CN109207523A - 基于ucp1基因的人类肥胖症斑马鱼模型的建立与应用 - Google Patents
基于ucp1基因的人类肥胖症斑马鱼模型的建立与应用 Download PDFInfo
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Abstract
本发明涉及基于UCP1基因的人类肥胖症斑马鱼模型的建立与应用,具体步骤包括针对斑马鱼ucp1基因编码关键功能结构域的外显子设计CRISPR序列,筛选确认可有效引导Cas9切割目标基因的活性sgRNA,然后运用有活性的sgRNA,制备ucp1基因敲除的初建者,待初建者生长至性成熟,令其自由交配,产生F1代;通过对F1个体的基因型鉴定,筛选fto基因敲除的斑马鱼突变体;通过对ucp1基因纯合突变的分析,确立ucp1的功能缺失有益于斑马鱼仔鱼的脂肪累积的减少,建立基于ucp1基因的人类肥胖症的斑马鱼模型,进而将ucp1作为标识基因应用于筛选与建立治疗人类肥胖症的新药物与新方法。
Description
技术领域
本发明属于生物技术领域,涉及筛选确定可对斑马鱼ucp1基因进行基因组编辑的sgRNA序列、制备斑马鱼ucp1基因靶向突变的优选方案,以及利用斑马鱼ucp1作为标志性基因筛选治疗人类肥胖症药物的斑马鱼模型的应用。
技术背景
肥胖是体内多余的脂肪堆积超过一定程度对人类健康造成困扰的能量代谢失衡的慢性疾病。人们通常用BMI(body mass index,体重指数)来判断个体是否肥胖。因体脂增加使体重超过标准体重20%或体重指数【BMI=体重(Kg)/(身高)2(m2)】大于24者称为肥胖症。肥胖会增加其它疾病的患病几率,尤其是心脏病、II型糖尿病、阻塞性睡眠呼吸窘迫、癌症和骨关节炎等疾病。近几十年来,肥胖症患者在全球范围内以惊人的速度增加。尤其是儿童、青少年肥胖增长日益迅猛(1,2)。在2014年约有600万成年人(13%) 和4200万5岁以下儿童患有肥胖症。肥胖被认为是21世纪最为严重的影响健康的疾病。
肥胖的病因主要分为外因和内因,外因以饮食过多,活动过少为主,热量摄入多余热量消耗,使脂肪合成增加(3,4)。而内因是要是由于遗传易感性引起的。类似于其它许多疾病,肥胖是遗传因素和环境因素之间相互作用的结果。人类的单纯性肥胖的发病有一定的遗传背景。许多基因存在的多态性控制着食欲和代谢从而在充足能量存在时更容易使体重增加从而易患肥胖症。在对家庭以及双胞胎的研究中表明, 40-70%的普通肥胖症患者属于遗传性肥胖(5,6)。
已有的研究数据表明,UCP1(热解偶联蛋白1)基因与肥胖症密切相关,UCP1存在于褐色脂肪组织内 (BAT)的线粒体膜上,棕色脂肪组织(BAT)线粒体与其他组织的对应物不同,因为ATP生产不是其主要的生理作用,BAT线粒体内有UCP1蛋白质,UCP1使电子传递链短路,使线粒体膜电位转变为热,使 BAT成为能够改变哺乳动物能量消耗和燃料代谢而不增加身体活动的组织(7,8)。之前的研究认为在人类中,BAT在妊娠期间的胎儿体内存在,胎儿发育期间UCP1的量增加,在出生前达到峰值,而在出生后前9个月开始下降(9,10)。直到2009年才发现BAT广泛存在于成年人的锁骨、颈、脊椎旁和肾上腺体中 (11)。BAT是哺乳动物不发热的主要部位,啮齿类动物研究表明,BAT产热活动可以保护免受肥胖。Ucp1 活性高可以有效避免肥胖,而Ucp1功能受损的小鼠中产热机制受损从而会导致肥胖(12)。2015年研究发现,Cnot7-/-和Tob-/-的小鼠中脂肪含量特别少,即使喂食高脂肪食物依然能够保持苗条,在这些小鼠的组织中发现,Ucp1的表达是增强的,Ucp1可通过把脂肪细胞内的脂肪转化成热能来减少脂肪囤积(13)。这些数据表明FTO基因的突变跟肥胖症密切相关。
