CN118421746A - 细胞表面粘附分子cd22的用途 - Google Patents
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Abstract
本发明公开了细胞表面粘附分子CD22的用途,具体为细胞表面粘附分子CD22在筛选用于镇痛的非成瘾性药物中的应用。本发明通过遗传学手段敲除CD22,发现敲除CD22的小鼠机械性痛觉过敏;通过鞘内注射靶向CD22的siRNA,发现抑制CD22表达会导致小鼠机械性痛觉过敏;通过尾静脉注射靶向CD22抗体,发现抑制CD22也会导致小鼠机械性痛觉过敏;通过尾静脉注射CD22过表达病毒,再进行SNL手术,可减轻SNL引起的神经病理性疼痛。本发明有助于更好地理解细胞表面粘附分子在疼痛发展中的重要作用,为临床镇痛药物研制以及预防和治疗提供了新的可能性。
Description
技术领域
本发明属于生物医药技术领域,具体涉及细胞表面粘附分子CD22的用途。
背景技术
疼痛作为躯体感觉的一种,可以在机体受到伤害时起到警示作用,引起机体一系列防御性保护反应避免受到进一步伤害。但当疼痛失去了正常保护功能,数月或数年的持续存在,就变成了危害身心健康的病理性疼痛(慢性疼痛)。据世界卫生组织报告,慢性疼痛是最常见也是疾病负担最重的疾患。有资料统计,大约30%的成年人患有慢性疼痛。慢性疼痛是临床上常见症状,如炎症、手术、神经创伤、病毒感染(如带状疱疹)、癌症、代谢性疾病(如糖尿病)、免疫性疾病(如多发性硬化、艾滋病)等都可以引起慢性疼痛。患者会产生对伤害性刺激敏感性增强和反应阈值降低的痛觉过敏,以及非痛刺激(如触摸)引起的触诱发痛表现,严重影响患者的身心健康和生活质量。
另外,镇痛也是临床难点,现有的镇痛药物多是阿片类镇痛药、非阿片类辅助镇痛药(非甾体类抗炎药、抗抑郁药、抗惊厥药)。阿片类镇痛药如吗啡、美沙酮、芬太尼、羟考酮等,常伴随有呼吸抑制和循环抑制副作用,常见为便秘、头晕、恶心、呕吐,瘙痒、口干等症状。非甾体类抗炎药常用环氧化酶-2抑制剂塞来昔布,其伴随有消化道副作用,包括腹痛、消化不良和恶心等症状。抗惊厥药、抗精神病药物为主的辅助性镇痛药,如卡马西平、可乐定、氯胺酮等,都有不同种类的不良反应,如疲乏、头疼、血小板减少、皮肤瘙痒、充血性皮疹、失眠、便秘等症状。因此进一步研究慢性疼痛的发生发展机制,从新的角度寻找有效的药物作用靶点具有重要的学术价值和现实意义。
CD22,又被称为Siglec-2,属于唾液酸结合免疫球蛋白型凝集素家族(Siglecs)成员,是一种I型跨膜蛋白,可特异性结合含唾液酸(Sia)的聚糖,并通过其免疫受体酪氨酸抑制性基序(ITIM)抑制B细胞受体(BCR)信号传导,发挥维持体液免疫稳态的作用,普遍存在于正常B细胞和B细胞恶性肿瘤中。CD22主要表达于成熟B细胞,是具有调控B细胞激活作用的细胞表面粘附分子,有助于B细胞对抗原反应敏感性的控制。目前认为CD22表达在B细胞中具有特异性,并且在小鼠和人类中都受到发育调控,CD22可在原B细胞和前B细胞中表达,并逐渐转移到细胞表面,但是表达水平低;在IgM+和IgD+的成熟B细胞中高表达,在滤泡性B细胞、套细胞、边缘区B细胞中也高表达,但最终在CD27+记忆B细胞,尤其是浆细胞中,表达下调。此外,最近的研究发现,在小鼠肠酸性粒细胞也观察到了CD22表达,这是一种新型的表达模式,表明CD22可能具有嗜酸性粒细胞调节功能。
CD22具有多个配体,其胞外域能与多种细胞表面糖蛋白上存在的α2-6唾液酸残基特异性结合。CD22可以与含唾液酸的细胞相互作用,包括T细胞、B细胞、中性粒细胞、单核细胞和红细胞。其中能与CD22胞外配体结合域相互作用的B细胞表面唾液酸配体为顺式配体,其他细胞表面配体则为反式配体。CD22通过顺式作用与B细胞表面的配体结合,通过反式作用与其他细胞表面配体、可溶性糖蛋白或细胞连接的抗原。
