本文選題：丙型肝炎病毒　切入點(diǎn)：toll樣受體2　出處：《寧夏醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的觀察HCV陽(yáng)性血清處理后BV-2細(xì)胞TLR2 mRNA及蛋白的表達(dá),以及TLR2介導(dǎo)的細(xì)胞因子TNF-α、IFN-α、MCP-1、IL-6、IL-12的表達(dá),初步探討中樞神經(jīng)系統(tǒng)小鼠小膠質(zhì)細(xì)胞BV-2對(duì)HCV陽(yáng)性血清刺激的免疫反應(yīng),為進(jìn)一步探討TLR2在中樞神經(jīng)系統(tǒng)HCV感染發(fā)病機(jī)制及組織損傷中的作用奠定基礎(chǔ)。 方法細(xì)胞分為空白對(duì)照組、正常對(duì)照組及實(shí)驗(yàn)組,分別給予常規(guī)培養(yǎng)用DMEM高糖培養(yǎng)液、含有200mL/L正常血清的DMEM高糖培養(yǎng)液及含有200mL/L HCV陽(yáng)性血清的DMEM高糖培養(yǎng)液,采用RT-qPCR方法和FCM方法分別檢測(cè)各組處理后4 h、8 h、16 h及24 h BV-2細(xì)胞TLR2 mRNA及蛋白的表達(dá)變化,并應(yīng)用ELISA方法檢測(cè)各組細(xì)胞培養(yǎng)上清液中TNF-α、IFN-α、MCP-1、IL-6、IL-12的表達(dá);同時(shí)設(shè)TLR2阻斷組及其對(duì)照組,首先均給予BV-2細(xì)胞TLR2單克隆阻斷抗體孵育30 min后分別給予含有200mL/L HCV陽(yáng)性血清的DMEM高糖培養(yǎng)液及200mL/L正常血清的DMEM高糖培養(yǎng)液培養(yǎng),24 h后收集上清液應(yīng)用ELISA方法檢測(cè)TNF-α、IFN-α、MCP-1、IL-6、IL-12的表達(dá)。所有數(shù)據(jù)均采用SPSS 13.0統(tǒng)計(jì)軟件進(jìn)行分析,組間和組內(nèi)比較采用單因素方差分析,組間兩兩比較采用LSD分析,以P0.05為差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果HCV陽(yáng)性血清處理后BV-2細(xì)胞TLR2 mRNA的表達(dá)顯著升高,其4 h、8 h、16 h及24 h與空白及正常血清對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),空白對(duì)照組與正常血清對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);正常BV-2細(xì)胞使用PE標(biāo)記的TLR2單克隆抗體同型對(duì)照抗體標(biāo)記后形成熒光峰值,實(shí)驗(yàn)組各時(shí)間點(diǎn)BV-2細(xì)胞使用PE標(biāo)記的TLR2單克隆抗體標(biāo)記未形成熒光峰值;HCV陽(yáng)性血清處理后BV-2細(xì)胞培養(yǎng)上清液中TNF-α、IFN-α、MCP-1、IL-6、IL-12的表達(dá)水平不同,均有不同程度升高,分別與相應(yīng)的對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05),TLR2蛋白阻斷后細(xì)胞因子TNF-α、IFN-α、MCP-1、IL-6、IL-12的表達(dá)均有不同程度的下降,分別與其對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),TLR2阻斷對(duì)照組與24 h(-)比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論BV-2細(xì)胞存在TLR2表達(dá),HCV陽(yáng)性血清處理可引起B(yǎng)V-2細(xì)胞形態(tài)發(fā)生改變,且TLR2 mRNA表達(dá)及細(xì)胞因子表達(dá)發(fā)生變化,提示HCV陽(yáng)性血清處理可能激活小膠質(zhì)細(xì)胞TLR2,并介導(dǎo)大量細(xì)胞因子的釋放,TLR2可能通過(guò)下游大量炎性細(xì)胞因子如TNF-α、IFN-α、MCP-1、IL-6、IL-12等機(jī)制介導(dǎo)BV-2抗HCV免疫及中樞神經(jīng)系統(tǒng)HCV可能的發(fā)病及損傷機(jī)制。
[Abstract]:Objective to investigate the expression of TLR2 mRNA and protein in BV-2 cells treated with HCV positive serum, and the expression of TLR2 mediated cytokine TNF- 偽 -IFN- 偽 MCP-1 / IL-6IL-12 in BV-2 cells, and to explore the immunoreaction of BV-2 in mouse microglia to HCV positive serum stimulation. To further explore the role of TLR2 in the pathogenesis of central nervous system HCV infection and the role of tissue injury. Methods cells were divided into blank control group, normal control group and experimental group. The cells were treated with DMEM high glucose culture medium, DMEM high glucose culture medium containing 200 mL / L normal serum and DMEM high glucose culture medium containing 200 mL / L HCV positive serum, respectively. The expression of TLR2 mRNA and protein in BV-2 cells were detected by RT-qPCR and FCM respectively at 4 h, 8 h, 16 h and 24 h after treatment, and the expression of TNF- 偽, IFN- 偽, MCP-1 and IL-6 IL-12 in the supernatant of each group were detected by ELISA method, and the TLR2 blocking group and its control group were set up at the same time. First, BV-2 cells were incubated with monoclonal antibody against TLR2 for 30 min. Then they were incubated with DMEM high glucose medium containing 200 mL / L HCV positive serum and DMEM high glucose culture medium containing 200 mL / L normal serum for 24 h. Then the supernatant was collected and detected by ELISA method. The expression of IL-12 in MCP-1 of TNF- 偽 and IFN- 偽 was analyzed by SPSS 13.0 software. Single factor ANOVA was used for inter-group and intra-group comparison, and LSD analysis was used for inter-group comparison. The difference was statistically significant with P0.05. Results the expression of TLR2 mRNA in BV-2 cells was significantly increased after treatment with HCV positive serum. There were significant differences between 4 h and 8 h and 24 h compared with the control group and the control group (P 0.05). There was no significant difference between the blank control group and the normal serum control group (P 0.05); the TLR2 monoclonal labeled with PE was used in the normal BV-2 cells. The fluorescence peak value was formed after the antibody was labeled with the same type of control antibody. The expression of IL-12 in the supernatant of BV-2 cells treated with PE labeled TLR2 monoclonal antibody was different and increased in different degree after BV-2 cells were treated with PE-labeled TLR2 monoclonal antibody and the positive serum of BV-2 cells was not formed. The expression level of TNF- 偽, IFN- 偽, MCP-1, IL-6 and IL-12 in the culture supernatant of BV-2 cells were increased in different degrees. Compared with the corresponding control group, the expression of cytokine TNF- 偽, IFN- 偽, MCP-1, IL-6 and IL-12 decreased in different degrees after the blocking of TLR2 protein. There was no significant difference between the control group and the control group (P 0.05). There was no significant difference between the control group and the control group (P 0.05). Conclusion the expression of TLR2 mRNA and cytokines in BV-2 cells can be changed by TLR2 positive serum treatment. These results suggest that HCV positive serum treatment may activate microglia TLR2 and mediate the release of a large number of cytokines. TLR2 may mediate the pathogenesis and damage of BV-2 against HCV immunity and HCV in central nervous system through a number of downstream inflammatory cytokines, such as TNF- 偽, IFN- 偽, MCP-1, IL-6, IL-12, and so on.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 呼建民;腦脊液細(xì)胞學(xué)電鏡研究方法的改進(jìn)[J];白求恩軍醫(yī)學(xué)院學(xué)報(bào);2004年02期

