IP-10 in astrocytes. Coxsackievirus A16 could also up-regulate the mRNA levels of pro-inflammatory cytokines IL-1,IL-6 and TNF- 伪 and chemokine CCL5,IP-10 in astrocytes, but not as much as Coxsackievirus B3. 5. ELISA quantitative detection of IP-10 content in culture medium showed that Coxsackievirus B3 could induce more IP-10, inactivated Coxsackievirus B3 and produce a small amount of IP-10. compared with Coxsackievirus A16. 6. The eukaryotic expression vector IP-10 (CXCL10) was successfully constructed and transformed into primary astrocytes to express IP-10..
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373
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