但目前尚未有文献报道CD22与病理性疼痛是否存在相关性的研究。
发明内容
针对现有技术的不足,本发明提供细胞表面粘附分子CD22的用途,为镇痛药物研制以及预防和治疗疼痛提供了新的可能性。
本发明是通过以下技术方案实现的:
细胞表面粘附分子CD22在筛选用于镇痛的非成瘾性药物中的应用,所述CD22的全长序列的NCBI编号为NM_001043317.2。
优选地,所述药物为促进CD22的表达或功能的试剂。
优选地,所述试剂为CD22的过表达载体或CD22蛋白。
优选地,所述CD22的过表达载体为包含CD22全长序列的慢病毒或腺相关病毒。
优选地,所述镇痛的疼痛类型为神经病理性疼痛。
一种治疗神经病理性疼痛的药物组合物,包括上述的试剂,以及药学上可接受的载体。
本发明的有益效果如下:
本发明利用qRT-PCR、Western blot、免疫组化和荧光原位杂交,发现CD22在小鼠DRG神经元和坐骨神经中表达;通过遗传学手段敲除CD22,发现敲除CD22的小鼠机械性痛觉过敏;通过鞘内注射靶向CD22的siRNA,发现抑制CD22表达会导致小鼠机械性痛觉过敏;通过尾静脉注射靶向CD22抗体,发现抑制CD22也会导致小鼠机械性痛觉过敏;通过尾静脉注射CD22过表达病毒,再进行脊神经结扎(Spinal Nerve Ligation,SNL)手术,可减轻SNL引起的神经病理性疼痛。本发明有助于更好地理解细胞表面粘附分子在疼痛发展中的重要作用,为临床镇痛药物研制以及预防和治疗提供了新的可能性。
附图说明
图1为实施例1中CD22在DRG神经元中表达:A为免疫荧光FISH检测DRG神经元中CD22 mRNA水平;B为免疫荧光双标检测DRG神经元CD22蛋白表达水平;C为Western Blot检测DRG神经元及坐骨神经中CD22的表达;D为免疫荧光双标检测CD22在坐骨神经中DRG神经元轴突上的表达;
图2为实施例2中敲除CD22的小鼠行为实验结果,***P<0.001,T test,n=8;
图3为实施例3中抑制DRG CD22 mRNA水平的实验结果:A为体外培养DRG神经元,转染三种CD22 siRNA,PCR检测CD22 mRNA水平;B为鞘内注释siRNA,72h后处死小鼠,收集DRG,实时荧光定量PCR检查CD22 mRNA表达;C为正常C57BL/6J小鼠鞘内注射CD22 siRNA,在注射后6h、24h、48h和72h检测机械性痛觉过敏行为;
图4为实施例4中正常小鼠尾静脉注射CD22抗体或等量IgG,在1h、2h、3h、5h和24h检测足底机械性痛觉过敏行为;
图5为实施例5中SNL小鼠模型过表达CD22的实验结果:A为小鼠在静脉注射AAV-PHP.S-CD22或AAV-PHP.S-Negative control,4周后实时荧光定量PCR检测DRG中CD22 mRNA表达,***P<0.001,T test,n=8;B为与AAV-PHP.S-Negative control小鼠相比,AAV-PHP.S-CD22小鼠在SNL后13天机械性痛觉过敏阈值,**P<0.01,T test,n=8。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明。
若无特殊说明,以下实施例中所用的技术手段,均为本领域技术人员所熟知的常规手段,未注明具体条件的实验方法,均为本领域常规方法。
以下实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中所用实验动物为C57BL/6J小鼠,所有动物实验均按照动物护理指南进行,并经江苏省实验动物管理委员会伦理认可,动物实验许可证为20150304-004。
实施例1CD22在DRG神经元和坐骨神经中表达
1、实验方法
(1)RNA提取及实时荧光定量PCR
