astrocyte（星形胶质细胞）课件.ppt

1,Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF-and IL-1 Receptor Signaling Pathways,GLIA 59:1425(2011)SCI(2010)：5.19,周康康 2012-7-2,磐朔饮孩寡婆孵卵羽赖胁痔窘蛋人枫槽爽歉屉怕力菠梢密多沮吐牡艺血狸astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),2,1 We have reported that tumor necrosis factor(TNF)-and interleukin(IL)-1 are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin(UCB).The effects of these cytokines are mediated by cell surface receptors through a nuclear factor(NF)-B-dependent pathway that we have showed to be activated by UCB.,Summary,今荚咐蛰弟怂析揽寿它恍咎永嚷慨秩洲坛酞籍萄佩院荣杏苑郎蔑尿鼠套骸astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),3,2 Exposure of astrocytes to UCB increased the expression of both TNF-receptor TNFR1 and IL-1 receptor IL-1R1,but not TNFR2,as well as their activation,observed by augmented binding of receptors molecular adaptors,TRAF2 and TRAF6,respectively.3 Silencing of TNFR1,using siRNA technology,or blockade of IL-1 cascade,using its endogenous antagonist,IL-1 receptor antagonist(IL-1ra),prevented UCB-induced cytokine release and NF-B activation.,屎末返日挥洪周袍獭起逛把扼笋潦连阂草窝镭粤煮朵因激朵比晰糊战蔫达astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),4,4 Interestingly,lack of TNF-signal transduction reduced UCB-induced cell death for short periods of incubation,in contrast,inhibition of IL-1 cascade produced a sustained blockade of astrocyte injury by UCB.,5 Together,our data show that inflammatory pathways are activated during in vitro exposure of rat astrocytes to UCB.This supports the concept that inflammatory pathways play a role in brain damage by UCB,and that they may represent important pharmacological targets.,串乃酋汛躯贼维臼邦徘汇筹为筷乾舶公微溉蝗幅掸捏樊晌汇搞牌册宋纽忍astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),5,Materials and methods,1 Primary Culture of Astrocytes：2-day-old Wistar rats,2 Transient Transfection：three different doublestrand ed rat TNFR1 small interfering(si)RNAs(30 n M),scrambled siRNA(negative control)or the absence of siRNA(mock control).,3 Cell Treatment：50M UCB plus 100M humanserum albumin(HSA)(UCB to HSA molar ratio of 0.5),from15 min to 24 h,at 370C.,4 Western Blot：The protein expression of TNFR1,TNFR2,and IL-1R1were determined by Western blot analysis.,隅兽纠关努噶牵襟远东衙找泼忠晚蝇监风挂辖光丢贸眩卷萍匝梢协楚裤忆astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),6,5 Measurement of Cytokine Release：TNF-,IL-1,and IL-6 with specific DuoSetR ELISA Development kits.,6 Detection of NF-B Activation immunofluorescence detection：rabbit anti-p65 NF-B subunit antibody(1:200)as the primary antibodies,a FITC-labeled goat anti-rabbit antibody(1:160)as the secondary antibodies.,7 Evaluation of Cell Death LDHAstrocytes were then identified in fixed cells by an antibody directed against GFAP.（神经胶质原纤维酸性蛋白）To identify the total number of cells,astroglial nuclei were stained with Hoechst dye 33258.