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1.
目的研究H2S对缺血再灌注损伤后神经元存活信号转导通路ERK1/2/P^90RSK的影响。方法将培养7d的海马神经元随机分为5组:正常培养组(C组)、缺血再灌注组(I/R组)、缺血再灌注+NaHS组fNaHS组)、缺血再灌注+NaHS+U-0126(ERK抑制剂1组(U组)、缺血再灌注+NaHS+Rapamycin(RS6K抑制剂)组(R组)。C组神经元按正常培养方法培养。NaHS组神经元在神经元进行缺血再灌注时加入NaHS使其终浓度为150μmol/L。U组和R组在加入150μmol/LNaHS的同时分别加入U-012610μmol/L或Rapamycin 10nmol/L.各组行细胞存活力、神经元凋亡、cAMP、磷酸化ERK1/2(PERK1/2)和磷酸化P^90RSK(PP^90RSK]蛋白表达的检测。结果NaHS显著增加了cAMP的浓度(与FR组比较,P〈0.01)、PERK1/2蛋白(与FR组比较,P〈0.05)和PP^90rks蛋白(与I/R组比较,P〈0.05)表达,同时增加了神经元存活率(与I/R组比较,P〈0.05)、降低了神经元凋亡率(与隙组比较,P〈0.05);U-0126抑制了PERK1/2蛋白(与NaHS组比较,P〈0.05)和PP^90RSK蛋白(与NaHS组比较,P〈0.05)表达同时使神经元存活率降低(与NaHS组比较,P〈0.05)、神经元凋亡率升高(与NaHS组比较,P〈0.05);Rapamycin抑制了PP^90RSK蛋白(与NaHS组比较,P〈0.05)表达同时使神经元存活率降低(与NaHS组比较,P〈0.05)、神经元凋亡率升高(与NaHS组比较,P〈0.051而不影响PERK1/2的表达(与NaHS组比较,P〉0.05)。结论H2S通过cAMP激活了ERK1/2/P^90RKS信号通路,在海马神经元缺血再灌注时抑制了神经元的凋亡,保护了神经元。  相似文献   

2.
下调Homer—1 b/c基因对机械性损伤神经元存活率的影响   总被引:1,自引:1,他引:0  
目的既往研究表明,Homer-1b/c在神经元损伤后恒定表达,但下调Homer—1b/c能否对损伤的神经元具有保护作用尚不清楚,这也是本实验中待研究的问题。方法采用RNA干涉(RNAi)技术,抑制神经元Homer-1b/c基因及蛋白表达。通过Westernblot法分析神经元转染小干扰RNA(siRNA)后Homer—1b/c基因抑制效果。建立神经元机械性损伤模型,通过细胞存活率、乳酸脱氢酶(LDH)活性测定,研究下调Homer-1b/c基因对神经元损伤的保护作用。结果siRNA转染神经元后36h,Homer-1b/cmRNA和蛋白质表达明显被抑制。siRNA转染组神经元损伤后细胞存活率明显比阴性对照组、空载体组高(P〈0.05)。阴性对照组、空载体组和siRNA转染组神经元损伤前培养液LDH性无统计学差异(P〉0.05),机械性损伤后24h,与阴性对照组及空载体组比较,siRNA转染组LDH活性明显降低(P〈0.05)。结论Homer—1b/csiRNA转染神经元效率较高,基因抑制效果显著。降低Homer-1b/c蛋白表达能够减少机械性损伤后继发性神经元损害,对神经元具有一定的保护作用。  相似文献   

