柯薩奇病毒感染星形膠質(zhì)細(xì)胞及細(xì)胞因子變化研究
發(fā)布時(shí)間：2019-05-27 11:26
【摘要】： 研究目的和意義 柯薩奇病毒(Coxsackievirus)是引起人類感染的最常見的致病原之一,其感染可以導(dǎo)致許多疾病和癥狀,如呼吸道感染疾病,典型癥狀為皰疹性咽峽炎,手足口病,嚴(yán)重的心肌炎、心包炎及神經(jīng)系統(tǒng)疾病。近年來在我國(guó)出現(xiàn)了幾起暴發(fā)流行,以無菌性腦膜腦炎為主要特點(diǎn)。病人的腦脊液中促炎癥細(xì)胞因子濃度出現(xiàn)異常升高。神經(jīng)膠質(zhì)細(xì)胞,尤其是小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)(CNS)細(xì)胞因子產(chǎn)生的主要來源,但其在Coxsackievirus感染CNS時(shí)的功能仍不清楚。因此,本實(shí)驗(yàn)研究的主要目的在于探索星形膠質(zhì)細(xì)胞在Coxsackievirus感染后所發(fā)生的病理變化及相關(guān)免疫反應(yīng)如細(xì)胞因子變化,為明確膠質(zhì)細(xì)胞在感染中的作用、理解CNS感染Coxsackievirus后的發(fā)病機(jī)制提供必要的實(shí)驗(yàn)依據(jù)。 運(yùn)用基因克隆的方法構(gòu)建趨化因子IP-10(CXCL10)真核表達(dá)載體,為進(jìn)一步研究趨化因子IP-10的生物學(xué)功能提供幫助。 材料和方法 新生(1d)的BALB/C小鼠,常規(guī)皮膚消毒,斷頭殺鼠,在無菌條件下迅速剝除硬腦膜,鈍性分離腦組織制成混合細(xì)胞懸液。培養(yǎng)20-25天,用溫和胰酶消化結(jié)合定軌搖床振蕩的方法分離純化星形膠質(zhì)細(xì)胞。用Coxsackievirus中具有代表性的病毒Coxsackievirus A16和B3分別感染星形細(xì)胞,檢測(cè)病毒復(fù)制、病毒生長(zhǎng)曲線、細(xì)胞病變、細(xì)胞存活率、細(xì)胞因子基因轉(zhuǎn)錄水平及蛋白分泌變化。運(yùn)用基因克隆的方法構(gòu)建IP-10(CXCL10)真核表達(dá)載體,轉(zhuǎn)染細(xì)胞使之過表達(dá)IP-10研究其對(duì)細(xì)胞生存狀態(tài)的影響。 結(jié)果及結(jié)論 1.成功分離并純化星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞。采用免疫熒光和流式細(xì)胞技術(shù)檢測(cè)純度在90%以上,方法成熟可重復(fù)性高,滿足實(shí)驗(yàn)要求。 2. Coxsackievirus B3可以感染星形膠質(zhì)細(xì)胞并在其中增殖產(chǎn)生子代病毒。星形膠質(zhì)細(xì)胞對(duì)Coxsackievirus A16不敏感,不能在其中有效復(fù)制產(chǎn)生子代病毒。 3. Coxsackievirus B3感染星形膠質(zhì)細(xì)胞可引起明顯的細(xì)胞病變效應(yīng)(CPE),培養(yǎng)上清中的病毒滴度在24h內(nèi)達(dá)到高峰。Coxsackievirus A16不引起星形細(xì)胞CPE出現(xiàn)。 4. Coxsackievirus B3感染可誘導(dǎo)星形膠質(zhì)細(xì)胞的促炎癥細(xì)胞因子IL-1、IL-6及TNF-α及趨化因子CCL5、IP-10的mRNA水平上調(diào);Coxsackievirus A16同樣可以引起星形膠質(zhì)細(xì)胞的促炎癥細(xì)胞因子IL-1、IL-6及TNF-α及趨化因子CCL5、IP-10的mRNA水平上調(diào),但沒有Coxsackievirus B3明顯。 5. ELISA定量檢測(cè)培養(yǎng)上清中的IP-10含量結(jié)果表明,與Coxsackievirus A16相比,Coxsackievirus B3感染星形膠質(zhì)細(xì)胞能夠誘導(dǎo)產(chǎn)生更多的IP-10,滅活的Coxsackievirus B3也可以誘導(dǎo)星形膠質(zhì)細(xì)胞產(chǎn)生少量IP-10。 6.成功構(gòu)建IP-10(CXCL10)真核表達(dá)載體,轉(zhuǎn)染原代星形膠質(zhì)細(xì)胞,使之表達(dá)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- 偽 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- 偽 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..
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373
本文編號(hào)：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- 偽 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- 偽 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..
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R373
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