CN114480259B - 一种诱导小鼠全能样干细胞的培养基及诱导方法 - Google Patents
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Abstract
本发明提供了一种诱导小鼠全能样干细胞的培养基及诱导方法。所述诱导小鼠全能样干细胞的培养基,包括诱导培养和维持培养基和维持培养基。本发明提供了一种利用mESC中含有低比例的小鼠二细胞胚胎样细胞(2C‑like cell,2CLC)的异质性特性,将其体外诱导为全能样干细胞的方法。该方法通过利用专用培养基来诱导细胞达到一种全能样状态,使细胞不仅在转录组和表观遗传中与体内2C胚胎更为相似,而且具有分化形成胚胎和胚外组织(胎盘、卵黄囊等)的能力,是一种全能样的干细胞。本发明方法操作简单,成本低廉,为小鼠早期胚胎发育的研究及构建各组织或细胞的疾病模型提供了基础。
Description
技术领域
本申请属于细胞培养技术领域,具体涉及一种诱导小鼠全能样干细胞的培养基及诱导方法。
背景技术
干细胞因具有自我更新及多向分化潜能,在生命科学、生物、医疗等各个领域的研究中备受关注。干细胞是组织器官再生的种子细胞,是实践再生医学的重要先决条件,其中全能性干细胞比其他任何类型的细胞都具有更强大发育潜能,其可产生胚胎及胚外组织(胎盘、卵黄囊等),并最终以一种高度有序的方式形成完整的生物个体,是一类举足轻重的特殊类型的干细胞。
小鼠受精卵和2C阶段的细胞是具有全能性的细胞,但受限于来源且不能稳定培养而无法得到广泛应用。小鼠胚胎干细胞(Mouse embryonic stem cell,mESC)虽不具备全能性,但具有异质性,其培养过程中有少比例的小鼠二细胞胚胎样细胞(2C-like cell,2CLC)(0.5-1%)出现,这些细胞表达小鼠二细胞胚胎(2C)特异性基因,可通过基于MERVL/Zscan4的荧光报告系统得到富集。基于以上,2CLC可作为一种全能性的替代模型,帮助了解早期小鼠胚胎全能性的分子特征及其生物学意义。但2CLC所占比例非常少,且不能形成稳定的细胞系,因此也极大的限制了2CLC的研究和应用。
近年来越来越多的课题组鉴定出一些促进或抑制全能性的调控因子,希望以此获得长期稳定的全能样干细胞。目前获得全能样细胞的方式主要分为两种:一种是通过过表达一些关键因子如Dux等提高mESC中2CLC的比例,但该方法获得的细胞存在以下缺陷:1.关键因子的过表达只能提高mESC中2CLC的比例,但总体效率仍然较为低下;2.获得的细胞为一种瞬时的状态,无法长期稳定维持;3.2CLC形态、转录组以及代谢等特征与小鼠二细胞时期的卵裂球不完全一致,所以目前广泛使用的2CLC可能是一种中间状态或不成熟状态。另一种获得全能性干细胞的方式则为化学诱导法,如2017年liu和Deng等人先后通过一些小分子化合物将小鼠8C或mESC诱导为超潜能多能干细胞(Extended pluripotent stemcell,EPSC),但该方法也引起了研究者的广泛争议,包括:1.根据EPSC所展示出的转录组及基因调控网络的数据,这种状态的细胞分别与E4.5和E5.5上胚层更为相似而非全能性状态;2.EPSC对胚外谱系贡献有待商榷,这些细胞在嵌合体实验中虽然可以定位在相应位置,但未能检测到胚外谱系的特异性基因。因此,目前仍然缺乏与2C相似度高的全能样干细胞的诱导及维持培养方法。
综上所述,开发新型的全能样干细胞诱导和培养方法意义重大,这不仅有助于其做为模型来研究小鼠胚胎发育过程以及由于不正常细胞分化或增值所引起的疾病,也有利于通过分化为特定组织和细胞而实现疾病模型的构建。