3.
目的探讨体外培养鼠骨髓间充质干细胞对谷氨酸诱导大脑皮质神经元凋亡的影响。方法分离、培养、传代Wistar大鼠骨髓间充质干细胞,细胞融合达90%时更换培养基,继续培养24h,收集细胞培养液即为骨髓间充质干细胞条件培养基;培养新生Wistar大鼠大脑皮质神经元,第8d随机分为对照组、谷氨酸损伤组和骨髓间充质干细胞条件培养基处理组。采用台盼蓝染色计算神经元存活率,同时用流式细胞仪和透射电镜技术检测各组神经元凋亡情况。结果谷氨酸(0.8mmol/L)可诱导细胞凋亡,骨髓间充质干细胞条件培养基处理组较谷氨酸损伤组细胞成活率明显升高(P〈0.001);流式细胞仪检测见骨髓间充质干细胞条件培养基处理组细胞凋亡率明显降低(P〈0.001);而电镜检测发现对照组无明显的凋亡细胞,谷氦酸组有典型凋亡神经元,骨髓间充质干细胞条件培养基处理组凋亡细胞数明显减少。结论骨髓间充质干细胞条件培养基对谷氨酸神经元毒性具有拮抗作用。  相似文献   

4.
目的探讨大黄素甲醚对PC12细胞缺氧损伤的影响。方法体外培养PC12细胞,缺氧处理后给予小同溶浓度大黄素甲醚下预;四甲基偶氮唑盐(MTT)法检测PCI2细胞增殖活性的变化,油镜观察PC12细胞核形态变化,测定上清液中超氧化物歧化酶(SOD)含量,PCR分析丝裂原活化蛋白激酶p38(p38MAPK)和半胱氨酸蛋白酶-3(Caspase-3)的表达。结果缺氧24h后,大黄素甲醚干预后细胞核形态较清楚,偶有肿胀皱缩,少见颗粒样物质形成;大黄素甲醚干预后细胞活性明显增加(P〈0.05);大黄素甲醚显著增加上清液中SOD含量(P〈0.05)。缺氧12h后PCR结果分析显示,大黄素甲醚显著降低p38MAPK和Caspase-3mRNA表达量(P〈0.01)。结论大黄素甲醚能增强缺氧所致神经元抗损伤能力,对神经元起保护作用。  相似文献   

5.
目的探讨胶质瘤细胞异柠檬酸脱氢酶1(IDHl)基因突变对替莫唑胺(TMZ)细胞毒性的影响。方法将培养的U87胶质瘤细胞分组转染含空载体(空载体组)、野生型IDHl基因(野生组)和R132H突变型IDHl基因(突变组;IDHl第132位的精氨酸被组氨酸所取代)质粒,利用液相色谱串联质谱法检测细胞上清液中2-羟基戊二酸(2-HG)水平、噻唑蓝法检测细胞增殖活性、实时荧光定量PCR法和免疫印迹法检测细胞caspase3mRNA和蛋白质表达;将含三种不同质粒的U87细胞接种大鼠建立荷瘤大鼠模型并观察大鼠经TMZ治疗后60d存活率。结果与空载体组和野生组比较,突变组细胞上清液中2-HG水平、细胞caspase3mRNA和蛋白质表达水平均显著升高(P〈0.05),而细胞增殖活性显著下降(P〈0.05);TMZ治疗后60d,突变组大鼠存活率显著升高(P〈O.05);空载体组和野生组之间均无统计学差异(P〈0.05)。结论IDHlR132H基因能够增加TMZ对神经胶质瘤细胞的细胞毒性作用并提高其治疗荷瘤大鼠的存活率。  相似文献   

6.
目的 探讨通心络对体外培养SH-SY5Y细胞Aβ25-35损伤的保护作用。方法采用细胞培养法,以神经母细胞瘤SH~SY5Y细胞系为材料制备Aβ25-35损伤的离体细胞损伤模型。采用细胞形态学观察细胞形态及以MTT法测定细胞存活率。结果 不同浓度通心络作用不同时间其MTT代谢率均高于损伤对照组(P〈0.01),高、中浓度通心络治疗组均高于低浓度治疗组(P〈0.05),而高、中浓度治疗组间差异无统计学意义;相同浓度通心络作用不同时间其MTT代谢率相比较,36、24h组优于12h组(P〈0.05),而24h与36h组间比较差异无统计学意义。结论 通心络可促进神经细胞生存,具有神经细胞保护作用。  相似文献   

