【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,嚴重的心肌炎、心包炎及神經(jīng)系統(tǒng)疾病。近年來在我國出現(xiàn)了幾起暴發(fā)流行,以無菌性腦膜腦炎為主要特點。病人的腦脊液中促炎癥細胞因子濃度出現(xiàn)異常升高。神經(jīng)膠質(zhì)細胞,尤其是小膠質(zhì)細胞和星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)(CNS)細胞因子產(chǎn)生的主要來源,但其在Coxsackievirus感染CNS時的功能仍不清楚。因此,本實驗研究的主要目的在于探索星形膠質(zhì)細胞在Coxsackievirus感染后所發(fā)生的病理變化及相關(guān)免疫反應(yīng)如細胞因子變化,為明確膠質(zhì)細胞在感染中的作用、理解CNS感染Coxsackievirus后的發(fā)病機制提供必要的實驗依據(jù)。 運用基因克隆的方法構(gòu)建趨化因子IP-10(CXCL10)真核表達載體,為進一步研究趨化因子IP-10的生物學功能提供幫助。 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)細胞。用Coxsackievirus中具有代表性的病毒Coxsackievirus A16和B3分別感染星形細胞,檢測病毒復(fù)制、病毒生長曲線、細胞病變、細胞存活率、細胞因子基因轉(zhuǎn)錄水平及蛋白分泌變化。運用基因克隆的方法構(gòu)建IP-10(CXCL10)真核表達載體,轉(zhuǎn)染細胞使之過表達IP-10研究其對細胞生存狀態(tài)的影響。 結(jié)果及結(jié)論 1.成功分離并純化星形膠質(zhì)細胞和小膠質(zhì)細胞。采用免疫熒光和流式細胞技術(shù)檢測純度在90%以上,方法成熟可重復(fù)性高,滿足實驗要求。 2. Coxsackievirus B3可以感染星形膠質(zhì)細胞并在其中增殖產(chǎn)生子代病毒。星形膠質(zhì)細胞對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)細胞可引起明顯的細胞病變效應(yīng)(CPE),培養(yǎng)上清中的病毒滴度在24h內(nèi)達到高峰。Coxsackievirus A16不引起星形細胞CPE出現(xiàn)。 4. Coxsackievirus B3感染可誘導(dǎo)星形膠質(zhì)細胞的促炎癥細胞因子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)細胞的促炎癥細胞因子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)細胞產(chǎn)生少量IP-10。 6.成功構(gòu)建IP-10(CXCL10)真核表達載體,轉(zhuǎn)染原代星形膠質(zhì)細胞,使之表達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..
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前9條

1 鄭煥英;劉冷;郭雪;柯昌文;肖紅;李柏生;王雅靜;柯碧霞;王冰姝;張欣;;2008年廣東省手足口病實驗室檢測結(jié)果分析[J];華南預(yù)防醫(yī)學;2009年06期

2 苗麗,楊冀洲,王麗;酶標儀自校驗方法探討[J];中國國境衛(wèi)生檢疫雜志;2003年04期

3 羅雙龍;酶標儀雙波長與單波長比色測定抗HCV的比較[J];臨床檢驗雜志;1998年06期

4 石小華;胡靜;喻文亮;于毅;;648例手足口病回顧性分析[J];南京醫(yī)科大學學報(自然科學版);2010年01期

5 劉順愛;徐道振;張劍平;黃克林;姚均;許麗鋒;袁正宏;聞玉梅;;治療性乙型肝炎疫苗的免疫效應(yīng)[J];中華肝臟病雜志;2006年02期

6 成軍,孫關(guān)忠,鄭懷競,李早榮,倪方榮;酶標儀性能評價與鑒定的基本方法及其初步應(yīng)用[J];中華醫(yī)學檢驗雜志;1998年01期

7 王巍;辛嘵燕;劉玉;李璞媛;丁燁;;Herceptin與卡鉑聯(lián)合應(yīng)用對宮頸癌細胞的抑制及其機制[J];中國腫瘤生物治療雜志;2007年01期

8 Sk Md Fazle Akbar;Norio Horiike;Morikazu Onji;;Immune therapy including dendritic cell based therapy in chronic hepatitis B virus infection[J];World Journal of Gastroenterology;2006年18期

9 郭迎春;;手足口病并發(fā)病毒性腦炎107例臨床分析[J];中國中西醫(yī)結(jié)合兒科學;2009年06期

，

本文編號：2486106