在分子生物学水平上,小鼠、非洲爪蛙、斑马鱼、果蝇、和线虫等模式动物内的是生物学事件与人享有相同的分子机制。因此,利用模式动物可以制作人类疾病的有效在体模型。在所有模式动物中,斑马鱼是连接无脊椎动物和哺乳动物模型之间的桥梁,人类基因中的87%在其中能找到同源基因。目前,全球有超过1000家实验室选择以斑马鱼作为模式动物开展从基础生物学、环境毒理评价到药物研发等不同研究领域的研究,国内有超过100家实验室以斑马鱼为主要研究对象。斑马鱼饲养简单,饲养环境控制在一定产卵量多,每次一对鱼每周可产卵200-300枚,胚胎发育透明,且发育周期较短,是进行胚胎发育机理,基因组研究以及疾病机理研究的好材料。
就与人类肥胖症密切相关的易感基因UCP1而言,人UCP1基因位于第4号染色体,UCP1蛋白由307 个氨基酸组成;而在斑马鱼中,跟人UCP1同源基因ucp1位于斑马鱼1号染色体,蛋白由309个氨基酸组成。生物信息学分析发现,ucp1基因和人UCP1基因为直系同源基因。在其表达产物蛋白质的氨基酸组成上,发现UCP1与Ucp1的相同性(identity)高,三个功能域(分别是Mito_carr)相同性更高,进一步支持UCP1基因在人和斑马鱼基因组中高度保守。
基于以上原因,我们以斑马鱼为模型,采用基因组编辑技术,靶向突变ucp1基因。对突变体的分析发现,ucp1的纯合突变导致脂肪细胞在斑马鱼体内的堆积显著减少。结果表明ucp1是一个与肥胖相关基因,可以用作筛选治疗肥胖症的标识基因。
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发明内容
为建立人类肥胖症的斑马鱼模型,我们利用CRISPR/Cas9基因组编辑技术靶向敲除ucp1基因,确认ucp1 基因对以脂肪堆积为特征的肥胖症发生的影响。CRISPR/Cas9技术是否能成功编辑ucp1基因,实现斑马鱼 ucp1的基因敲除,关键在于是否能筛选到可以有效引导Cas9识别目标基因序列(CRISPR序列)的sgRNA。当sgRNA能够有效引导Cas9靶向识别目标基因,其中的Cas9就可以将CRISPR序列之PAM前的3至4核苷酸间的3′-5′磷酸二酯键切断,从而形成基因组DNA的双链断裂(DSB)。一旦DSB形成,胚胎细胞将启动非同源末端连接修复或微同源末端连接修复机制,修复基因组的DSB,导致在DSB处引入插入缺失(Indel)突变。通过在F1代筛选携带具移码突变基因的突变体,就可以获得ucp1基因敲除的斑马鱼突变体,从而有望了解ucp1在肥胖症发生中的作用。为实现这一目标,我们根据Ucp1的功能域的分析,将CRIPSR序列设计在第一个功能域所对应的第4外显子上。我们共设计了4个CRISPR序列(表一)。依据上述CRISPR 序列,我们制备了相应的sgRNA(sgRNA1至sgRNA4)。运用显微注射技术,将获得的4个sgRNA与体外转录获得的Cas9mRNA共同注射到斑马鱼受精卵中,对所述sgRNA进行活性验证。依据活性验证的结果,本发明首先提供了两个可以有效引导Cas9识别目标CRISPR序列的sgRNA1和sgRNA3。
将sgRNA1和sgRNA3与Cas9蛋白共注射入斑马鱼受精卵(F0),待注射胚胎发育至性成熟时,令其自由交配,获得F1个体。通过剪取尾鳍,对F1个体进行基因型鉴定。依据基因型鉴定的结果,本发明又提供了携带ucp1基因敲除突变的斑马鱼突变体的制备方案。
待F1杂合子生长至性成熟时,将F1自交,获得F2代胚胎。待胚胎发育至受精后4天(4dpf),以油红对胚胎进行染色分析,依据分析结果,我们发现:当斑马鱼ucp1功能缺失时,胚胎的脂肪堆积量显著下降。由此,本发明再提供了基于斑马鱼ucp1基因的人类肥胖症模型,斑马鱼ucp1可作为筛选治疗肥胖症药物的标志基因,即能够使ucp1表达下降的任何物质将具备抑制肥胖症的作用。