7.
刺五加多糖对H2O2损伤的海马神经元表达Fas和Fasl的影响   总被引:1,自引:0,他引:1  
目的探讨刺五加多糖(ASPS)对氧化应激损伤的海马神经元表达Fas和Fasl的影响,进一步研究ASPS抑制海马神经元凋亡的机制。方法从新生大鼠脑分离海马,其神经元经原代培养后分为阴性对照组、损伤模型组(1mmol/LH2O2)和ASPS干预组(该组按ASPS的干预剂量的不同又分为3个亚组,即终浓度为10、5、2.5μg/ml的3个亚组)。用H2O2建立海马神经元应激损伤模型(阴性对照组除外)。倒置显微镜下观察各组细胞形态学变化;免疫组化法检测其Fas和Fasl蛋白的表达,TUNEL法检测海马神经元凋亡,并计算各组的凋亡率。结果与阴性对照组比较,模型组细胞损伤严重,并高表达Fas和Fasl蛋白(P〈0.05),神经元凋亡率明显升高(P〈0.01)。与损伤模型组相比,各药物干预组受损伤海马神经元Fas和Fasl蛋白的表达水平和凋亡率均明显降低(P〈0.05),并表现一定的浓度依赖性。结论 ASPS能减少受损海马神经元细胞中Fas和Fasl蛋白的表达,抑制细胞的凋亡,对受损的海马神经元有明显的保护作用。  相似文献   

8.
脂多糖预处理改善脂多糖诱导的中脑脑片神经元损伤   总被引:1,自引:0,他引:1  
丁晔  李良 《神经科学通报》2008,24(4):209-218
目的探讨脂多糖(lipopolysaccharide,LPS)预处理对脂多糖所致中脑脑片多巴胺能神经元炎性损伤的影响及其可能的机制。方法建立大鼠中脑脑片体外培养体系,于体外培养14d后以不同剂量脂多糖(0、1、3、6及10ng/mL)预处理24h,然后用大剂量脂多糖(100ng/mL)作用72h,观察脂多糖预处理对脑片乳酸脱氢酶(lactic acid dehydrogenase,LDH)活性的影响。通过免疫组化检测酪氨酸羟化酶(Tyrosine hydroxylase,TH)和OX-42的阳性细胞数变化,应用酶联免疫吸附法测定培养液上清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。结果100ng/mL多糖作用72h后引起脑片TH阳性细胞数从对照组的191±12减少到46±4,LDH活性明显升高(P〈0.01),小胶质细胞大量激活,TNF-α水平显著增高(P〈0.01)。脂多糖预处理能减少神经细胞的丢失(3ng/mL和6ng/mL的LPS处理后TH阳性细胞数分别为126±12和180±13),降低脑片LDH活性(P〈0.05),并有效地抑制小胶质细胞的激活,明显减少TNF-α的生成(P〈0.05)。结论小剂量脂多糖预处理可改善脂多糖对大鼠中脑脑片多巴胺能神经元的损伤,其作用机制可能是通过抑制小胶质细胞的激活,减少TNF-α的释放,减轻炎症反应对神经元的损伤。这种保护作用可为帕金森氏病的治疗提供新思路。  相似文献   

9.
目的探讨重组人促红细胞生成素(r—HuEPO)对外伤性脑损伤大鼠凋亡基因表达的影响。方法将48只健康成年雄性SD大鼠随机分成4组:r-HuEPO1000U/kg、3000U/kg、5000U/kg治疗组和生理盐水对照组。采用改良的Feeney氏法制作大鼠自由落体脑创伤模型,r—HuEPO干预。一周后麻醉取脑冻存:免疫组织化学法测定脑组织中NF—κB、C-myc和Fas/Fasl的阳性细胞及凋亡细胞数。结果与对照组相比,各组中NF—KB、Fas和Fasl阳性细胞及凋亡细胞数均有显著减少(P〈0.05),尤其是5000U/kgr—HuEPO组减少最显著(P〈0.01)。结论r—HuEPO可抑制创伤性脑损伤大鼠NF—KB、Fas/Fasl的促凋亡作用,减轻迟发性神经元损伤,从而起到神经保护的作用。  相似文献   