按照Trizol法抽提总RNA,测定浓度后取1μg总RNA,参考Vazyme公司HiScript IIIRT SuperMix Kit的说明进行逆转录反应。得到的cDNA产物稀释8倍用于实时荧光定量PCR的模板,每个PCR体系加入2μL模板,参考Vazyme公司AceQ实时荧光定量PCR SYBR GreenMaster Mix的说明进行,在NCBI中查找靶基因CD22 ID(NM_001043317.2),选择全部mRNA或CDS序列,采用Primer Premier 5.0软件分别设计正义引物(SEQ ID NO.1:5’-GCCAAGCGTGTGAGACTTTT-3’)和反义引物(SEQ ID NO.2:5’-CCTCAACCCCAGATTCCCAC-3’),并委托生工生物工程(上海)有限公司合成引物序列。按参考Vazyme公司AceQ实时荧光定量PCR SYBR Green Master Mix的说明进行,配制10μL反应体系,置于Bio-Rad CFX实时荧光定量PCR扩增仪上进行反应。采用2-ΔΔCT方法对结果进行计算和统计,以GAPDH作为内参校正PCR模板的拷贝数,消除组间的加样误差。
(2)蛋白质提取及Western Blot
C57BL/6J小鼠用生理盐水经心脏灌注后,取出脊髓腰段(L4、L5)背根神经节和坐骨神经,放入含有蛋白酶抑制剂的组织裂解液中,冰上电动匀浆,经离心后取上清,即得组织蛋白质溶液。BCA蛋白含量检测试剂盒测定样本总蛋白质浓度,加入蛋白上样缓冲液,标定蛋白统一浓度。经沸水煮5min后蛋白质变性,经SDS-聚丙烯酰胺凝胶电泳和湿法转膜后,将膜置入5%脱脂奶粉封闭液中室温封闭2h。封闭结束孵育5%脱脂奶粉稀释的一抗,4℃过夜,经洗膜后孵育二抗,洗膜后使用成像系统成像。图像蛋白条带分析使用ImageJ软件。
(3)免疫荧光染色
C57BL/6J小鼠经4%多聚甲醛灌注固定,取出腰段(L4、L5)背根神经节,经过含30%蔗糖溶液脱水,沉底后行冰冻切片,片厚12μm。0.01M PBS溶液漂洗切片3次,10min/次,1% BSA(含0.5% Triton X-100)室温孵育封闭1.5h,孵育一抗:CD22(rabbit,1:200,Bioss)、神经元标记物TUJ1(mouse,1:800,Millipore),4℃孵育过夜。0.01M PBS溶液漂洗切片3次,10min/次,孵育二抗:Cy3-羊抗兔(1:1000,Jackson)、Alexa Fluor 488-驴抗山羊(1:1000,Jackson),室温孵育2h。0.01M PBS溶液漂洗切片3次,10min/次。裱片后荧光封片剂封片,荧光显微镜下观察拍照。
(4)原位杂交染色(FISH)
C57BL/6J小鼠经4%多聚甲醛灌注固定,取出腰段(L4、L5)背根神经节,经过含30%蔗糖溶液脱水,沉底后行冰冻切片,片厚12μm,将组织切片封装在SUPERFROST PLUS载玻片上。60℃下烤片30min,然后在4℃下将载玻片浸入预冷的4% PFA溶液,孵育15min,进行后固定。切片经50% EtOH、200mL 70%EtOH和400mL 100% EtOH依次脱水,1×抗原修复液修复,RNAscope双氧水及蛋白酶III处理。使用3-plex Mm-荧光阳性对照探针及RNAscope Probe-Mm-CD22荧光探针按照多通道二代荧光试剂盒(货号323100)进行原位杂交以及免疫荧光共染。裱片后荧光封片剂封片,荧光显微镜下观察拍照。
2、实验结果
C57BL/6J小鼠用生理盐水经心脏灌注后,取出脊髓腰段(L4、L5)背根神经节和坐骨神经进行检测,如图1所示。
免疫荧光FISH检测DRG神经元中CD22 mRNA水平如图1中A所示,免疫荧光双标检测DRG神经元CD22蛋白表达水平如图1中B所示,显示DRG神经元中存在CD22 mRNA和蛋白表达;如图1中C所示,Western Blot证实DRG神经元及坐骨神经中存在CD22表达;如图1中D所示,免疫荧光双标实验明确了CD22在坐骨神经中DRG神经元的轴突上表达。