附图说明
图1斑马鱼Ucp1蛋白质的保守结构功能域分析。斑马鱼Ucp1包含三个保守功能域,均为Mito_carr superfamily。
图2可有效引导Cas9靶向切割ucp1基因的sgRNA活性验证分析。利用Vector NTI软件对重组子测序序列进行比对分析(共10个重组子),表明sgRNA1、sgRNA3有活性,且活性率均不低于11%。
图3斑马鱼F1代突变体3号携带的ucp1突变等位基因部分序列的测序峰图。
图4斑马鱼F1代突变体3号携带的ucp1突变等位基因预测编码的截短蛋白结构示意图。WT:野生型Ucp1 蛋白质结构示意图;MUT1:其中一个ucp1突变等位基因突变后预测形成的蛋白质结构示意图,MUT2:第二个ucp1突变等位基因突变后预测形成的蛋白质结构示意图。
图5斑马鱼F1代突变体15号携带的ucp1突变等位基因部分序列的测序峰图。
图6斑马鱼F1代突变体15号携带的ucp1突变等位基因预测编码的截短蛋白结构示意图。WT:野生型 Ucp1蛋白质结构示意图;MUT2:第二个ucp1突变等位基因突变后预测形成的蛋白质结构示意图。
图7携带ucp1纯合突变的斑马鱼突变体仔鱼内脂质储存量减少。油红O对发育到4天的斑马鱼仔鱼进行染色,发现与野生型斑马鱼相比较,ucp1突变体斑马鱼仔鱼体内脂肪含量明显减少。
具体实施方式
实施例中的实验方法,如无特殊说明,均为常规方法。
实施例1活性sgRNA的筛选
(一)靶向突变斑马鱼ucp1基因的CRISPR的设计
1、基因信息分析
1.1斑马鱼ucp1基因的CDS序列
ATGGTGGGTCTGAAGCCGTCAGATGTTCCTCCTCCTCTGACTGTGAAGGTGTTGAGTGCAGGAACGGCCGCCTGCATCGCTGACCTCGTCACCTTCCCGCTGGACACGGCCAAAGTCCGCCTGCAGATCCAGGGGGAGAAAGCGGTGACAGGAGCCGCTAAAGGCATCCGCTACAAAGGTGTTTTCGGGACCATCAGCACCATGATGAGGACGGAGGGTCCGCGCTCGCTCTACAACGGCCTGGTCGCCGGCCTACAGAGACAGATGGCCTTCGCCTCCATCCGCATTGGCCTCTACGACAACGTCAAGAGCTTCTACACGCGTGGAAAAGACAACCCTAATGTGGCGGTGCGAATCCTGGCGGGCTGTACTACCGGAGCGATGGCCGTGTCCATGGCTCAGCCCACAGACGTGGTAAAGGTGCGTTTCCAGGCCCAAATGAACCTCCAGGGTGTGGGCAGACGATACAACGGCACCATGCAGGCCTACAGGCAGATCTTCCAGCTTGAGGGACTCCGTGGTCTCTGGAAAGGAACTCTGCCGAACATCACGAGGAACGCTCTGGTCAACTGCACAGAACTGGTGTCTTACGATCTGATCAAAGAGGCTATCCTTAAACACAGACTCCTGTCAGACAATCTCCCGTGTCACTTTGTGTCTGCGTTCGGCGCGGGCTTCATCACGACGGTGATCGCGTCTCCTGTGGATGTGGTAAAAACACGGTACATGAACTCTCCACCGGGACAGTACAGCAGCTCCACCAACTGCGCCTGGACCATGCTCACTAAAGAGGGACCCACAGCTTTCTACAAAGGTTTTGTCCCGTCATTCCTGCGGCTGGGCTCCTGGAACGTGGTGATGTTCGTGTCGTTTGAGCAGCTCAAACGGGCCATGATGGTGTCCAGAAACAGAATCGAAGCCGCTGCGTAG
1.2斑马鱼Ucp1蛋白质序列
