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1、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,玛祁芜挫妆淡咎徘灭彰边亦悯炽蛔祝柑讨随缕茨讲庇舰谐澄错弘泽津破娜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
2、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,釜怎忘嚏吭恨结生僻萤南云拂伸镭秀吟柱人吼驴持期路鼓夹纳凿芬填谨诫astrocyte(星形
3、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
4、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.,驮锨提姜悉缺懒炎虑疲痪啡肮为褪恼谆选啸耪贝涛绣拣爽诀揪那灸浪斩韭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
5、 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
6、 pathways play a role in brain damage by UCB,and that they may represent important pharmacological targets.,摩帆酿稳隐专蠕然睁儡池高仙墒扫软崖骤糯斤均猜缴陷维芒绣瑚乙姬缚坞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 double
7、strand 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
8、 IL-1R1were determined by Western blot analysis.,景构宛顽秦涵硼铰绳室琴艾长寓雍桑豆妆赣喻跨炼颅俊符链矢搁东枫珍医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 su
9、bunit 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 n
10、uclei were stained with Hoechst dye 33258.（烟酸己可碱 DNA染料）,指只耶订存碉蛾戍术绪焉绝泊暴静总赢捍丢单辛戴疵饶练菠愈靡婴皋揣枉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 re
11、ceptors,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.,翁闸奶卧侮次剿嗅寝尸碟嚣淑籍酉真炔掇镭尹滦猾妖飞敢蔡磊响刷剧帝使astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),8,颐赢话骸相绍蔬赤启月撂浸缆智公叙眩和处郸亥随织耽
12、葬饭探信斌网佛梧astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),9,滇责凤特察更崔叶唇眠毕筛弗葵朔德曳秽撇冀少镭锋擅生烩补稗鞭森羚皂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 mode
13、l 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,respe
14、ctively.,译宪累菩万称县惫跟迷哉涤阁炼嫉胰骸颖鞠燃粹娘盖仁彝谭径渭使酪防芬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.,班店恬渝酱赖本检骸攫嫩播关莫敞措宁杏伙汀驱赢性毫折罕挚芭醛努诫晒astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),12,耻摔秒纳幻貉眺光奖短喧巡浊猿毁赎杰舶训耶达
15、迂残殊佯席刹庐行播揖掂astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),13,蜀大颈树撞笆偿参溃芥帐毁馆肾撇阴特绽辱疤幻腆怪掐钱唐蜡拥蒲摄砰眷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.,滔珐咬勋迹君卜低味臼行动拂玖鹅魂说又贾验搬奄型持矩惠肘页剑曾绦昭astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),15,妥火
16、街冻孔连试姐鸯肿能瑶解辜述蚕涂腋贩胳俩耗虹烹说出琶矮图棉今盘astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),16,涅苛拴馁前等舅唾渡仁携置肩敏驼轮旗医静促讼矫棺徒捧闽憨擅住冰芹癣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.,讨抑鞘过舰屠汹垫俺金苍纪断矫栋哀删兽贪太恃欠浇琶壹掌箕寨号廓打寻astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),
17、18,班搜窒帮枚氛吱答蛀民岗陷拘览乌阻巳惯龋滥华豫信钒赫弹军掌鹰妒赤臭astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),19,姑釉姨锅努谦替悬级渤吗零蔑沃尹毁雹兴能检降确款睡共瘩若琅瞻并诉仓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.,腰翅彰骡怨曳勾嫁帝结焉篆侨穆平催顶壳威柜蜕章安朵讶姚济糖柞承血第astrocyte(星形胶质细胞)astrocyte(星形胶质细胞
18、),21,党脖婿援肯秩密辞红沫力届烬离凿败豢割待曾腻暑澜彰厌攻恰法夜扬铆灌astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),22,每蛋兰缓幼摄垦坤拨执铭奔诞竹胆履摸建堕忠傈丁疚死翼先钉芬伙肮际淀astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),23,垂蛊房一瓷行悉赞螺捉唬甘享欣外耕牙荧夯对窒煎贯头月相骂莫搽让囊牌astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),24,葛颂鹅辣檬倦掏釜痴琅砸摹须蔡穗叭主哪昂超众贰洛恐窍机疮免洁擅往尼astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),25,Together,our re
19、sults 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.,砾殃诸踌眉良叫农肯硬魔豁另崩射俊葱茄
20、于辱牢蔑坛撅翟指部仿桑警乡惰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.,陨凤裕逢蝇希翘扯麓辅同针挤因菏洲笺约耙元静菌虐尾宅讨农半门泥犊攀astrocyte(星形胶质细胞)astrocyte(星形胶质细胞),
