【作者】 郭远瑾；

【导师】 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2010， 博士

【摘要】 目的研究柯里拉京对HSV-1感染后小胶质细胞表达炎性细胞因子及炎性介质的影响。方法小胶质细胞系BV2细胞接种HSV-1制造细胞模型,MTT法选择合适的细胞用药浓度,细胞分为正常对照组、HSV-1感染组、柯里拉京组(20ng/ml)、黄芪多糖组(100ug/ml)、地塞米松组(0.5ug/ml)。使用ELISA法检测各组白细胞介素1β(IL-1β)、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)的表达,采用Griess reagentNO试剂盒检测NO表达。结果病毒感染后,各细胞因子均明显上升；柯里拉京干预后,小胶质细胞IL-1β、TNF-α及NO的表达与病毒组比较减少差异有统计学意义(P<0.01),IL-10升高不显著。结论柯里拉京通过减少小胶质细胞促炎因子及炎性介质的分泌从而减缓病毒诱导的免疫炎性反应。目的研究柯里拉京干预对单纯疱疹病毒(HSV-1)诱导的BV2及PC12细胞凋亡的影响。方法PC12细胞及小胶质细胞系BV2细胞接种HSV-1制造细胞模型,MTT法选择合适的细胞用药浓度,细胞分为正常对照组、HSV-1病毒感染组、柯里拉京组(20ng/ml)、黄芪多糖组(100ug/ml)、地塞米松组(0.5ug/ml),采用流式细胞术检测12、24、48h时间点各组BV2及PC12细胞的凋亡率。结果1.BV2细胞被病毒感染后,细胞凋亡率显著增加,与正常组比较(P<0.05)；与病毒组比较,柯里拉京组与地塞米松组细胞凋亡率显著增加(P<0.05)；APS组对BV2细胞的凋亡影响较小,表现出其阴性对照的作用。12、24及48h时间点均显示出相似的结果。2.病毒感染PC12细胞后,细胞凋亡率显著增加,与正常组比较(P<0.05)；与病毒组比较,柯里拉京组、地塞米松组及APS组细胞凋亡率降低(P<0.05)。48h时间点与24h显示出相似的结果。而12h时间点各药物组未显示出明显的抑制作用。结论柯里拉京可以促进HSV-1诱导的小胶质细胞系BV2的凋亡,并且能够抑制病毒诱导的PC12细胞的凋亡。目的探讨柯里拉京促进单纯疱疹病毒(HSV-1)诱导的小胶质细胞凋亡途径。方法BV2细胞接种HSV-1制造细胞模型,MTT法选择合适的药物浓度,细胞分为正常对照组、HSV-1病毒感染组、柯里拉京组(20ng/ml)、黄芪多糖组(100ug/ml)、地塞米松组(0.5ug/ml),干预24h后流式细胞学检测各组caspase-3、caspase-8、caspase-9、caspase-12的活性,Western-blot法检测细胞色素C含量。结果1.病毒感染BV2细胞后,caspase-3的表达显著升高,与正常组相比(P<0.05)；与病毒组相比,柯里拉京组及地塞米松组表达水平显著增高(P<0.05)；作为阴性对照,APS干预后caspase-3表达降低。2.病毒组与正常组比较caspase-8的表达率没有明显差异；与病毒组比较,柯里拉京组及地塞米松组caspase-8的表达率均明显提高(P<0.05); APS组仍然表现出阴性对照的作用,caspase-8的表达水平是降低的。3.与正常组相比较,病毒组caspase-9的表达率显著升高(P<0.05)；而且柯里拉京组、地塞米松组及APS组caspase-9的表达率均比病毒组升高(P<0.05)：各组细胞色素C的表达与caspase-9表达的趋势一致。4.与正常组比较,病毒组caspase-12的表达没有明显的升高；地塞米松组与病毒组比较其表达也没有明显的差异；而柯里拉京组与病毒组比较caspase-12的表达显著增加(P<0.05)；APS组与病毒组比较caspase-12也是显著增加的(P<0.05)。结论凋亡的三个主要途径：线粒体途径、死亡受体途径及内质网途径,在柯里拉京促进HSV-1诱导的小胶质细胞凋亡的过程中均发挥着重要的作用。而HSV-1诱导小胶质细胞凋亡主要是通过细胞色素C及caspase-9介导的线粒体途径来实现的。目的研究柯里拉京治疗单纯疱疹病毒性脑炎的可行性,为其临床应用提供实验依据。方法Balb/c小鼠颅内注射HSV-1制造小鼠单纯疱疹病毒性脑炎(herpes simplex encephalitis, HSE)模型,随机分成5组：正常组、感染组、柯里拉京组(0.4mg/只)、地塞米松组(2ug/只)、黄芪多糖组(0.8mg/只)。采用ELISA试剂盒检测脑组织TNF-a表达,硝酸还原酶法检测NO含量,通过HE染色观察脑组织形态学,TUNEL法观察脑细胞的凋亡。结果病毒感染后第四天：1.感染组、柯里拉京组、地塞米松组及黄芪多糖(APS)组TNF-a表达较正常对照组明显增加(P<0.01)；与感染组比较,柯里拉京组、地塞米松组TNF-a表达降低有统计学差异(分别为P<0.01、P＜0.05)。2.感染组、柯里拉京组、地塞米松组及黄芪多糖(APS)组NO表达较正常对照组明显增加(P<0.01)：与感染组比较,柯里拉京组、地塞米松组、APS组NO表达降低有统计学差异(分别为P<0.01、P<0.01、P<0.05)。3.各药物干预组小鼠脑组织病理改变减轻。4.各药物干预组较病毒组TUNEL阳性细胞明显减少。结论柯里拉京能够有力的防治HSV-1病毒感染继发的脑损伤,改善单纯疱疹病毒性脑炎的预后。更多还原

【Abstract】 Aims:1.Assessment of regulation and expression of cytokines IL-1β、IL-10、TNF-αand NO in HSV-1 infected microglia 2. Effect of Corilagin on microglial production of cytokines Methods:Microglial cells were divided in 5 groups namely,1. control group,2.HSV-1 infected group,3.HSV-1 infected+ Coraligin (20ng/ml)group,4. HSV-1 infected+ Dexamethasone (0.5ug/ml)group,5.HSV-1 infected+APS(100ug/ml) group. ELISA was used to detect IL-1β、IL-10、TNF-αexpression and Griess reagent NO to detect NO.Results:HSV-1 infected group showed increase in expression of all cytokines named above.HSV-1 infected group with Coraligin showed significant decrease in IL-1β、TNF-αand NO expressions(P<0.01).while IL-10 increase was