1.3蛋白质的保守结构功能域
见图1。
2、CRISPR的设计
2.1斑马鱼ucp1基因的外显子3序列
黄色字体为设计的CRISPR。下划线的序列为PAM序列NGG(RC:CCN)。
表一、靶向突变斑马鱼ucp1基因之CRISPR序列
(二)靶向突变斑马鱼ucp1基因的sgRNA活性的鉴定
1、目标基因的基因型鉴定
1.1待扩增的目标基因组序列
黄色高亮标记为设计的CRISPR,带下划线的为PAM序列NGG(RC:CCN),绿色高亮标记为引物序列。
1.2PCR扩增的引物设计
序列名称 | 序列 | 用途 |
F-ucp1-1 | cagtgcacacacattatgtcaa | PCR正向引物 |
R-ucp1-1 | gcatatttcagcatccattgc | PCR反向引物 |
2、sgRNA的体外合成
2.1sgRNA模板的制备(PCR法)
2.1.1PCR引物
正向引物序列如下:
注:灰色高亮部分为T7启动子部分,下划线部分为ucp1特异的CRISPR序列,小写字母为部分与sgRNA 骨架模板互补的序列。
反向引物(为通用引物)R-Common:AAAAAAAGCACCGACTCGGTGCCAC
2.1.2预计获得的sgRNA体外转录模板序列
注:红色高亮部分为T7启动子部分,下划线部分为ucp1特异的的CRISPR序列,小写字母为sgRNA骨架模板序列。
2.2sgRNA的体外转录
2.2.1体外转录过程
将上述纯化后的模板用T7RNA聚合酶进行体外转录。使用RNA体外转录试剂盒(MAXIscript SP6/T7, Ambion,USA),按试剂盒说明书要求依次加入10×TranscriptionBuffer 2μl、10mM ATP 1μl、10mM CTP 1μl、 10mM GTP 1μl、10mM UTP 1μl、T7RNApolymerase mix 2μl、模板DNA 12μl,轻弹混匀离心后,37℃水浴1h。
加入DNase I(Ambion,USA)1μl,37℃水浴15min,以去除模板。
之后加入30μl DEPC水扩大体积至50μl,同时加入5μl无核酸酶3M醋酸钠(pH5.2)和3倍体积的无水乙醇(国产分析纯),-80℃沉淀过夜。4℃12,000g离心20min,去除上清后,沉淀在通风橱晾干,然后以 15μl无核酸酶超纯水重悬后储存于-80℃冰箱备用。
2.2.2sgRNA序列
UCP1-sgRNA1:
GGAUUCGCACCGCCACAUUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
UCP1-sgRNA2:
GGGCUGUACUACCGGAGCGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
UCP1-sgRNA3:
GACCACGGAGUCCCUCAAGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
UCP1-sgRNA4:
GGUGUGGGCAGACGAUACAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU
3、Cas9蛋白
将从商业公司(金丝瑞)购买的Cas9蛋白用DEPC水稀释为400ng/μl,分装后置于-20℃冰箱中保存。
4、斑马鱼受精卵的显微注射
按常规方法收取斑马鱼受精卵。将上述sgRNA1、sgRNA2、sgRNA3、sgRNA4(终浓度为50ng/μl)和 Cas9蛋白(终浓度为200ng/μl)混合后,显微注入斑马鱼受精卵,注射量为1nl每胚胎。
5、目标基因编辑子(Edits)的重组确认
5.1目标基因组片段模板的制备