10.
目的观察西酞普兰和氟西汀两种药物对PC12细胞活力及酪氨酸羟化酶(TH)和磷酸化细胞外信号调节激酶(pERK1/2)表达的影响。方法以NGF诱导后的PC12细胞作为细胞模型,给予5,10,20,50gm不同剂量西酞普兰和氟西汀,分别进行直接作用和保护作用处理24或48h(直接作用为直接给予不同剂量齐拉西酮,保护作用为直接作用后再进行12h的去血清损伤)。采用细胞计数试剂盒-8(CCK-8)检测细胞活性,免疫组织化学检测TH和pERK1/2的表达水平的变化。结果分别处理24h后,两种药物在剂量为20μm时均可促进PC12细胞的活性(与对照组相比较,P〈0.01),而且相同浓度的两种药物对细胞活力的作用没有统计学差异(P〉0.05)。药物作用48h后,西酞普兰10μm组对PC12细胞活力具有保护作用(与对照组相比较,P〈0.05),西酞普兰20μm组对PC12细胞活力的促进作用和保护作用均高于氟西汀20μm组(P〈0.05),而且氟西汀在作用48h后对细胞表现出毒性作用(与对照组相比较P〈0.01);PC12细胞TH和pERK1/2的表达随着药物浓度5μm到20μm逐渐升高,但是在药物浓度为50μm时表达下降,其中氟西汀50μm时TH和pERK1/2的表达低于对照组(P〈0.01);西酞普兰20μm组TH和pERK1/2的表达均高于氟西汀20μm组(P〈0.05)。结论中剂量的西酞普兰和氟西汀两种药物对PC12细胞活力都有促进作用,都可促进TH的表达,而且这种作用可能是通过ERK途径产生的;西酞普兰对PC12细胞的保护作用优于氟西汀,而且高剂量的氟西汀表现出细胞毒性作用。  相似文献   

11.
We have previously shown that tissue plasminogen activator (tPA) participates in the neurotoxicity of microglial conditioned medium (MgCM). Killing of hippocampal neurons by MgCM was prevented by both plasminogen activator inhibitor-1 (PAI-1) and anti-tPA antibody. An N-methyl-D-aspartate (NMDA) receptor blocker protected neurons from MgCM, suggesting that this subtype of glutamate receptor is involved. Whereas glutamate receptor-mediated events are important in cerebral ischemia and tPA has previously been shown to enhance excitotoxicity in hippocampus, we hypothesized that tPA would exaggerate oxygen glucose deprivation (OGD) injury in cultures of hippocampal neurons. Dissociated rat hippocampal cells were grown under conditions designed to optimize neuronal growth while minimizing glial replication. At 7--10 days, cultures were subjected to OGD for 2.5 hr. Recombinant human tPA (1,000 IU) was added immediately after OGD. Viability was assessed 24 hr later. Viable, apoptotic, and necrotic cells were classified and quantified based on staining patterns of acridine orange and ethidium bromide under fluorescence microscopy. tPA alone did not alter neuronal integrity. OGD produced significant neuronal death (viability reduced by 45%, P < 0.001). tPA completely protected OGD-exposed cultures. Potential mechanisms of tPA protection were explored. Whereas tPA antibody abolished the protective effect of tPA, its proteolytic inhibitor PAI-1 did not alter the effect. The effect of tPA was tested in separate free radical and excitatory amino acid insults. It did not protect neurons from hydrogen peroxide (1 microM), S-nitro-acetylpenicillamine (10 microM), glutamate (50 microM), or NMDA (10 microM) damage but significantly attenuated injury caused by 250 microM kainate. We conclude that tPA is capable of protecting hippocampal neurons from OGD by a nonproteolytic action. The mechanism of protection was not defined, although attenuation of AMPA/kainate glutamate receptors may play a role.  相似文献   