发明内容
本发明要解决的技术问题是克服上述现有技术的缺陷和不足,提供诱导小鼠全能样干细胞的培养基,以及一种新的体外诱导全能样干细胞的方法。本发明所述的mESC诱导获得的全能样干细胞的方法,可在短期内获得大量、较高比例、与小鼠二细胞胚胎在转录组及表观遗传等方面更为相似的全能样干细胞,为其在组织工程方面的应用及疾病模型建立创造了条件。
本发明采用以下技术方案实现本发明的目的:
本发明提供一种诱导小鼠全能样干细胞的培养基,包括全能性诱导培养基和全能性维持培养基;
所述全能性诱导培养基的配方为:
在基础培养基中(如KnockOutTM DMEM/F12培养基),加入以下成分:10~20%KnockOutTM血清替代物(KnockOutTM serum replacement),1×非必需氨基酸溶液,1~5mML-谷氨酰胺,50~100μg/ml维生素C(又名L-抗坏血酸),10~100μg/ml牛血清白蛋白,50~110μM 2-巯基乙醇,0.5~5μM组蛋白甲基转移酶DOT1L高效选择性抑制剂,0.5~5μM组蛋白去甲基酶KDM5A和KDM5B抑制剂,5~25ng/ml白介素-6,5~25ng/ml可溶性白介素-6受体α。
作为本发明所述的诱导小鼠全能样干细胞的培养基的优选实施方式,所述全能性维持培养基的配方为:
在基础培养基中(如KnockOutTM DMEM/F12培养基),加入以下成分:10~20%KnockOutTM血清替代物(KnockOutTM serum replacement),1×非必需氨基酸溶液,1~5mML-谷氨酰胺,50~100μg/ml维生素C,10~100μg/ml牛血清白蛋白,50~110μM 2-巯基乙醇,0.5~5μM组蛋白甲基转移酶DOT1L高效选择性抑制剂,0.5~5μM组蛋白甲基转移酶G9a和GLP抑制剂,0.5~5μM组蛋白去甲基酶KDM5A和KDM5B抑制剂,5~25ng/ml白介素-6,5~25ng/ml可溶性白介素-6受体α。
优选地,所述组蛋白甲基转移酶DOT1L高效选择性抑制剂为SGC0946、EPZ004777、DotL-IN-4、Pinometostat(EPZ5676)中的至少一种。
优选地,所述全能性诱导培养基的配方为:KnockOutTM DMEM/F-12培养基中,加入以下浓度的成分:
20%KnockOutTM血清替代物(KnockOutTM serum replacement),1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C,50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα;
优选地,所述全能性维持培养基的配方为:KnockOutTM DMEM/F12培养基中,加入以下浓度的成分:
20%KnockOutTM血清替代物(KnockOutTM serum replacement),1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C,50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,2μM A366,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα。