待注射胚胎发育至24hpf时,各取5枚胚胎,使用南京尧顺禹公司生产的《斑马鱼基因型鉴定试剂盒》制备基因组DNA模板,具体反应条件为:65℃30min,95℃5min,16℃1min,4℃
5.2PCR扩增反应体系(20μl)
2×Mastermix(Transgene)10μl、7μl的超纯水、1μl的正反向(F1-ucp1-1和R1-ucp1-1)引物(5μM)、和1μl的由5.1制备好的基因组DNA模板。
5.3PCR反应条件
95℃3min,30×(95℃30s,56℃30s,72℃1min),72℃10min,4℃。
5.4目标基因PCR扩增产物的重组
将F1-ucp1和R1-ucp1的PCR扩增产物重组入pGEM-T Easy载体(5μl总体积的反应),反应组成为:
1.5μl PCR扩增产物(取自5.5.2)
2.5μl 2×Ligation Buffer
0.5μl T4DNA连接酶
0.5μl pGEM-T Easy载体(Promega)
反应时间为:室温孵育60min
5.5重组子转化
将上述5μl连接产物用100μl大肠杆菌DH5α感受态细胞(pfu≥108)进行转化。然后涂布于含氨苄(50 μg/ml)的LB板,37℃下倒置培养过夜。
5.6目标基因组DNA编辑子(Edits)的重组确认
依据条带大小,选取克隆直接送测序,测序引物为T7。送测10个克隆,测序结果反馈10个,具体序 列比对见图2。结果显示sgRNA1和sgRNA3有活性,且活性率均不低于11%。
图2目标基因组DNA编辑子(Edits)序列比对图sgRNA1、sgRNA3有活性,且活性率均不低于11%。
6、结论:
综合以上实验结果,可以确认所述的sgRNA1和sgRNA3可有效引导Cas9靶向切割目标基因。
实施例2斑马鱼ucp1基因敲除突变体制作方案
(一)初建者的制备
按实施例1中方案(二)之4描述的方法,按常规方法收取斑马鱼受精卵。将上述sgRNA1和sgRNA3 (终浓度为50ng/μl)和Cas9蛋白(终浓度为200ng/μl)混合后,显微注入斑马鱼受精卵(注射量为1nl 每胚胎),制备ucp1基因敲除突变体之初建者(Founder)。
(二)F1突变体的筛选
1、F1代个体模板基因组DNA制备
1.1F1代斑马鱼的繁育与饲养
将注射了Cas9/sgRNA的F0胚胎饲养至性成熟,然后令其自由交配,获得F1胚胎。将F1胚胎按常规饲养至性成熟,进行基因型鉴定,筛选基因编辑突变体。
1.2组织无创取材
取3~4月龄的F1成鱼,剪取部分尾鳍组织,按顺序分别放入200μl PCR管中,然后置于冰上存放。
1.3基因组模板DNA制备
向含有尾鳍组织的200μl PCR管中加入南京尧顺禹生产的YSY buffer 10μl。快速离心后,将PCR管放入PCR仪中,进行如下反应:65℃30min,95℃5min,16℃1min,4℃。
2、目标基因组DNA的扩增
2.1含sgRNA识别位点的基因组序列
绿色高亮字体标识用于PCR扩增的引物序列,黄色高亮标识sgRNA识别位点序列。
2.2基因组片段扩增引物设计
F-ucp1-1:CAGTGCACACACATTATGTCAA
R-ucp1-1:GCATATTTCAGCATCCATTGC(RC:GCAATGGATGCTGAAATATGC)
2.3基因组DNA片段PCR扩增
2.3.1反应的组成
2×Mastermix(Vazyme)10μl、7μl的超纯水、1μl的正反向(F-phf8-1和R1R-phf8-1)引物(5μM) 和1μl的由1.3.2步骤获得的细胞基因组DNA模板。
2.3.2PCR反应条件
95℃3min,35×(95℃30s,56℃30s,72℃50s),72℃10min,4℃。
3、携带ucp1突变等位基因的F1突变体的筛选鉴定
将ucp1突变体的PCR产物直接送测序,通过峰图读取确认其突变体携带的突变等位基因的基因型。