12.
Effect of tissue-type plasminogen activator (tPA) on oxygen-glucose deprivation (OGD) was studied in cultured cortical neurons prepared from tPA gene knockout (tPA-KO) and wild-type (Wt) mice. Three hours of OGD induced 45% and 23% of neuronal death in Wt and tPA-KO mice, respectively. Neuronal death in tPA-KO mice was increased to 42% by additional tPA. Six hours of OGD induced 80% and 40% of neuronal death in Wt and tPA-KO mice, respectively, whereas the addition of tPA increased to 62% in tPA-KO mice. These results suggest that tPA is directly involved in the process of neuronal death induced by ischemia-mimic stress without involving vascular or circulatory components.  相似文献   

13.
目的:研究人参皂甙(Ginsenosides,Gs)的三种单体成分GSRb_1、GSRb_3、GSRg_1对培养小鼠皮层神经细胞缺血损伤的保护作用。方法:①将离体培养的ICR小鼠胎鼠神经细胞缺血培养以模仿缺血对中枢神经元的损伤,观察不同缺血时间神经元活性变化。②利用MTT比色法观察不同终浓度(0、20、40、60、80、100μmol/L)的GS Rb_1、Rb_3、Rg_1对缺血神经细胞的作用。③检测细胞外液LDH释放量以观察60μmol/L浓度的GSRb_1、Rb_3、Rg_1对缺血神经细胞存活的影响。结果:①培养的小鼠胎鼠皮层神经元随着缺血时间的延长,神经元活性逐渐降低,缺血时间越长,神经元活性下降越多、细胞受损越明显、死亡率增加。②在所研究GS浓度范围(20~60μmol/L)中,对神经细胞的保护作用呈现浓度依赖性,以60μmol/L终浓度的GS单体能明显提高缺血神经元的活性和生存能力,减轻细胞的形态学损伤,且这种保护作用以GSRb_2最明显,随着浓度的再进一步升高,保护作用减弱,100μmol/L的GS单体对缺血神经元无保护作用。结论:适宜浓度人参皂甙单体对缺血培养的小鼠胎鼠皮层神经细胞具保护作用。  相似文献   

14.
The ability to sense and adapt to hypoxic conditions plays a pivotal role in neuronal survival. Hypoxia induces the release of tissue-type plasminogen activator (tPA) from cerebral cortical neurons. We found that the release of neuronal tPA or treatment with recombinant tPA promotes cell survival in cerebral cortical neurons previously exposed to hypoxic conditions in vitro or experimental cerebral ischemia in vivo. Our studies using liquid chromatography and tandem mass spectrometry revealed that tPA activates the mammalian target of rapamycin (mTOR) pathway, which adapts cellular processes to the availability of energy and metabolic resources. We found that mTOR activation leads to accumulation of the hypoxia-inducible factor-1α (HIF-1α) and induction and recruitment to the cell membrane of the HIF-1α-regulated neuronal transporter of glucose GLUT3. Accordingly, in vivo positron emission tomography studies with 18-fluorodeoxyglucose in mice overexpressing tPA in neurons show that neuronal tPA induces the uptake of glucose in the ischemic brain and that this effect is associated with a decrease in the volume of the ischemic lesion and improved neurological outcome following the induction of ischemic stroke. Our data indicate that tPA activates a cell signaling pathway that allows neurons to sense and adapt to oxygen and glucose deprivation.  相似文献   

15.
In vitro cultures of primary cortical neurons are widely used to investigate neuronal function. However, it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons, and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions. Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods, our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice. We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons, and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey. Although cultured monkey cortical neurons developed slowly in vitro, they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin(HTT), the Huntington's disease protein. A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites. Therefore, compared with the primary cortical neurons of mice, cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity, such as electrophysiological activity. Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease, and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration. All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China(IACUC Approval No. 20200512-04) on May 12, 2020. All monkey experiments were approved by the IACUC protocol(IACUC Approval No. LDACU 20190820-01) on August 23, 2019 for animal management and use.  相似文献   