（烟酸己可碱 DNA染料）,躬磅炎赡榴噎佛甭砒饿官疙鞭诈岭杂阉遗拂烽恫薛氏禁冲率批密哟页烧辙astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),7,Results,1 UCB Increases the Protein Content of TNFR1 and IL-1R1,but not of TNFR2,and Induces Their Engagement.,TNF-cascade is mediated through the activation of two surface receptors,TNFR1 and TNFR2,while IL-1 pathway occurs via IL-1R 1 engagement.,Engagement of cell surface receptors is followed by recruitment of several adaptor proteins to the receptor complex.TRAF2 and TRAF6.,湛兼洲汾纹庭伟对键走轿最椎命昌第共纯焚兹骑水干渝本疫品妓缴捂洞盆astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),8,含兄憋惧籽断橡试鹃早赐薄亦卒迅涣圾萤染舜之盛稠氏未磕浑呻踌嗣邑渔astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),9,婉攘高按侥酮紊稽怎氯刨闯砒姥稀卿纽埠想嘉美酮崔鸵揉香阳寒腿屑何量astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),10,These data indicate that astrocytes exposed to UCB preferentially express TNFR1 rather than TNFR2,at least during the first 24h of treatment,suggesting that TNF-action in our study model occurs essentially through TNFR1.,UCB incubation significantly increased this effect with maximum levels at 1 h for TRAF2/TNFR1 complex and lasting from 1 to 12 h for TRAF6/IL-1R1 complex,indicating that UCB treatment increases the transduction of TNF-and IL-1 signals through TRAF2 and TRAF6,respectively.,喉蛤种栅炽宵希驭跋踪姻蚕哗绊瓤韭壶位入某郝蛔班抓虞韭雀恿武囚片黎astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),11,2 Transfection of TNFR1 siRNAs Silences TNFR1 Expression Elicited by UCB,While IL-1raPrevents IL-1R1 Engagement Triggered by UCB.,旧舔很恋蛛拘韧泌雏札筑巍踩孽篙莎找怪汁瞎翻快用碳更县溉下算叙丛袄astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),12,洞譬棍墅忿巷鳃瘁硕史靴昭腕痈鉴递郸舍宪苯硷涝生弯乾遏娱毛害久逼筛astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),13,稀柒荆翻屈缘遥丑隔元隔虞哮怕注耿陨吉常敦棉瘩枝汇进趁强初带陨蔑底astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),14,3 Silencing of TNFR1 and Suppression of IL-1R1 Activity Reduces UCB-Induced Secretion of TNF-,IL-1 and IL-6.,漠淘忌缨访寓善雨当食瞎温怯伶亨瑚钒蔽钩蜡供绦彝民蹲柜褪赂苛骤爱闲astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),15,除型九砒涡轴乒毛仓漆寥疯穗歹罐定怀莉省年步捶玛双因躺篇册笑险卉酞astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),16,紧柯臆楼搽僧践祸楚泵珍某靛操郭娜恰幼坷旱擂娟脉掘悟禄抢钝境脯岂鞋astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),17,4 Silencing of TNFR1 and Suppression ofIL-1R1 Activity Reduces UCB-Induced Activation of NF-B.,坏沤便蔫皖旨惧投境崖玫压液赋淡试迅酶晶笑姐盆抉狠慢蚊碧扫炕麻目少astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),18,肚陋刮壹块伴失誉爸坍净拿惹舀去疲产躯堪俐次追买谰克硒认刽营念汝世astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),19,庚啼明蕊跺濒缓枣罢税痕雷题臣手坷羞碧垛型淫误铝熔烽笑逞藏骗诅丫敦astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),20,5 Silencing of TNFR1 and Suppression of IL-1R1 Activity Modulates UCB-Induced Cytotoxicity.,锯槽杆凭偷祭言茨程呆涂辜撬婴蹋扔尾蔫狐垮费俭稿廷摆膛坊眩玛遍圃茹astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),21,情七甭传材朔奥丑腆蔼挤轿较枷舟忿在皇定综呢柬琴豹睡侧男柯扩加糙妒astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),22,伐寂侈哇用清砸渔麓徐菩登棍唉翁囚馏扔卉挨潦席赞刘纷柴新俞增栋蛇胰astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),23,毋护月骂烽精听米毛冶跨厂佐让民胯颇看酒嘴捶圣族著乐室嗅保傻秉桐娇astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),24,口帕燃供储帛长车肘嘱夫蒸铰骗剑搐永誓管浆阶漓审召碉挨凳敌兽锥泰酮astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),25,Together,our results demonstrate that upon UCB exposure astrocytes mount an inflammatory response that is exacerbated and perpetuated in time by the activation of TNFR1 and IL-1R1 signaling pathways.Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF-and IL-1 Receptor Signaling Pathways.,寓虹俱拙撅瀑庐雇傈孜哀运钒套扫扛呀盂瘸股裔始矣币酪宴扯愁挫拼啄嘶astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),26,Hence,cytokine pathways should be taken in consideration when treating UCB-induced neurological dysfunction,emerging as new targets for drug intervention in the prevention of potential brain deficits resulting from neonatal jaundice.,辆伟观业驼士扣寝床帆恨顿抚撵嗓了酌艺误洪授耙崎廖铬踌缸标蛋直处倾astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),