实施例2敲除CD22基因诱发小鼠机械性痛觉过敏
1、实验方法
(1)实验动物:8周龄雄性野生型C57BL/6J小鼠,8周龄雄性CD22-/-小鼠各86只,使用von Frey filament评估小鼠机械性痛觉过敏行为。
(2)机械性痛觉过敏检测方法
行为检测前,小鼠单独放在机械性痛觉测试架上的有机玻璃盒子内适应环境至安静,室温在23±1℃,连续适应3天。按照Dixon等报道的“up and down”方法,使用von Freyfilament检测小鼠左后爪底机械性触诱发痛感受阈值。测量时从0.16g的von Freyfilament开始,刺激小鼠左后爪底,以filament稍弯曲作为完全受力的标准,持续刺激2s,小鼠如出现缩爪、甩爪、舔爪等现象记为疼痛,再使用小一级克数的filament刺激,如不痛则使用高一级克数的filament刺激,刺激间隔为3min,记录6个刺激结果,最后对照阈值表查出缩爪阈值(threshold),单位为“g”。缩爪阈值越低说明机械性痛觉过敏越严重。
2、实验结果
如图2所示,与野生型(WT)小鼠相比,CD22-/-小鼠对于von Frey filament刺激产生的机械性痛觉过敏阈值显著降低,差别具有统计学差异(****P<0.0001)。说明CD22基因敲除诱发小鼠产生了机械性痛觉过敏。
实施例3抑制DRG中CD22表达诱发机械性痛觉过敏
1、实验方法
(1)DRG神经元的分离与培养
准备解剖液2~3mL,放于置于冰上的中皿中;取新生P1小鼠消毒后处死;沿背部打开髓腔,取出DRG,置于冰上培养皿中;添加3mg/mL胶原酶37℃消化30min;0.25%胰酶37℃消化20min;然后加入完全培养基(DMEM+10%FBS)终止消化,成单细胞悬液,经筛网,1000rpm/min离心5min;弃上清,加入15%BSA,重悬沉淀,900rpm/min离心5min;完全培养基重悬细胞,接种至中皿;4~6h后,加入神经元培养基(Neuronbasal Medium+B27+Glutamine+Pen-Strep双抗),并加入Arac以去除非神经元细胞。
(2)设计合成小鼠CD22 siRNA
siRNA设计合成由广州市锐博生物科技有限公司完成:通过美国国立生物技术信息中心(NCBI)数据库中获取小鼠CD22 mRNA的序列全长(NM_001043317.2),跟据RNAi原理,结合设计软件,设计针对小鼠CD22mRNA基因转录本的3个候选siRNA,经BLAST比对检查以保证和其他基因没有同源性,再进行化学合成。3个候选siRNA的靶序列信息如下表1所示。
表1候选siRNA的靶序列信息
候选siRNA编号 | SEQ ID | 靶序列 |
si-mouse-CD22_001(siRNA-1) | 3 | GTCTACTACAGTCCAGAGA |
si-mouse-CD22_002(siRNA-2) | 4 | GCAATGGATGACACCGTTA |
si-mouse-CD22_003(siRNA-3) | 5 | CTTACTCGGTGATACAGAA |
(3)CD22 siRNA干预实验
配制转染试剂,取1.5mL EP管,其中加入Lipofectamine RNAiMAX、miRNA mimics和Opti-MEMTM无血清培养基,置于冰上孵育10~15min;培养的DRG神经元分别用RNAMAX转染不同siRNA,48h后,裂解细胞提取RNA、逆转后采用实时荧光定量PCR检测转染效率。
(4)总RNA提取及实时荧光定量PCR分析
