TLR2介导的小鼠小胶质细胞抗HCV免疫应答初探

发布时间：2018-03-11 08:08

本文选题：丙型肝炎病毒　切入点：toll样受体2　出处：《宁夏医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的观察HCV阳性血清处理后BV-2细胞TLR2 mRNA及蛋白的表达,以及TLR2介导的细胞因子TNF-α、IFN-α、MCP-1、IL-6、IL-12的表达,初步探讨中枢神经系统小鼠小胶质细胞BV-2对HCV阳性血清刺激的免疫反应,为进一步探讨TLR2在中枢神经系统HCV感染发病机制及组织损伤中的作用奠定基础。方法细胞分为空白对照组、正常对照组及实验组,分别给予常规培养用DMEM高糖培养液、含有200mL/L正常血清的DMEM高糖培养液及含有200mL/L HCV阳性血清的DMEM高糖培养液,采用RT-qPCR方法和FCM方法分别检测各组处理后4 h、8 h、16 h及24 h BV-2细胞TLR2 mRNA及蛋白的表达变化,并应用ELISA方法检测各组细胞培养上清液中TNF-α、IFN-α、MCP-1、IL-6、IL-12的表达;同时设TLR2阻断组及其对照组,首先均给予BV-2细胞TLR2单克隆阻断抗体孵育30 min后分别给予含有200mL/L HCV阳性血清的DMEM高糖培养液及200mL/L正常血清的DMEM高糖培养液培养,24 h后收集上清液应用ELISA方法检测TNF-α、IFN-α、MCP-1、IL-6、IL-12的表达。所有数据均采用SPSS 13.0统计软件进行分析,组间和组内比较采用单因素方差分析,组间两两比较采用LSD分析,以P0.05为差异具有统计学意义。结果HCV阳性血清处理后BV-2细胞TLR2 mRNA的表达显著升高,其4 h、8 h、16 h及24 h与空白及正常血清对照组比较差异均有统计学意义(P0.05),空白对照组与正常血清对照组比较差异无统计学意义(P0.05);正常BV-2细胞使用PE标记的TLR2单克隆抗体同型对照抗体标记后形成荧光峰值,实验组各时间点BV-2细胞使用PE标记的TLR2单克隆抗体标记未形成荧光峰值;HCV阳性血清处理后BV-2细胞培养上清液中TNF-α、IFN-α、MCP-1、IL-6、IL-12的表达水平不同,均有不同程度升高,分别与相应的对照组比较差异具有统计学意义(P0.05),TLR2蛋白阻断后细胞因子TNF-α、IFN-α、MCP-1、IL-6、IL-12的表达均有不同程度的下降,分别与其对照组比较差异均有统计学意义(P0.05),TLR2阻断对照组与24 h(-)比较差异无统计学意义(P0.05)。结论BV-2细胞存在TLR2表达,HCV阳性血清处理可引起BV-2细胞形态发生改变,且TLR2 mRNA表达及细胞因子表达发生变化,提示HCV阳性血清处理可能激活小胶质细胞TLR2,并介导大量细胞因子的释放,TLR2可能通过下游大量炎性细胞因子如TNF-α、IFN-α、MCP-1、IL-6、IL-12等机制介导BV-2抗HCV免疫及中枢神经系统HCV可能的发病及损伤机制。
[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- 伪 -IFN- 伪 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- 伪, IFN- 伪, 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- 伪 and IFN- 伪 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- 伪, IFN- 伪, 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- 伪, IFN- 伪, 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- 伪, IFN- 伪, MCP-1, IL-6, IL-12, and so on.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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