16.
Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal development and synaptic plasticity. Although the physiological effects of BDNF have been examined in detail, target proteins which mediate its actions remain largely unknown. Here, we report that BDNF stimulates the expression of tissue-type plasminogen activator (tPA) in primary cultures of cortical neurons in a time- and concentration-dependent manner. Among the other members of the neurotrophin family, neurotrophin-4 (NT-4) and to a lesser extent neurotrophin-3 (NT-3) also increased tPA mRNA expression, while nerve growth factor (NGF) was devoid of any effect. Induction of tPA expression by BDNF is accompanied by an increase in the proteolytic activity of tPA associated with cortical neurons and a release of tPA into the extracellular space. Release of tPA induced by BDNF depends on extracellular Ca2+ since it is markedly reduced in the presence of ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Up-regulation of tPA expression by BDNF is followed by the induction of plasminogen activator inhibitor 2 (PAI-2), an inhibitor of tPA. Together these results suggest that activation of tPA by BDNF may contribute to structural changes associated with neuronal development or synaptic plasticity.  相似文献   

17.
目的 探讨γ-Adducin蛋白是否可减轻Aβ1-42纤维诱导的神经元突起损伤。方法 实验一:培养原代神经元,用10 μmol/L Aβ1-42纤维处理神经元24 h,采用神经元特异性标记物MAP2检测神经元突起损伤,用TUNEL染色检测神经元凋亡; 实验二:分别用对照病毒和表达γ-Adducin的腺相关病毒感染神经元,蛋白表达5 d后再分别加入Aβ1-42纤维处理,用免疫荧光染色法观察γ-Adducin蛋白过表达对Aβ1-42纤维引起的神经元突起损伤的影响,用Dil染色检测神经元树突棘密度的变化。结果 Aβ1-42纤维对神经元的突起具有损伤作用,并且引起神经元凋亡; 过表达γ-Adducin蛋白对Aβ1-42纤维引起的神经元突起和树突棘损伤均有保护作用。结论 γ-Adducin蛋白可减轻Aβ1-42纤维导致的神经元突起损伤,它可能是1个新的阿尔茨海默病治疗靶点。  相似文献   

18.
Tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which are both used for thrombolytic treatment of acute ischemic stroke, are serine proteases that convert plasminogen to active plasmin. Although recent experimental evidences have raised controversy about the neurotoxic versus neuroprotective roles of tPA in acute brain injury, uPA remains unexplored in this context. In this study, we evaluated the effect of uPA on neuronal death in the hippocampus of mice after kainate-induced seizures. In the normal brain, uPA was localized to both nuclei and cytosol of neurons. Following severe kainate-induced seizures, uPA completely disappeared in degenerating neurons, whereas uPA-expressing astrocytes substantially increased, suggesting reactive astrogliosis. uPA-knockout mice were more vulnerable to kainate-induced neuronal death than wild-type mice. Consistent with this, inhibition of uPA by intracerebral injection of the uPA inhibitor UK122 increased the level of neuronal death. In contrast, prior administration of recombinant uPA significantly attenuated neuronal death. Collectively, these results indicate that uPA renders neurons resistant to kainate-induced excitotoxicity. Moreover, recombinant uPA suppressed cell death in primary cultures of hippocampal neurons exposed to H2O2, zinc, or various excitotoxins, suggesting that uPA protects against neuronal injuries mediated by the glutamate receptor, or by oxidation- or zinc-induced death signaling pathways. Considering that tPA may facilitate neurodegeneration in acute brain injury, we suggest that uPA, as a neuroprotectant, might be beneficial for the treatment of acute brain injuries such as ischemic stroke.  相似文献   

19.
20.
Newborn rat dorsal root ganglia release two different plasminogen activators (PAs): the urokinase (UK) and the tissue (tPA) type. The former is secreted by neurons while the latter is secreted by Schwann cells. tPA release by Schwann cells is modulated by choleratoxin, a known mitogen for these cells. UK but not tPA stimulates in a dose-dependent fashion the proliferation of Schwann cells. This effect is observed in the absence of plasminogen, suggesting that the substrate for PAs in the developing nervous system is not plasminogen. Since UK is secreted by neurons, our data suggest a new mechanism for neuronal control of Schwann cell proliferation.  相似文献   

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