采用Trizol法提取4组DRG神经元总RNA,用核酸蛋白测定仪检测吸光度(A260/280nm),计算RNA浓度及纯度。参照诺唯赞公司第三代高效cDNA一链合成试剂盒对总RNA进行逆转录,产物cDNA可直接用于PCR反应或-80℃保存。在NCBI中查找靶基因CD22 ID(NM_001043317.2),选择全部mRNA或CDS序列,采用Primer Premier 5.0软件分别设计正义引物(SEQ ID NO.1)和反义引物(SEQ ID NO.2),并委托生工生物工程(上海)有限公司合成引物序列。按参考Vazyme公司AceQ实时荧光定量PCR SYBR Green Master Mix的说明进行,配制10μL反应体系,置于Bio-Rad CFX实时荧光定量PCR扩增仪上进行反应。采用2-ΔΔCT方法对结果进行计算和统计,以GAPDH作为内参校正PCR模板的拷贝数,消除组间的加样误差。
(5)鞘内注射药物
此实验从3个候选siRNA中筛选出高效针对小鼠CD22基因的siRNA后,再由广州市锐博生物科技有限公司对此siRNA进行甲氧基修饰,以提高在活体内的稳定性,同时进行胆固醇修饰。修饰后的siRNA不需借助转染载体复合物,直接进入细胞发挥干扰基因表达作用,用于在体鞘内注射后行为学检测。
电推剪剃除小鼠腰背部毛后,经异氟烷吸入麻醉,酒精消毒皮肤,使用30gauge(BD公司)注射针在第四、五腰椎棘突间注射10μL药物到小鼠蛛网膜下隙,以尾巴出现颤动或突然侧向甩动为穿刺成功标志。吸入麻醉停止后,小鼠可以很快清醒,对随后的疼痛行为学检测及其它研究无影响,保证了实验结果的可靠性。
2、实验结果
(1)筛选出高效针对小鼠CD22基因的siRNA
利用体外培养的DRG神经元进行实验,筛选出最有效的干扰CD22表达的siRNA。如图3中A所示,3个候选siRNA均有效果,其中siRNA-3有最显著干扰效果(***P<0.001与siRNA-NC组相比,n=4,单因素方差分析和Bonferroni校正检验)。因此选用此siRNA-3进行甲氧基修饰和胆固醇修饰,使siRNA在体内更稳定且不用借助于转染试剂直接在体注射发挥干扰作用,用于行为学实验。
(2)siRNA抑制CD22 mRNA表达能够增强机械性痛觉过敏的敏感性
鞘内注射针对CD22基因的经甲氧基修饰和胆固醇修饰的siRNA-3,特异性抑制CD22表达,von Frey filament检测小鼠左后爪底机械性触诱发痛感受阈值。
如图3中B所示,鞘内注射siRNA-3-2OMe+5Chol高剂量(5μg)在72h后实时荧光定量PCR检查干扰效果,发现siRNA-3-2OMe+5Chol高剂量(5μg)显著抑制DRG中CD22 mRNA。
如图3中C所示,鞘内注射siRNA-003-2OMe+5Chol(5μg)后,小鼠DRG中的CD22 mRNA水平显著下降(**P<0.01与siRNA-NC-2OMe+5Chol组相比,n=4-5,Student’s t-test)。
以上结果表明抑制CD22 mRNA表达能够增强机械性痛觉过敏的敏感性。
实施例4抑制CD22的功能诱发机械性痛觉过敏
1、实验方法
8周龄雄性C57BL/6J小鼠,尾静脉注射CD22抗体(10μg)或等量IgG,在1h、2h、3h、5h和24h采用von Frey filament检测小鼠左后爪底机械性触诱发痛感受阈值。本实施例中使用的CD22抗体来自于BioXCell(Cy34)公司。该抗体特异靶向CD22的胞外结构域,抑制其与配体结合。
2、实验结果
如图4所示,与注射IgG的小鼠相比,尾静脉注射CD22抗体的小鼠在1h缩爪阈值明显降低,持续到24h,表明CD22抗体诱发小鼠机械性痛觉过敏(*P<0.05,**P<0.01,***P<0.001,与IgG组相比,n=8-10,双因素重复测量方差分析,Bonferroni校正检验进行两两比较)。
以上实验结果说明抑制CD22的功能诱发机械性痛觉过敏。
实施例5过表达CD22抑制SNL小鼠机械性痛觉过敏
1、实验方法
(1)小鼠脊神经结扎(Spinal Nerve Ligation,SNL)神经病理性疼痛模型的建立
