本文選題：乙型肝炎病毒 + Toll樣受體2��； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：目的： 1.在HBV慢性復(fù)制小鼠模型中，探索TLR2活化能否抑制體內(nèi)HBV復(fù)制。 2.在HBV慢性復(fù)制小鼠模型中，探討TLR2活化抑制HBV復(fù)制的機(jī)制。 方法： 1. TLR2配體Pam3CSK（P3C）小鼠尾根皮下注射。在不同時(shí)間點(diǎn)，定量ELISA檢測(cè)小鼠血清中炎癥因子：IL-6、TNF-α和IL-1β等的表達(dá)。 2. C57BL/6小鼠高壓尾靜脈注射pAAV/HBV1.2質(zhì)粒建立慢性HBV復(fù)制小鼠模型。在pAAV/HBV1.2質(zhì)粒注射后的早期（day0, day7和day14）或晚期（day14, day21和day28），連續(xù)3次尾根皮下注射P3C處理小鼠。 3.按既定實(shí)驗(yàn)設(shè)計(jì)對(duì)小鼠進(jìn)行眼眶采血，并在固定時(shí)間點(diǎn)留取肝脾組織樣本。通過(guò)定量ELISA檢測(cè)小鼠血清炎癥因子（IL-6、TNF-α和IL-1β等）和HBV血清標(biāo)志物（如HBsAg、HBeAg）；用免疫組織化學(xué)方法檢測(cè)肝組織中HBcAg的表達(dá)；用realtime PCR和real time RT-PCR分別檢測(cè)血清中HBV DNA水平和肝組織相關(guān)分子mRNA的表達(dá)水平；用ELISPOT檢測(cè)脾淋巴細(xì)胞中抗原特異性的分泌IFN-γ的細(xì)胞數(shù)；通過(guò)細(xì)胞內(nèi)因子染色檢測(cè)PBMCs和脾淋巴細(xì)胞中HBs/HBc peptide特異性的分泌IFN-γ的CD8+T細(xì)胞陽(yáng)性百分率；用Dimer染色檢測(cè)脾細(xì)胞中HBs/HBcpeptide特異性的CTLs情況。 4.使用SPSS18.0對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析，使用GraphPad Prism5作圖。 結(jié)果： 1. naive小鼠和HBV慢性復(fù)制小鼠經(jīng)尾根皮下注射P3C后，血清中IL-6的產(chǎn)生在注射后3h達(dá)到最高，在24h內(nèi)降至正常水平。并且，血清中IL-6的表達(dá)水平與P3C的處理劑量成正相關(guān)。 2. HBV慢性復(fù)制小鼠經(jīng)早期應(yīng)用P3C后，，血清中HBsAg、HBeAg及HBV DNA水平明顯降低，小鼠肝內(nèi)HBcAg的表達(dá)也顯著減少。而在P3C晚期處理小鼠中，未發(fā)現(xiàn)明顯的抗HBV效應(yīng)。 3.在早期應(yīng)用P3C組和未處理組的小鼠中，脾淋巴細(xì)胞和PBMCs均未能檢測(cè)到HBV特異性的細(xì)胞免疫應(yīng)答。僅在第10天，早期應(yīng)用P3C組小鼠的脾淋巴細(xì)胞經(jīng)rHBcAg刺激后分泌IFN-γ的T細(xì)胞數(shù)量少于對(duì)照組。 4.通過(guò)對(duì)小鼠肝組織相關(guān)分子mRNA水平進(jìn)行real time RT-PCR檢測(cè)，我們發(fā)現(xiàn)：早期應(yīng)用P3C組和對(duì)照組小鼠相比，IFN-β和IFN-γ mRNA無(wú)明顯差異；促炎因子IL-6和TNF-α mRNA在第4天明顯高于對(duì)照組；抑炎因子IL-10mRNA在第4天和第10天明顯高于對(duì)照組。 結(jié)論： 1. naive小鼠和HBV慢性復(fù)制小鼠經(jīng)TLR2配體P3C處理后，血清IL-6等炎癥因子的產(chǎn)生是一個(gè)較快的過(guò)程，一般在幾個(gè)小時(shí)內(nèi)達(dá)到最高峰，且炎癥因子的濃度與P3C的劑量成正相關(guān)。 2.在慢性HBV復(fù)制小鼠模型中，早期應(yīng)用P3C能發(fā)揮明顯的抗HBV效應(yīng)，而晚期應(yīng)用則無(wú)此明顯效應(yīng)。 3.在慢性HBV復(fù)制小鼠模型中，早期應(yīng)用P3C產(chǎn)生明顯的抗HBV效應(yīng)的機(jī)制可能是通過(guò)TLR2介導(dǎo)的天然免疫應(yīng)答對(duì)HBV的早期復(fù)制產(chǎn)生抑制，而TLR2活化誘導(dǎo)的特異性免疫應(yīng)答在其中發(fā)揮的作用十分有限。
[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- 偽 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- 偽 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- 緯 secreting cells in spleen lymphocytes. The positive percentage of CD8 T cells secreting IFN- 緯 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- 緯 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- 緯 mRNA between the early P3C group and the control group, and the level of IL-6 and TNF- 偽 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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373.21

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