insignificant. Conclusions:Coraligin has inhibitory effects on microglial chemokines production following HSV-1 central nervous infectionAims:To study the effects of Corilagin on the apoptosis of HSV-1 stimulated BV2 cells and HSV-1 infected PC 12 cells. Methods:PC 12 and BV2 cells were divided into 5 groups namely,1.Normal control group (non-HSV-1 infected),2.HSV-1 infected group 3.HSV-1 infected+ Corilagin (20ng/ml))group 4. HSV-1 infected+ APS(100ug/ml) group.5. HSV-1 infected+ Dexamethasone(0.5ug/ml) group. Apoptotic rate of these BV2 and PC12 cells were determined at 12h,24h,48h intervals. Flow cytometry assay for examining apoptosis rate. Results:1. Compared to the control group, the HSV-infected group showed significant BV2 cells apoptosis(P<0.05).When the same HSV-infected group was compared to the Corilagin group and Dexamethasone group, the latter 2 groups displayed even more significant BV2 apoptosis(P<0.05).Nevertheless, comparison between APS group and HSV-infected group showed little difference of BV2 cells apoptosis; hence, this group served as a negative control in this experiment.All results were uniformly present during each of the 12h,24h and 48 h cells viability testing. 2. Following infection with HSV-1, PC12 cells death in the HSV-1 infected group was significant as compared to the control group(P<0.05).However, PC12 cells death was significantly reduced in the Corilagin group and Dexamethasone group (P<0.05)as compared to the HSV-1 infected group. These results were present at the 24h and 48h cells count intervals.At the 12h cells count interval no significant difference in the apoptotic ratee of the PC12 cells was noted between all the treated groups and the HSV-1-infected group.Conclusions:Corilagin can induce apoptosis of HSV-1 stimulated microglial BV2 cells and at the same time can inhibit HSV-1 induced PC12 cells apoptosisAims:To investigate the apoptotic pathway involved in Hsv-1 stimulated microglias following corilagin intervention. Methods:Model of HSV-1 stimulated BV2 cells was set-up.These BV2 cells were then divided into the following groups:1.Normal control group,2.HSV-1 infected group,3.HSV-1 infected+ Corilagin (20ng/ml)group,4. HSV-1 infected+ APS (100ug/ml)) group,5.HSV-1 infected+Dexamethasone(0.5ug/ml) group.A control group consisting of normal BV2 cells was also included. After a time lapse of 24h, cysteine proteases:caspase-3,caspase-8,caspase-9 and caspase-12 activities were analysed.using Flow cytometry assay., cytochrome c level was also determined by western-blot. Results:1. In the HSV-1 infected group, caspase-3 expression was markedly increase as compared to the control group (P<0.05).In the Corilagin group and Dexamethasone group expression of caspase-3 was more than the HSV-1 infected group(P<0.05).Acting as a negative control caspase 3 expression in the APS group was decreased.2. Caspase-8 expression between the control group and HSV-1 infected group was insignificant(P>0.05)In the Corilagin group and Dexamethasone group, expression of caspase-8 was significantly increased