优选地,所述培养基还包括小鼠胚胎干细胞培养基,所述培养基的配方为:体积1:1的DMEM/F12和Neurobasal medium中,加入1%N2 supplement、2%B27 supplement、1×非必需氨基酸溶液,2mM L-谷氨酰胺,50~100μg/ml维生素C、50~110μM 2-巯基乙醇、5~20ng/ml LIF、0.5~2μM PD0325901、0.5~5μM CHIR99021;或
所述培养基的配方为:KnockOutTM DMEM/F-12培养基中,加入10~20%KnockOutTM血清替代物(KnockOutTM serum replacement)、1×非必需氨基酸溶液、1~5mM L-谷氨酰胺、50~100μg/ml维生素C(又名L-抗坏血酸)、10~100μg/ml牛血清白蛋白、50~110μM 2-巯基乙醇和5~20ng/ml LIF。
本发明提供一种诱导全能样干细胞的方法,包括以下步骤:
(1)将小鼠胚胎干细胞消化为单细胞作为种子细胞,将所述种子细胞接种培养板,培养20~30小时;
(2)取步骤(1)培养的细胞,清洗后,加入所述的全能性诱导培养基,培养2~5天,隔天更换所述全能性诱导培养基;
(3)取步骤(2)培养的细胞,进行单细胞传代,接种培养板,更换为所述的全能性维持培养基,隔天更换所述全能性维持培养基,每2~5天传代一次,即成。
本发明将mESC以单层培养的方式,通过直接更换培养基快速获得大量,高比例的与小鼠二细胞胚胎相似的全能样干细胞。
作为本发明所述的诱导全能样干细胞的方法的优选实施方式,在所述步骤(1)中,所述消化采用的方法为:取汇合度60%~80%的小鼠胚胎干细胞,加入0.05%Trypsin-EDTA消化4~5min,即成。
作为本发明所述的诱导全能样干细胞的方法的优选实施方式,在所述步骤(1)和所述步骤(3)中,所述接种培养板采用3×105~4×105个/cm2的密度接种。
作为本发明所述的诱导全能样干细胞的方法的优选实施方式,在所述步骤(3)中,所述单细胞传代采用1:(4~6)的分瓶比例。
本发明提供另一种诱导全能样干细胞的方法,包括以下步骤:
(1)小鼠二细胞胚胎分离成单个卵裂球,接种,加入所述的全能性诱导培养基,培养10~15天,隔天更换所述全能性诱导培养基;
(2)取步骤(1)培养的细胞,清洗,消化为单细胞作为种子细胞,接种,加入所述的全能性维持培养基培养,隔天更换所述全能性维持培养基;
(3)取步骤(2)培养的细胞,进行单细胞传代,接种培养板,加入所述的全能性维持培养基,隔天更换所述全能性维持培养基,每2~5天传代一次,即成。
作为本发明所述的诱导全能样干细胞的方法的优选实施方式,在所述步骤(2)中,所述消化采用的方法为:取步骤(1)培养的细胞,加入0.05%Trypsin-EDTA消化4~5分钟,即成。
优选地,所述的培养条件为37℃、5%CO2、饱和湿度下进行培养。
本发明的有益效果为:
本发明提供了一种利用mESC中含有低比例的2CLC的异质性特性,将其体外诱导为全能样干细胞的方法。该方法通过利用专用培养基来诱导细胞达到一种全能样状态,使细胞不仅在转录组和表观遗传中与小鼠二细胞胚胎更为相似,而且具有分化形成胚胎和胚外组织(胎盘、卵黄囊等)的能力,是一种全能样的干细胞。本发明方法操作简单,成本低廉,为小鼠早期胚胎发育的研究及构建各组织或细胞的疾病模型提供了基础。
附图说明
图1为小鼠胚胎干细胞转变为全能样干细胞的流程。
图2为小鼠胚胎二细胞胚胎时期的卵裂球转变为全能样干细胞的流程。
图3为体外诱导小鼠二细胞胚胎时期的卵裂球为全能样干细胞的细胞培养照片。
图4为核心多能性和全能性基因在小鼠胚胎干细胞、全能样干细胞中的表达水平比较.
