柯里拉京对单纯疱疹病毒性脑炎发病过程中TLR2通路的干预作用机制研究

发布时间：2018-04-02 11:12

本文选题：单纯疱疹病毒1型　切入点：小胶质细胞系BV2　出处：《华中科技大学》2014年博士论文

【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(P0.01)。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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05); MyD88、TRAF6表达上升(P0.05); P38、NEMO、NF-κBp65、IL-6、TNF-α表达下降(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。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。
[Abstract]:The effect of HSV-1 infection on Toll like receptors in the first part of microglia
The expression of Toll like receptors in microglia after HSV-1 infection. Objective to study the method of microglial cell line BV2 cells were inoculated with HSV-1 cell manufacturing model, using real-time fluorescence quantitative polymerase chain reaction for detection of HSV-1 infection group and control group TLR2, TLR3, TLR9mRNA. Results the expression of virus infection, TLR2, TLR3, TLR9mRNA were significantly increased, there were compared with the control group (P0.01). Conclusion: TLR2, TLR3, TLR9 in recognition of BV2 microglia to HSV-1 virus.
The effect of HSV-1 infected BV2 cells on the signal molecules of TLR2 and downstream pathway in the second part
The effect of TLR2 signal pathway of HSV-1 infected microglia. Method of microglial cell line BV2 were inoculated with HSV-1 producing cell model, Malp2 stimulation of BV2 cells as the positive control group, the expression of TLR2 protein detected by flow cytometry; Western blot MyD88, TRAF6, detection of TIRAP, P38, NEMO, expression of NF- kappa Bp65 protein; real-time fluorescence quantitative polymerase chain reaction for detection of TLR2, IRAP, MyD88, TRAF6, P38, NEMO expression of mRNA; ELISA to detect IL-6 expression of TNF-. Results 1, BV2 cells were stimulated by Malp2, TLR2 and downstream signaling factor expression were significantly increased, and the gap was significantly lower than the control group (P0.05.2 HSV-1), BV2 cells infected with TLR2 and downstream signaling pathway factor expression was significantly increased, and the gap was significantly lower than the control group (P0.05). Conclusion TLR2 and downstream signaling pathways mediated by sub HSV-1 infection of BV2 cells Immunological response.
The third part of corilagin on HSV-1 infection of BV2 cells after TLR2 pathway regulation experiment 1 corilagin of HSV-1 infected normal BV2 cell TLR2 pathway regulation
Objective to study the corilagin regulating effect on BV2 cells of the TLR2 pathway after HSV-1 infection. Methods the microglia cell line BV2 were inoculated with HSV-1 producing cell model, Malp2 stimulation of BV2 cells as the positive control group, HSV-1 and Malp2 cells by intervention treatment by Currie La Gin 24h, TLR2 protein expression was detected by flow cytometry; Western blot detection of TIRAP, MyD88 TRAF6, P38, NEMO, NF-, the expression of NF kappa Bp65 protein; real-time fluorescence quantitative polymerase chain reaction for detection of TLR2, TIRAP, MyD88, TRAF6, P38, NEMO expression of mRNA; ELISA to detect IL-6 expression of TNF-. Results 1, Currie pull the treatment of a Malp2 stimulated BV2 cells, expression of TLR2 and its downstream signal the molecular pathways were decreased, compared with saline treatment group (.2, P0.05) treatment corilagin HSV-1 infected BV2 cells, the expression of TLR2 and downstream signaling molecules were decreased, with statistical difference compared with the normal saline treatment group Significance (P0.05). Conclusion the corilagin TLR2 signaling pathways could inhibit HSV-1 infection of BV2 cells after activation.
Study on BV2 cells of TLR2 pathway in Experiment 2 corilagin inhibited HSV-1 infection
Objective to study the mechanism of corilagin inhibit HSV-1 infection of BV2 cells after TLR2 pathway. Methods by lentiviral vector to BV2 cells overexpression of TLR2, 24h and Currie La Gin treatment of HSV-1 infected BV2 cells; the expression silencing of BV2 TLR2 cells by small molecule RNA, HSV-1 infection of BV2 cells after treatment of 24h Currie pull the results by Western blot, expression Real-time PCR and ELISA TLR2 detection of downstream signaling molecules. Results 1, overexpression of TLR2, treatment of corilagin HSV-1 infected BV2 cells, compared with the saline treated group, the expression of TLR2 downstream signaling molecules were decreased (P0.05).2, TLR2 silencing, corilagin treatment of HSV-1 infected BV2 after cells compared with the saline treated group: TTRAP did not change significantly (P0.05); MyD88, TRAF6; P38 (P0.05) increased expression of NEMO, NF-, IL-6, alpha kappa Bp65, TNF- (P0.05). Conclusion the decreased expression of corilagin on T IRAP, MyD88, the inhibitory effect of TRAF6 TLR2 is required at the same time, corilagin may through Toll like receptors or other directly to P38, NEMO, NF-, IL-6, alpha kappa Bp65, TNF- inhibit.
The fourth part effect of corilagin on mice with herpes simplex virus encephalitis TLR2 pathway
Objective to study the feasibility of corilagin by inhibiting the TLR2 pathway in the treatment of herpes simplex virus encephalitis. Methods Balb/c mice were randomly divided into model, herpes simplex virus encephalitis after intracranial injection of HSV-1 manufacturing: normal group, corilagin group, Malp2+ saline group, Malp2+ Ke Gratin treatment group, HSV-1+ infection group, saline treatment, HSV-1+ treatment of corilagin group. HE staining was used to observe the morphology of brain tissue; immunohistochemical detection of TLR2 expression; real-time fluorescence quantitative polymerase chain reaction for detection of TLR2, TIRAP, MyD88, TRAF6, P38, NEMO mRNA Western blot expression; MyD88, TRAF6, TIRAP detection, P38, NEMO, expression of NF- kappa Bp65 protein; ELISA detection of IL-6, TNF- expression alpha. Results 1, corilagin intervention group mice brain tissue pathological changes alleviated 2 Malp2+ saline treatment group and saline treatment HSV-1+ infection group compared with normal group, TLR2 and the downstream signaling pathway factor The expression of.3 was significantly increased (P0.05), corilagin treatment induced by Malp2 in mice, compared with the saline treated group, the expression of TLR2 and downstream signaling molecules were decreased (P0.05.4), corilagin treatment by HSV-1 infected mice, compared with the saline treated group, the expression of TLR2 and downstream signaling molecules are decreased (P0.05). Conclusion corilagin can reduce brain damage by inhibiting the TLR2 pathway in HSV-1 infection excitation.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.3

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