【作者】 罗涛；

【导师】 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2014， 博士

【摘要】 第一部分HSV-1感染小胶质细胞后对Toll样受体的影响目的研究HSV-1感染小胶质细胞后Toll样受体表达情况。方法小胶质细胞系BV2细胞接种HSV-1制造细胞模型,使用实时荧光定量聚合酶链反应检测HSV-1感染组与对照组TLR2、TLR3、TLR9mRNA表达。结果病毒感染后,TLR2、TLR3、 TLR9mRNA均明显上升,与对照组比较差异有统计学意义(P<0.01)。结论TLR2、TLR3、TLR9参与了BV2小胶质细胞对HSV-1病毒的识别。第二部分HSV-1感染BV2细胞后对TLR2及下游通路信号分子的影响目的研究HSV-1感染小胶质细胞后对TLR2信号通路的影响。方法小胶质细胞系BV2接种HSV-1制造细胞模型,Malp2刺激BV2细胞作为阳性对照组,采用流式细胞术检测TLR2蛋白表达；Western blot检测TIRAP、MyD88、TRAF6、P38、 NEMO、NF-κBp65蛋白表达；实时荧光定量聚合酶链反应检测TLR2、IRAP、MyD88、 TRAF6、P38、NEMO的mRNA表达；ELISA检测IL-6、TNF-α的表达。结果1、BV2细胞被Malp2刺激后,TLR2及下游信号通路因子表达均明显升高,与空白对照组比较差异有统计学意义(P<0.05)。2、HSV-1感染BV2细胞后,TLR2及下游信号通路因子表达明显升高,与空白对照组比较差异有统计学意义(P<0.05)。结论TLR2及下游信号通路因子介导了HSV-1感染BV2细胞的免疫炎性反应。第三部分柯里拉京对HSV-1感染后BV2细胞TLR2通路的调节作用实验1柯里拉京对HSV-1感染正常BV2细胞TLR2通路的调节作用目的研究柯里拉京对HSV-1感染后BV2细胞TLR2通路的调节作用。方法小胶质细胞系BV2接种HSV-1制造细胞模型,Malp2刺激BV2细胞作为阳性对照组,细胞受HSV-1和Malp2干预后柯里拉京治疗24h,采用流式细胞术检测TLR2蛋白表达；Western blot检测TIRAP、MyD88、TRAF6、P38、NEMO、NF-κBp65蛋白表达；实时荧光定量聚合酶链反应检测TLR2、TIRAP、MyD88、TRAF6、P38、 NEMO的mRNA表达；ELISA检测IL-6、TNF-α的表达。结果1、柯里拉京治疗受Malp2刺激的BV2细胞后,TLR2及下游信号通路分子表达均下降,与生理盐水治疗组比较(P<0.05)。2、柯里拉京治疗受HSV-1感染的BV2细胞后,TLR2及下游信号通路分子表达均下降,与生理盐水治疗组比较差异有统计学意义(P<0.05)。结论柯里拉京可以抑制HSV-1感染BV2细胞后激活的TLR2信号通路。实验2柯里拉京抑制HSV-1感染后BV2细胞TLR2通路机制研究目的研究柯里拉京抑制HSV-1感染后BV2细胞TLR2通路的机制。方法通过慢病毒载体使BV2细胞TLR2过表达,HSV-1感染BV2细胞后柯里拉京治疗24h；通过小分子RNA使BV2细胞TLR2表达沉默,HSV-1感染BV2细胞后柯里拉京治疗24h结果,采用Western blot、Real-time PCR和ELISA检测TLR2下游信号通路分子表达情况。结果1、TLR2过表达时,柯里拉京治疗受HSV-1感染的BV2细胞后,与生理盐水治疗组比较,TLR2下游信号通路分子表达均下降(P<0.05)。2、TLR2表达沉默时,柯里拉京治疗受HSV-1感染的BV2细胞后,与生理盐水治疗组比较：TTRAP变化不明显(P>0.05); MyD88、TRAF6表达上升(P<0.05); P38、NEMO、NF-κBp65、IL-6、TNF-α表达下降(P<0.05)。结论柯里拉京对TIRAP、MyD88、TRAF6的抑制作用需要TLR2的参与,同时柯里拉京可能通过其他的Toll样受体或者直接对P38、NEMO, NF-κBp65、IL-6、TNF-α产生抑制作用。第四部分柯里拉京对单纯疱疹病毒性脑炎小鼠TLR2通路的影响目的研究柯里拉京通过抑制TLR2通路治疗单纯疱疹病毒性脑炎的可行性。方法Balb/c、鼠颅内注射HSV-1制造小鼠单纯疱疹病毒性脑炎模型,随机分成：正常组、柯里拉京组、Malp2+盐水治疗组、Malp2+柯里拉京治疗组、HSV-1+盐水治疗感染组、HSV-1+柯里拉京治疗组。采用HE染色观察脑组织形态学；免疫组化检测TLR2表达；实时荧光定量聚合酶链反应检测TLR2、TIRAP、MyD88、TRAF6、 P38、NEMO的mRNA表达；Western blot检测TIRAP、MyD88、TRAF6、P38、NEMO、 NF-κBp65蛋白表达；ELISA检测IL-6、TNF-α的表达。结果1、柯里拉京干预组小鼠脑组织病理改变减轻2、Malp2+盐水治疗组和HSV-1+盐水治疗感染组与正常组比较,TLR2及下游信号通路因子表达明显升高(P<0.05)。3、柯里拉京治疗受Malp2刺激的小鼠后,与生理盐水治疗组比较,TLR2及下游信号通路分子表达均下降(P<0.05)。4、柯里拉京治疗受HSV-1感染的小鼠后,与生理盐水治疗组比较,TLR2及下游信号通路分子表达均下降(P<0.05)。结论柯里拉京可以通过抑制TLR2通路减轻HSV-1感染激发的脑损伤。更多还原