图5为MERVL-GFP做为小鼠胚胎干细胞转变为全能样干细胞的全能性荧光报告。
图6为小鼠胚胎、胚胎干细胞(mESC)、全能样干细胞(TLSC)、超潜能干细胞(EPSC)的转录组比较。
图7为小鼠胚胎、胚胎干细胞(mESC)、全能样干细胞(TLSC)的染色质可及性比较。
图8为小鼠胚胎、胚胎干细胞(mESC)、全能样干细胞(TLSC)的H3K4me3全基因组分布的比较。
图9为免疫荧光染色显示mCherry标记的全能样干细胞(TLSC)广泛地向胚胎和胚外组织发育。
图10为全能样干细胞来源的嵌合小鼠胚胎的单细胞转录组分析。
图11为mCherry标记的全能样干细胞(TLSC)分化成多种胚胎和胚外细胞类型及其比例。
图12为mCherry标记的全能样干细胞(TLSC)向生殖嵴嵌合。
图13为mCherry标记的全能样干细胞(TLSC)向生殖嵴嵌合产生的小鼠的后代(F2)。
图14为全能样干细胞(TLSC)形成类囊胚结构。
图15为免疫荧光显示类囊胚含有内细胞群(OCT4标记)和滋养层细胞(GATA2标记)。
图16为全能样干细胞(TLSC)形成的类囊胚结构诱导蜕膜化反应。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例详细说明本发明的技术方案。如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。
下述实施例培养基添加试剂中,KnockOutTM serum replacement为KnockOutTM血清替代物;SGC0946、EPZ004777、DotL-IN-4和Pinometostat(EPZ5676)是组蛋白甲基转移酶DOT1L高效选择性抑制剂;A366是竞争性G9a/GLP抑制剂;AS8351是组蛋白去甲基酶KDM5A和KDM5B抑制剂;CHIR99021是GSK3α和GSK3β高效选择性抑制剂;PD0325901是选择性MEK抑制剂;LIF是白血病抑制因子(Leukemia Inhibitory Factor);IL-6是白介素-6;sIL-6Rα是可溶性白介素-6受体α。
实施例1:体外诱导小鼠胚胎干细胞(mESC)为全能样干细胞(TLSC)
(1)本实施例中使用以下培养基:
mESC培养基:为2iL培养基;包括以下浓度的成分:
所述培养基的配方为:体积1:1的DMEM/F12和Neurobasal medium中,加入1%N2supplement、2%B27 supplement、1×非必需氨基酸溶液,2mM L-谷氨酰胺,50~100μg/ml维生素C(又名L-抗坏血酸)、50~110μM 2-巯基乙醇、5~20ng/ml LIF、0.5~2μMPD0325901、0.5~5μM CHIR99021;或
所述培养基的配方为:KnockOutTM DMEM/F-12培养基中,加入10~20%KnockOutTMserum replacement、1×非必需氨基酸溶液、1~5mM L-谷氨酰胺、50~100μg/ml维生素C(又名L-抗坏血酸)、10~100μg/ml牛血清白蛋白、50~110μM 2-巯基乙醇和5~20ng/mlLIF。
全能性诱导培养基:为KnockOutTM DMEM/F-12培养基中加入以下浓度的成分:
20%KnockOutTM serum replacement,1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C(又名L-抗坏血酸),50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα;
全能性维持培养基:为KnockOutTM DMEM/F-12培养基中加入以下浓度的成分:
20%KnockOut serum replacement,1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C(又名L-抗坏血酸),50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,2μM A366,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα。
(2)体外诱导mESC为全能样干细胞的流程如图1所示,培养条件为37℃、5%CO2、饱和湿度下进行培养。
具体包括以下步骤:
S1.取汇合度达70%的mESC,用0.05%Trypsin-EDTA消化4-5分钟,离心收集单细胞,用PBS清洗后得到种子细胞;将种子细胞以3×105~4×105个/cm2的密度接种于Gelatin包被并带有饲养细胞(feeder cell)的培养板,用mESC培养基培养24小时。
S2.取步骤S1培养的细胞,弃去其培养基,用PBS清洗一次,更换为全能性诱导培养基,培养3天,且隔天换液。
S3.将步骤S2的细胞按照1:4~1:6的分瓶比例进行单细胞传代,以3×105~4×105个/cm2的密度接种于Gelatin包被并带有饲养细胞(feeder cell)的培养板,更换为全能性维持培养基,培养3天,且隔天换液。
实施例2:体外诱导小鼠二细胞胚胎时期的卵裂球为全能样干细胞
(1)本实施例中使用以下培养基:
全能性诱导培养基:为KnockOutTM DMEM/F-12培养基中加入以下浓度的成分:
20%KnockOutTM serum replacement,1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C(又名L-抗坏血酸),50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα;
全能性维持培养基:为KnockOutTM DMEM/F-12培养基中加入以下浓度的成分:
20%KnockOut serum replacement,1×非必需氨基酸溶液,2mM L-谷氨酰胺,100μg/ml维生素C(又名L-抗坏血酸),50μg/ml牛血清白蛋白,100μM 2-巯基乙醇,2μMSGC0946,2μM A366,3μM AS8351,10ng/ml IL-6,10ng/ml sIL-6Rα。
(2)培养流程如图2所示,具体步骤如下:
S1.取小鼠二细胞胚胎分离成单个卵裂球,然后接种于饲养细胞(feeder cell)上,使用全能性诱导培养基培养两周,且隔天换液。
S2.取步骤S1培养的细胞,弃去其培养基,用PBS清洗一次,用0.05%Trypsin-EDTA消化4-5分钟,离心收集单细胞,用PBS清洗后得到种子细胞;将种子细胞接种于Gelatin包被并带有饲养细胞(feeder cell)的培养板中,继续使用全能性维持培养基培养,且隔天换液。
S3.将步骤S2的细胞进行1:4~1:6的单细胞传代,以3×105~4×105个/cm2的密度接种于Gelatin包被并带有饲养细胞(feeder cell)的培养板,更换为全能性维持培养基,培养3天,且隔天换液。
小鼠二细胞胚胎时期的卵裂球在培养的第1天、第4天、第7天、第12天,传代1代培养的第3天,以及传代8代培养的第4天的细胞形态如图3所示,荧光标记检测传代细胞中存在Zscan4表达,Zscan4在功能上可以启动二细胞胚胎基因的转录并维持小鼠胚胎干细胞基因组的完整性。
验证例:mESC体外诱导为TLSC的全能性鉴定
(1)基因Oct4、Sox2、Nanog是检测胚胎干细胞是否具备多能性的常用指标,基因Zscan4、Tcstv1、Tcstv3、Sp110、Obox3、Dux、Spz1是检测胚胎干细胞是否具备全能性的常用指标。通过对上述基因表达情况的检测,能够说明小鼠干细胞具备多能性或全能性。
因此,本实施例使用细胞免疫荧光染色的方法检测实施例1和2获得的干细胞中上述基因的表达情况。
结果如图4所示,检测结果表明实施例1和2获得的干细胞(TLSCESC和TLSCE2C)中的全能性基因Zscan4、Tcstv1、Tcstv3、Sp110、Obox3、Dux、Spz1的表达水平与小鼠二细胞期胚胎相似、且显著高于小鼠胚胎干细胞(mESC),但多能性基因Oct4、Sox2、Nanog的表达水平显著低于mESC。表明实施例1和2获得的干细胞具有全能性。
(2)以MERVL-GFP做为全能性荧光报告基因,可见阳性比例达到60%以上(见图5)。
(3)转录组分析对全能样干细胞进行了分析鉴定,结果显示出全能样干细胞与小鼠二细胞胚胎具有相似的转录组特征(见图6)。
(4)染色质可及性分析对全能样干细胞进行了分析鉴定,结果显示出全能样干细胞与小鼠二细胞胚胎具有相似的染色质开放状态(见图7)。
(5)对组蛋白修饰H3K4me3的全基因组景观图进行分析,结果显示出全能样干细胞与小鼠二细胞胚胎具有相似的H3K4me3全基因组景观图特征(见图8)。
(1)-(5)的检测结果表明,全能样干细胞具有与小鼠二细胞胚胎非常相似的分子特征。随后,进一步对全能样干细胞进行了发育潜能鉴定。