as compared to that of the HSV-1 infected group(P<0.05). Acting as a negative control caspase 8 expression in the APS group was again decreased.3. In the HSV-1 infected group caspase-9 expression was markedly increase as compared to the control group (P<0.05).By contrast, in the Corilagin group, Dexamethasone group and APS group expression of caspase-9 was increased as compared to that of the HSV-1 infected group(P<0.05).In each group, the level of cytochrome c was consistent with its respective caspase-9 expression level.4. Caspase-12 expression between the HSV-1 infected group and control group did not show any significant increase. Caspase-12 expression between the HSV-1 infected group and Dexamethasone was also insignificant. By contrast, in the Corilagin group, expression of caspase-12 was increased as compared to that of the HSV-1 infected group(P<0.05). Furthemore, in the APS group, expression of caspase-12 was also increased as compared to that of the HSV-1 infected group(P<0.05).Conclusions:Alone HSV-1 stimulated microglias apoptosis follows the cytc and caspase-9 initiated mitochondrial pathway.However,following Corilagin intervention, all the 3 main apoptotic pathways(mitochondrial,death receptor and endoplasmic) are involved. Aim:To provide laboratory evidence for possible clinical use of Coraligin through its application on HSV-1 infected murine brain. Methods:Murine model of HSV-1 encephalitis was set up following injection of HSV-1 samples in Balb/c mice brains.These subjects were then randomly divided in the following groups according to the various additional treatments given:1.virus group 2.infected+ Corilagin(0.4mg/per mouse) group 3. infected+ Dexamethasone(2ug/per mouse) group 4. infected+ APS (0.8mg/per mouse) group. A fifth group of uninfected mice acting as control was also included. Following sacrifice of a number of mice in each group at day 4 of experiment, ELISA method was used to detect TNF-a, NO level in the mice brains. HE stain was also used to study brain samples. Brain cells necrosis was observed using TUNEL method. Results:1.Of those sacrificed animals on day 4, levels of TNF-a in the infected group, Corilagin group, Dexamethasone group, APS group were greater when compared to the control group(P<0.01);when Corilagin group,Dexamethasone group were compared to the infected group, TNF-a levels were lower(P<0.01、P<0.05)2. Levels of NO in the infected group, Corilagin group Dexamethasone group,APS group were also greater when compared to the control group(P<0.01);when the Corilagin group, Dexamethasone group,and APS group were compared to the infected group,NO levels were lower(P<0.01、P<0.05)3.In all the intervened groups(Corilagin,APS,Dexamethasone), those sacrificed pathological changes in the brain tissues were minimum. When compared to the infected group, these intervened groups TUNEL positive cells were also lesser.Conclusions:Corilagin can effectively prevent HSV-1 induced brain damage and hence improve prognosis of HSV-1 encephalitis in this study of murine model.更多还原