3.1突变体3基因型的确认
3.1.1突变体3基因型及预测的突变等位基因编码的截短蛋白序列
3.1.2测序峰图(部分)
突变体3的目标基因组片段PCR扩增产物直接测序结果图(部分)见图3
3.1.3预测的突变基因编码的截短蛋白结构示意图
见图4。
3.2突变体15基因型的确认
3.2.1突变体15基因型及预测的突变等位基因编码的截短蛋白序列
3.2.2测序峰图(部分)
突变体15的目标基因组片段PCR扩增产物直接测序结果图(部分)见图5。
3.2.3预测的突变基因编码的截短蛋白结构示意图
突变体15携带的移码突变等位基因预测编码的截短蛋白结构示意图见图6。
4、结论:共筛选到2尾携带ucp1移码突变的F1突变斑马鱼。
实施例3斑马鱼ucp1功能缺失降低脂肪的累积
1、胚胎准备
携带ucp1突变的F1代杂合子斑马鱼养殖在南京尧顺禹生物科技有限公司斑马鱼独立养殖单元中,待其性成熟时,令F1代个体自由交配,按常规收集受精卵,所有胚胎及幼鱼养殖在室温28.5℃,待胚胎发育至4天时用于实验。
2、油红O的配制
购于Sigma公司,母液配置为0.5%(w/v),取0.5g油红O溶于100ml异丙醇中。
3、胚胎固定
取2g多聚甲醛在65℃水浴锅中溶于PBS中(76g NaCl,9.9g Na2HPO4,3.6gNaH2PO4,溶于10L水中, pH调至7.0),配置为4%PFA,用于固定4dpf胚胎。
4、油红O染色
1)取4天斑马鱼幼鱼,4%多聚甲醛4℃固定12小时
2)PBST洗2次
3)0.5%油红O室温染色4-6小时
4)PBST洗3次,60%异丙醇洗2次,每次5分钟
5)PBST洗一次,解剖镜下观测,数码照相机拍照记录。
5、基因型鉴定及结果分析
对经油红O染色的ucp1斑马鱼突变体,提取基因组,按实施例2所列写的方案对每个胚胎逐一进行 基因型鉴定。结果发现ucp1纯合突变的胚胎脂肪的积累明显减少(图7)。
Claims (10)
1.基于UCP1基因的人类肥胖症斑马鱼的建立与应用,其特征在首先提供了两个可有效引导Cas9识别斑马鱼ucp1基因的活性sgRNA,利用活性sgRNA制作了ucp1基因敲除的斑马鱼突变体,提供了作为人类肥胖症的斑马鱼模型,因而确立了斑马鱼ucp1基因可作为标识基因应用于筛选与建立治疗人类肥胖症的新药物与新方法。
2.如权利要求1所述的特征,可有效引导Cas9识别斑马鱼ucp1基因的活性sgRNA分别为sgRNA1和sgRNA3。
3.如权利1所述的特征,斑马鱼的突变体分别为携带了插入5bp和插入7bp的ucp1无效等位基因的杂合子,性成熟的杂合子自交产生的纯合子作为人类肥胖症的斑马鱼模型。
4.如权利3所述的斑马鱼模型,其斑马鱼仔鱼的年龄为受精后3天、4天、5天、6天和7天。
5.如权利3所述的斑马鱼模型,应用于筛选与建立治疗人类肥胖症的新药物与新方法。
6.如权利1所述的特征,ucp1基因作为人类肥胖症的标识基因,其纯合突变导致斑马鱼仔鱼脂肪堆积受到抑制。
7.如权利6所述的特征,斑马鱼ucp1基因表达水平的降低指示治疗人类肥胖症的新药物与新方法的筛选与建立。
8.如权利6所述的特征,斑马鱼ucp1基因可以进行各种基因修饰,包括但不仅限于在ucp1位点敲入荧光报告基因,以指示ucp1基因表达的减少,用于筛选与建立治疗人类肥胖症的新药物与新方法。
9.如权利6所述的特征,斑马鱼ucp1基因的调控序列可用于驱动包括但不限于荧光报告基因,以制备转基因斑马鱼,用于筛选与建立治疗人类肥胖症的新药物与新方法。
10.如权利要求1所述的特征,斑马鱼的ucp1基因可以是其它鱼类模式动物的ucp1基因作为标识基因,用于筛选与建立治疗人类肥胖症的新药物与新方法。
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