(6)嵌合体实验证明其可发育形成胚胎和胚外组织(胎盘、卵黄囊等)(见图9-11)。
(7)全能样干细胞也能向生殖系统嵌合并发育形成生殖细胞,产生小鼠后代(见图12和13)。
(8)全能样干细胞可以在体外形成类囊胚结构(blastoid),该类囊胚可以植入小鼠子宫内膜,并诱导小鼠子宫内膜发生蜕膜化反应(见图14-16)。
(6)-(8)的试验结果表明,全能样干细胞具有形成胚胎和胚胎组织的强大发育潜能。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种诱导小鼠全能样干细胞的培养基,其特征在于,由全能性诱导培养基和全能性维持培养基组成;
所述全能性诱导培养基的配方为:
在基础培养基中,加入以下成分:10~20%血清替代物,1×非必需氨基酸溶液,1~5mML-谷氨酰胺,50~100μg/ml维生素C,10~100μg/ml牛血清白蛋白,50~110μM 2-巯基乙醇,0.5~5μM组蛋白甲基转移酶DOT1L高效选择性抑制剂,0.5~5μM组蛋白去甲基酶KDM5A和KDM5B抑制剂,5~25ng/ml白介素-6,5~25ng/ml可溶性白介素-6受体α;
所述全能性维持培养基的配方为:
在基础培养基中,加入以下成分:10~20%血清替代物,1×非必需氨基酸溶液,1~5mML-谷氨酰胺,50~100μg/ml维生素C,10~100μg/ml牛血清白蛋白,50~110μM 2-巯基乙醇,0.5~5μM组蛋白甲基转移酶DOT1L高效选择性抑制剂,0.5~5μM组蛋白甲基转移酶G9a和GLP抑制剂,0.5~5μM组蛋白去甲基酶KDM5A和KDM5B抑制剂,5~25ng/ml白介素-6, 5~25ng/ml可溶性白介素-6受体α;
所述基础培养基为DMEM/F12培养基。
2.一种诱导全能样干细胞的方法,其特征在于,包括以下步骤:
(1)将小鼠胚胎干细胞消化为单细胞作为种子细胞,将所述种子细胞接种培养板,培养20~30小时;
(2)取步骤(1)培养的细胞,清洗后,加入权利要求1所述的全能性诱导培养基,培养2~5天,隔天更换所述全能性诱导培养基;
(3)取步骤(2)培养的细胞,进行单细胞传代,接种培养板,更换为权利要求1所述的全能性维持培养基,隔天更换所述全能性维持培养基,每2~5天传代一次,即成。
3.根据权利要求2所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(1)中,所述消化采用的方法为:取汇合度60%~80%的小鼠胚胎干细胞,加入0.05%Trypsin-EDTA消化4~5分钟,即成。
4.根据权利要求2所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(1)和所述步骤(3)中,所述接种培养板采用3×105~4×105个/cm2的密度接种。
5.根据权利要求2所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(3)中,所述单细胞传代采用1:(4~6)的分瓶比例。
6.一种诱导全能样干细胞的方法,其特征在于,包括以下步骤:
(1)小鼠二细胞胚胎分离成单个卵裂球,接种,加入权利要求1所述的全能性诱导培养基,培养10~15天,隔天更换所述全能性诱导培养基;
(2)取步骤(1)培养的细胞,清洗,消化为单细胞作为种子细胞,接种,加入权利要求1所述的全能性维持培养基培养,隔天更换所述全能性维持培养基;
(3)取步骤(2)培养的细胞,进行单细胞传代,接种培养板,加入权利要求1所述的全能性维持培养基,隔天更换所述全能性维持培养基,每2~5天传代一次,即成。
7.根据权利要求6所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(2)中,所述消化采用的方法为:取步骤(1)培养的细胞,加入0.05%Trypsin-EDTA消化4~5分钟,即成。
8.根据权利要求6所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(3)中,所述接种培养板采用3×105~4×105个/cm2的密度接种。
9.根据权利要求6所述的诱导全能样干细胞的方法,其特征在于,在所述步骤(3)中,所述单细胞传代采用1:(4~6)的分瓶比例。
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