TLR2活化对HBV持续复制的影响及其机制研究

发布时间：2018-05-30 07:03

本文选题：乙型肝炎病毒 + Toll样受体2　；参考：《华中科技大学》2012年硕士论文

【摘要】：目的： 1.在HBV慢性复制小鼠模型中，探索TLR2活化能否抑制体内HBV复制。 2.在HBV慢性复制小鼠模型中，探讨TLR2活化抑制HBV复制的机制。方法： 1. TLR2配体Pam3CSK（P3C）小鼠尾根皮下注射。在不同时间点，定量ELISA检测小鼠血清中炎症因子：IL-6、TNF-α和IL-1β等的表达。 2. C57BL/6小鼠高压尾静脉注射pAAV/HBV1.2质粒建立慢性HBV复制小鼠模型。在pAAV/HBV1.2质粒注射后的早期（day0, day7和day14）或晚期（day14, day21和day28），连续3次尾根皮下注射P3C处理小鼠。 3.按既定实验设计对小鼠进行眼眶采血，并在固定时间点留取肝脾组织样本。通过定量ELISA检测小鼠血清炎症因子（IL-6、TNF-α和IL-1β等）和HBV血清标志物（如HBsAg、HBeAg）；用免疫组织化学方法检测肝组织中HBcAg的表达；用realtime PCR和real time RT-PCR分别检测血清中HBV DNA水平和肝组织相关分子mRNA的表达水平；用ELISPOT检测脾淋巴细胞中抗原特异性的分泌IFN-γ的细胞数；通过细胞内因子染色检测PBMCs和脾淋巴细胞中HBs/HBc peptide特异性的分泌IFN-γ的CD8+T细胞阳性百分率；用Dimer染色检测脾细胞中HBs/HBcpeptide特异性的CTLs情况。 4.使用SPSS18.0对数据进行统计分析，使用GraphPad Prism5作图。结果： 1. naive小鼠和HBV慢性复制小鼠经尾根皮下注射P3C后，血清中IL-6的产生在注射后3h达到最高，在24h内降至正常水平。并且，血清中IL-6的表达水平与P3C的处理剂量成正相关。 2. HBV慢性复制小鼠经早期应用P3C后，，血清中HBsAg、HBeAg及HBV DNA水平明显降低，小鼠肝内HBcAg的表达也显著减少。而在P3C晚期处理小鼠中，未发现明显的抗HBV效应。 3.在早期应用P3C组和未处理组的小鼠中，脾淋巴细胞和PBMCs均未能检测到HBV特异性的细胞免疫应答。仅在第10天，早期应用P3C组小鼠的脾淋巴细胞经rHBcAg刺激后分泌IFN-γ的T细胞数量少于对照组。 4.通过对小鼠肝组织相关分子mRNA水平进行real time RT-PCR检测，我们发现：早期应用P3C组和对照组小鼠相比，IFN-β和IFN-γ mRNA无明显差异；促炎因子IL-6和TNF-α mRNA在第4天明显高于对照组；抑炎因子IL-10mRNA在第4天和第10天明显高于对照组。结论： 1. naive小鼠和HBV慢性复制小鼠经TLR2配体P3C处理后，血清IL-6等炎症因子的产生是一个较快的过程，一般在几个小时内达到最高峰，且炎症因子的浓度与P3C的剂量成正相关。 2.在慢性HBV复制小鼠模型中，早期应用P3C能发挥明显的抗HBV效应，而晚期应用则无此明显效应。 3.在慢性HBV复制小鼠模型中，早期应用P3C产生明显的抗HBV效应的机制可能是通过TLR2介导的天然免疫应答对HBV的早期复制产生抑制，而TLR2活化诱导的特异性免疫应答在其中发挥的作用十分有限。
[Abstract]:Objective: 1. To explore whether the activation of TLR2 can inhibit the replication of HBV in vivo in mice model of chronic HBV replication. 2. To explore the mechanism of TLR2 activation inhibiting HBV replication in chronic HBV mouse model. Methods: 1. TLR2 ligand Pam3 CSKG P3 C) mice were subcutaneously injected into caudal root. At different time points, quantitative ELISA was used to detect the expression of TNF- 伪 and IL-1 尾 in serum of mice. 2. C57BL/6 mice model of chronic HBV was established by injecting pAAV/HBV1.2 plasmid into high pressure caudal vein. Mice were treated with P3C subcutaneously at the early stage of pAAV/HBV1.2 plasmids injection, day7 and day14) or at the late stages of P3C, day21 and day28, and the mice were subcutaneously injected into the caudal root for 3 times. 3. The blood was collected from the orbit of mice according to the established experimental design, and the liver and spleen tissue samples were taken at fixed time point. The levels of IL-6 TNF- 伪 and IL-1 尾 in serum of mice were detected by quantitative ELISA, and the expression of HBcAg in liver tissue was detected by immunohistochemical method. Realtime PCR and real time RT-PCR were used to detect the level of HBV DNA in serum and the expression level of mRNA in liver tissue, and ELISPOT was used to detect the number of antigen-specific IFN- 纬 secreting cells in spleen lymphocytes. The positive percentage of CD8 T cells secreting IFN- 纬 in PBMCs and spleen lymphocytes was detected by intracellular factor staining, and the HBs/HBcpeptide specific CTLs in splenocytes was detected by Dimer staining. 4. Use SPSS18.0 to carry on statistical analysis to the data, use GraphPad Prism5 to make the map. Results: 1. After subcutaneous injection of P3C into the tail root of naive mice and HBV mice, the production of IL-6 in serum reached the highest level 3 hours after injection, and decreased to normal level within 24 hours. Furthermore, the expression of IL-6 in serum was positively correlated with the dose of P3C. 2. The levels of HBeAg and HBV DNA in serum of chronic replicating mice with HBV were significantly decreased after early application of P3C, and the expression of HBcAg in liver of mice was also significantly decreased. However, no significant anti-HBV effect was found in late P3C treated mice. 3. The spleen lymphocytes and PBMCs could not detect the specific cellular immune response of HBV in the mice treated with P3C and untreated at early stage. Only on the 10th day, the number of T cells secreting IFN- 纬 in spleen lymphocytes of P3C group was less than that of control group after rHBcAg stimulation. 4. The levels of mRNA in liver tissue of mice were detected by real time RT-PCR. We found that there was no significant difference in the levels of IFN- 尾 and IFN- 纬 mRNA between the early P3C group and the control group, and the level of IL-6 and TNF- 伪 mRNA was significantly higher than that of the control group on the 4th day. The IL-10mRNA of anti-inflammatory factor on the 4th and 10th day was significantly higher than that of the control group. Conclusion: 1.After the treatment of TLR2 ligand P3C in naive mice and HBV chronic replicating mice, the production of serum IL-6 and other inflammatory factors was a rapid process, which generally reached the peak within a few hours, and the concentration of inflammatory factors was positively correlated with the dose of P3C. 2. In chronic HBV induced mouse model, early application of P3C could play a significant role in anti-HBV effect, but late application did not. 3. In chronic HBV induced mouse model, the mechanism of early application of P3C to produce obvious anti-HBV effect may be the inhibition of early replication of HBV by innate immune response mediated by TLR2. The specific immune response induced by TLR2 activation plays a very limited role.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373.21

【共引文献】