【Abstract】 PART Ⅰ The change of toll-like receptors after HSV-1infected microgliaObjective:Study the expression of toll-like receptors after HSV-1infected microglia.Methods:BV2cells were infected by HSV-1, then Real-time PCR was used to detect the expression of TLR2/TLR3/TLR9mRNA.Results:HSV-1infected group showed increase in expression of TLR2/TLR3/TLR9mRNA, the difference were significant compare with that of in normal group (P<0.01).Conclusions:TLR2, TLR3and TLR9were involved in the recognition of Herpes simplex virus-1in BV2cells. PART Ⅱ The change of toll-like receptor2and downstream mediators after HSV-1infected microgliaObjective:Study the expression of toll-like receptor2and downstream mediators after HSV-1infected microglia. Methods:BV2cells were infected by HSV-1and Malp2, then Flow cytometry (FCM) was used to measure the levels of TLR2and Western blot was used to detect the levels of TIRAP、MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIRAP、 MyD88、TRAF6、P38and NEMO. ELISA was also used to measure the levels of IL-6and TNF-α. Results:1、TLR2and its downstream mediators were significantly increased after Malp2stimulated BV2cells, the difference were significant compare with that of in normal group (P<0.05).2、TLR2and its downstream mediators were significantly increased after HSV-1infected BV2cells, the difference were significant compare with that of in normal group (P<0.05). Conclusions:TLR2and its downstream mediators initiated innate responses to herpes simplex virus type1in BV2cells. Keywords:herpes simplex virus type1, microglia, toll-like receptor2signaling pathway PART Ⅲ The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microgliaExperiment1The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected normal microgliaObjective:Study the effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microglia. Methods:BV2cells were infected by HSV-1and Malp2, then cells treated by Corilagin for24h. Flow cytometry (FCM) was used to measure the levels of TLR2and Western blot was used to detect the levels of TIRAP、 MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIRAP、MyD88、TRAF6、P38and NEMO. ELISA was also used to measure the levels of IL-6and TNF-a. Results:1、After Corilagin treated BV2cells that stimulated by Malp2,TLR2and its downstream mediators were significantly decreased, the difference were significant compare with that of in normal saline group (P<0.05).2、After Corilagin treated BV2cells that infected by HSV-1,TLR2and its downstream mediators were significantly decreased, the difference were significant compare with that of in normal saline group (P<0.05)Conclusions:Corilagin can inhibit the activation of TLR2and its downstream mediators after HSV-1infected BV2cells. Experiment2The mechanism of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microgliaObjective:Study the mechanism of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microglia. Methods:When TLR2was excessive or TLR2was silent, BV2cells were infected by HSV-land Malp2, then treated by Corilagin for24h. Western blot、Real-time PCR and ELISA were used to measure the levels of TLR2downstream mediators. Results:1、When TLR2was excessive,after Corilagin treated BV2cells that stimulated by Malp2,TLR2downstream mediators were decreased, the difference were significant compare with that of in normal saline group (P<0.05).2、When TLR2was silent, after Corilagin treated BV2cells that infected by HSV-1,the levels of MyD88and TRAF6were increased compare with that of in normal saline group (P<0.05), but the difference of TRIAP was not significant (P>0.05).however, the levels of P38、NEMO、NF-κBp65、IL-6and TNF-α were decreased, the difference were significant compare with that of in normal saline group (P<0.05). Conclusions:Corilagin might inhibit NEMO, P38, p-P38, NF-κB, TNF-α, IL-6directly or via other TLRs and the inhibition of Corilagin to the TIRAP/MyD88-TRAF6pathway needed TLR2. PART IV The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected murine brainObjective:Study the effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected murine brain. Methods:Murine model of HSV-1was set up following injection of HSV-1samples in Balb/c mice brains. These subjects were randomly divided in the following groups:normal group, Corilagin group, Malp2+normal saline group, Malp2+Corilagin group, HSV-1+normal saline group, HSV-1+Corilagin group. HE stain was used to study brain samples, immunohistochemical staining was used to detect the expression of TLR2, Western blot was used to detect the levels of TIRAP、MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIAP、 MyD88、TRAF6、P38and NEMO,ELISA was also used to measure the levels of IL-6and TNF-α. Results:1、pathological changes in the brain tissues were minimum in Corilagin-treated group.2、Malp2+normal saline group and HSV-1+normal saline group compare with that of in normal group,TLR2and its downstream mediators were significant increased (P<0.05).3%Malp2+Corilagin group compare with Malp2+normal saline group, TLR2and its downstream mediators were significant decreased (P<0.05).4、HSV-1+Corilagin group compare with HSV-1+normal saline group, TLR2and its downstream mediators were significant decreased (P<0.05)Conclusions:Corilagin can effectively prevent HSV-1induced brain damage through TLR2signaling pathway.更多还原