8周龄雄性C57BL/6J小鼠腹腔注射复合麻药后,用剃毛器将腰背部毛发剃除,碘酒酒精消毒后,在后正中线左外侧约5mm处做一纵切口,长约1cm,2/3的切口在髂嵴之上。然后切开胸腰筋膜,钝性分离竖脊肌并向外牵拉,在手术显微镜下显露第6腰椎的横突并咬断,用玻璃分针轻柔地分离出L5脊神经,用6-0号的丝线紧紧结扎L5脊神经,然后逐层缝合切口。
(2)尾静脉注射病毒及机械性痛觉过敏检测方法
8周龄雄性C57BL/6J小鼠分为两组,每组8只,使用von Frey filament评估小鼠机械性痛觉过敏行为。行为检测前,小鼠单独放在机械性痛觉测试架上的有机玻璃盒子内适应环境至安静,室温在23±1℃,连续适应3天。按照Dixon等报道的“up and down”方法,使用von Frey filament检测小鼠左后爪底机械性触诱发痛感受阈值。测量时从0.16g的vonFrey filament开始,刺激小鼠左后爪底,以filament稍弯曲作为完全受力的标准,持续刺激2s,小鼠如出现缩爪、甩爪、舔爪等现象记为疼痛,再使用小一级克数的filament刺激,如不痛则使用高一级克数的filament刺激,刺激间隔为3min,记录6个刺激结果,最后对照阈值表查出缩爪阈值(threshold),单位为“g”。缩爪阈值越低说明机械性痛觉过敏越严重。正常小鼠在手术之前测试缩爪阈值,作为基础阈值(Baseline,BL)。
接下来,两组小鼠分别尾静脉注射200μL AAV-PHP.S-CD22病毒(包含CD22全长序列的腺相关病毒,由广州云舟生物科技股份有限公司包装)或对照病毒(AAV-PHP.S-Negative control,由广州云舟生物科技股份有限公司提供),病毒滴度为3×1013。三周后两组小鼠均进行SNL手术,诱发痛觉过敏。在手术后13天,使用von Frey filament评估小鼠机械性痛觉过敏行为,记录缩爪阈值。
2、实验结果
注射病毒的小鼠在4周后处死,收集DRG,实时荧光定量PCR检测CD22mRNA表达,如图5中A所示,与注射对照病毒(AAV-PHP.S-Negative control)的小鼠相比,注射AAV-PHP.S-CD22病毒的小鼠DRG中CD22 mRNA表达明显升高,差别具有统计学差异(***P<0.001)。
如图5中B所示,与注射对照病毒(AAV-PHP.S-Negative control)的SNL小鼠相比,注射AAV-PHP.S-CD22病毒的SNL小鼠对于von Frey filament刺激产生的机械性痛觉过敏阈值显著升高,差别具有统计学差异(**P<0.01)。
以上实验结果说明过表达CD22抑制SNL诱导的机械性痛觉过敏。
有研究报道CD22也是肿瘤治疗的重要靶点,而不论是肿瘤本身还是利用化疗药治疗肿瘤时都可导致痛觉过敏,产生神经病理性疼痛,本发明的实验结果说明,在治疗癌症引起的神经病理性疼痛中,以CD22作为药物靶点可能还会有一箭双雕的效果。
以上实施例仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明的保护范围。
Claims (6)
1.细胞表面粘附分子CD22在筛选用于镇痛的非成瘾性药物中的应用,其特征在于,所述CD22的全长序列的NCBI编号为NM_001043317.2。
2.根据权利要求1所述的应用,其特征在于,所述药物为促进CD22的表达或功能的试剂。
3.根据权利要求2所述的应用,其特征在于,所述试剂为CD22的过表达载体或CD22蛋白。
4.根据权利要求3所述的应用,其特征在于,所述CD22的过表达载体为包含CD22全长序列的慢病毒或腺相关病毒。
5.根据权利要求1所述的应用,其特征在于,所述镇痛的疼痛类型为神经病理性疼痛。
6.一种治疗神经病理性疼痛的药物组合物,其特征在于,包括权利要求2-4任一项所述的试剂,以及药学上可接受的载体。
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