本文選題：單純皰疹病毒1型　切入點：小膠質(zhì)細胞系BV2　出處：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分HSV-1感染小膠質(zhì)細胞后對Toll樣受體的影響 目的研究HSV-1感染小膠質(zhì)細胞后Toll樣受體表達情況。方法小膠質(zhì)細胞系BV2細胞接種HSV-1制造細胞模型,使用實時熒光定量聚合酶鏈反應(yīng)檢測HSV-1感染組與對照組TLR2、TLR3、TLR9mRNA表達。結(jié)果病毒感染后,TLR2、TLR3、 TLR9mRNA均明顯上升,與對照組比較差異有統(tǒng)計學(xué)意義(P0.01)。結(jié)論TLR2、TLR3、TLR9參與了BV2小膠質(zhì)細胞對HSV-1病毒的識別。 第二部分HSV-1感染BV2細胞后對TLR2及下游通路信號分子的影響 目的研究HSV-1感染小膠質(zhì)細胞后對TLR2信號通路的影響。方法小膠質(zhì)細胞系BV2接種HSV-1制造細胞模型,Malp2刺激BV2細胞作為陽性對照組,采用流式細胞術(shù)檢測TLR2蛋白表達；Western blot檢測TIRAP、MyD88、TRAF6、P38、 NEMO、NF-κBp65蛋白表達；實時熒光定量聚合酶鏈反應(yīng)檢測TLR2、IRAP、MyD88、 TRAF6、P38、NEMO的mRNA表達；ELISA檢測IL-6、TNF-α的表達。結(jié)果1、BV2細胞被Malp2刺激后,TLR2及下游信號通路因子表達均明顯升高,與空白對照組比較差異有統(tǒng)計學(xué)意義(P0.05)。2、HSV-1感染BV2細胞后,TLR2及下游信號通路因子表達明顯升高,與空白對照組比較差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論TLR2及下游信號通路因子介導(dǎo)了HSV-1感染BV2細胞的免疫炎性反應(yīng)。 第三部分柯里拉京對HSV-1感染后BV2細胞TLR2通路的調(diào)節(jié)作用實驗1柯里拉京對HSV-1感染正常BV2細胞TLR2通路的調(diào)節(jié)作用 目的研究柯里拉京對HSV-1感染后BV2細胞TLR2通路的調(diào)節(jié)作用。方法小膠質(zhì)細胞系BV2接種HSV-1制造細胞模型,Malp2刺激BV2細胞作為陽性對照組,細胞受HSV-1和Malp2干預(yù)后柯里拉京治療24h,采用流式細胞術(shù)檢測TLR2蛋白表達；Western blot檢測TIRAP、MyD88、TRAF6、P38、NEMO、NF-κBp65蛋白表達；實時熒光定量聚合酶鏈反應(yīng)檢測TLR2、TIRAP、MyD88、TRAF6、P38、 NEMO的mRNA表達；ELISA檢測IL-6、TNF-α的表達。結(jié)果1、柯里拉京治療受Malp2刺激的BV2細胞后,TLR2及下游信號通路分子表達均下降,與生理鹽水治療組比較(P0.05)。2、柯里拉京治療受HSV-1感染的BV2細胞后,TLR2及下游信號通路分子表達均下降,與生理鹽水治療組比較差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論柯里拉京可以抑制HSV-1感染BV2細胞后激活的TLR2信號通路。 實驗2柯里拉京抑制HSV-1感染后BV2細胞TLR2通路機制研究 目的研究柯里拉京抑制HSV-1感染后BV2細胞TLR2通路的機制。方法通過慢病毒載體使BV2細胞TLR2過表達,HSV-1感染BV2細胞后柯里拉京治療24h；通過小分子RNA使BV2細胞TLR2表達沉默,HSV-1感染BV2細胞后柯里拉京治療24h結(jié)果,采用Western blot、Real-time PCR和ELISA檢測TLR2下游信號通路分子表達情況。結(jié)果1、TLR2過表達時,柯里拉京治療受HSV-1感染的BV2細胞后,與生理鹽水治療組比較,TLR2下游信號通路分子表達均下降(P0.05)。2、TLR2表達沉默時,柯里拉京治療受HSV-1感染的BV2細胞后,與生理鹽水治療組比較：TTRAP變化不明顯(P0.05); MyD88、TRAF6表達上升(P0.05); P38、NEMO、NF-κBp65、IL-6、TNF-α表達下降(P0.05)。結(jié)論柯里拉京對TIRAP、MyD88、TRAF6的抑制作用需要TLR2的參與,同時柯里拉京可能通過其他的Toll樣受體或者直接對P38、NEMO, NF-κBp65、IL-6、TNF-α產(chǎn)生抑制作用。 第四部分柯里拉京對單純皰疹病毒性腦炎小鼠TLR2通路的影響 目的研究柯里拉京通過抑制TLR2通路治療單純皰疹病毒性腦炎的可行性。方法Balb/c、鼠顱內(nèi)注射HSV-1制造小鼠單純皰疹病毒性腦炎模型,隨機分成：正常組、柯里拉京組、Malp2+鹽水治療組、Malp2+柯里拉京治療組、HSV-1+鹽水治療感染組、HSV-1+柯里拉京治療組。采用HE染色觀察腦組織形態(tài)學(xué)；免疫組化檢測TLR2表達；實時熒光定量聚合酶鏈反應(yīng)檢測TLR2、TIRAP、MyD88、TRAF6、 P38、NEMO的mRNA表達；Western blot檢測TIRAP、MyD88、TRAF6、P38、NEMO、 NF-κBp65蛋白表達；ELISA檢測IL-6、TNF-α的表達。結(jié)果1、柯里拉京干預(yù)組小鼠腦組織病理改變減輕2、Malp2+鹽水治療組和HSV-1+鹽水治療感染組與正常組比較,TLR2及下游信號通路因子表達明顯升高(P0.05)。3、柯里拉京治療受Malp2刺激的小鼠后,與生理鹽水治療組比較,TLR2及下游信號通路分子表達均下降(P0.05)。4、柯里拉京治療受HSV-1感染的小鼠后,與生理鹽水治療組比較,TLR2及下游信號通路分子表達均下降(P0.05)。結(jié)論柯里拉京可以通過抑制TLR2通路減輕HSV-1感染激發(fā)的腦損傷。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R512.3

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