2 王振海;謝鵬;孔繁元;;丙型肝炎病毒對(duì)中樞神經(jīng)系統(tǒng)損傷的研究進(jìn)展[J];國(guó)際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2006年01期

3 趙承軍;張東梅;鄧其躍;陳鵬慧;蔡文琴;陶忠芬;張吉強(qiáng);;免疫電鏡技術(shù)運(yùn)用過(guò)程中電子染色方法的選用[J];局解手術(shù)學(xué)雜志;2008年02期

4 張亞飛,聶青和,羅新棟,黃曉峰,邵彬,茍艷子;人滋養(yǎng)層細(xì)胞內(nèi)丙型肝炎病毒樣顆粒的免疫電鏡形態(tài)學(xué)研究[J];醫(yī)學(xué)研究生學(xué)報(bào);2005年08期

5 朱長(zhǎng)庚;免疫細(xì)胞化學(xué)在神經(jīng)科學(xué)研究中的應(yīng)用[J];解剖學(xué)雜志;1986年01期

6 王英;顏永碧;湯瑩;;包埋前免疫金電鏡標(biāo)記的使用難點(diǎn)及解決方法[J];解剖學(xué)雜志;2006年04期

7 王得新;新發(fā)神經(jīng)系統(tǒng)病毒感染性疾病[J];臨床薈萃;2004年21期

8 楊蘭;卜志平;劉國(guó)敏;王曉鵬;;病毒的檢測(cè)方法[J];腦與神經(jīng)疾病雜志;2008年04期

9 吳洪娟,王尊哲,周力;免疫電鏡膠體金標(biāo)記技術(shù)應(yīng)用中關(guān)鍵環(huán)節(jié)的處理[J];濰坊醫(yī)學(xué)院學(xué)報(bào);2003年04期

10 陳健;李燕婷;王曄;高曄;;膠體金法與病毒分離鑒定在流感診斷中的比較[J];上海預(yù)防醫(yī)學(xué)雜志;2008年12期

，

本文編號(hào)：1597305