CN116622619A - 一种非动物源性的内皮细胞分化培养基及其应用 - Google Patents
一种非动物源性的内皮细胞分化培养基及其应用 Download PDFInfo
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Abstract
本发明涉及一种非动物源性的内皮细胞分化培养基,同时提供了其分化方法。本发明采用DMEM/F12并额外添加1×GlutaMAX,1×ITS‑X和维生素C作为基础培养基,用Wnt通路抑制剂CHIR99021作用3天,再用FGF2,VEGFA,EGF和SB431542作用3天,能有效诱导hPSCs向ECs分化。在该分化策略中,从干细胞培养至内皮细胞分化均处在无异种成分的培养基及基质中,批次之间差异较小,所得到的细胞成分明确,具有临床应用价值。
Description
技术领域
本发明属于细胞培养技术领域,具体涉及一种培养基,尤其是一种基于非动物源性诱导分化内皮细胞的培养基,及利用该培养基进行内皮细胞诱导分化方法。
背景技术
人胚胎干细胞(Human Embryonic Stem Cells,hESCs)的成功分离与培养以及人诱导多能干细胞(Human Induced Pluripotent Stem Cells,hiPSCs)技术的建立为科学研究与临床应用提供了大量细胞资源。近年来,利用干细胞体外定向分化获得具有正常生理功能的特定组织的细胞的方法不断建立,这些干细胞来源的组织细胞在构建疾病模型、药物筛选以及体内移植治疗疾病等方面均有广阔的应用前景。
ECs是构成血管和淋巴管的基本细胞类型,其在体内参与了多个生理学过程,如器官和组织间的互作,血管对外周血中物质的通透性,组织的血液灌注情况,炎症的发生发展以及血栓的形成。当ECs处于一些慢性病,如高血压和糖尿病的病理环境下时,其会出现代谢以及功能的紊乱,血管内血流动力学改变,进而导致血管内的凝血-纤溶平衡失调,使血管内形成血栓,阻碍了血液流动,最终导致组织缺乏血液灌注而缺血坏死。因此,如何从根本上改善ECs的功能是缺血性疾病对因治疗的关键。目前临床上以溶栓和支架等对症治疗为主,不能对ECs的功能做针对性的干预,尽管在治疗后一段时间内能恢复正常的血液流动,但部分患者治疗后出现再次梗阻,疗效不佳。干细胞技术近年来的发展为缺血性疾病的治疗提供了新的方案。通过将干细胞来源的内皮细胞(Stem-Cell-Derived EndothelialCells,SC-ECs)直接注射到缺血部位,能够有效促进血管新生,改善组织灌注情况。现有研究已表明注射SC-ECs的治疗方法在难治性心绞痛、缺血性心力衰竭、扩张性心肌病和下肢缺血等疾病中均能使患者受益。
将SC-ECs应用到临床治疗的一大难点是细胞的安全性问题。由于SC-ECs需要直接注射到人体内,培养基中的异种成分(Xenogeneic Components)可能作为潜在的变应原激活宿主的免疫反应,导致过敏。同时,一些来源于牛的异种成分可能携带朊病毒(Prion)、感染性牛鼻气管炎病毒(Infectious Bovine Rhinotracheitis)、副流感病毒(Parainfluenza)和牛病毒性腹泻病毒(Bovine Viral Diarrhea Virus,BVDV)等病原体,其中部分病原体如朊病毒导致的疾病为人畜共患病,会给接受细胞治疗的患者带来健康威胁。
在内皮细胞培养及分化过程中,常使用牛血清(Fetal Bovine Serum,FBS)来维持细胞的增殖及存活。由于FBS的组成成分不明确,批次之间差异大,很快出现了多种细胞培养添加剂来取代FBS,如Knockout血清替代物(Knockout Serum Replacement,KOSR)和B-27添加剂。这些添加剂参考了FBS中部分已知的成分,如BSA,ITS-X,必需氨基酸,维生素,生长因子和激素等,尽管化学成分确定,但仍包含异种成分。随后,一些商品化培养基如Advanced DMEM/F12进一步简化了培养基的组成,其中的异种成分只有BSA一种。BSA在培养基中可作为生长因子的载体蛋白(Carrier Protein),并能有效降低流体剪切力,从而促进细胞存活。但已有临床研究报导为患者注射在10% FBS中培养的树突状细胞后,在患者外周血中检测到了抗BSA的IgM、IgG和IgE。且患者出现了明显的I型超敏反应症状。因此BSA也是潜在的变应原,应在细胞培养中避免使用。
现有的SC-ECs分化策略常采用LaSR作为基础培养基,该培养基由Advanced DMEM/F12,1×GlutaMAX和60 μg/ml维生素C组成,其中包含BSA。现有的无异种成分分化生成内皮祖细胞(Endothelial Progenitor Cells,EPCs)的策略,该策略后续采用商品化的EGM2将EPCs分化为SC-ECs,而EGM2中含有FBS,未在SC-ECs分化中做到无异种成分。
如何进一步优化培养基的组成,达到完全的无异种成分培养是SC-ECs分化策略目前仍存在的问题。
发明内容
为了解决以上技术问题,本发明提供了一种基于非动物源性诱导分化内皮细胞的培养基。
根据本发明具体实施方式的非动物源性的内皮细胞分化培养基,所述培养基包括以下组分:
DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C和1×ITS-X。
优选的,维生素C的浓度为50-100 μg/ml,更为优选的,维生素C的浓度为100 μg/ml。
本发明选择丙氨酸-谷氨酸(丙谷二肽,商品名GlutaMAX)作为培养基的必要成分,其可以有效维持细胞蛋白合成和增殖的,并能够避免于自发分解等不稳定性因素。
在细胞分化过程中,由于细胞受到较强的外源信号刺激,本发明进一步选择维生素C(Vitamin C,Vc),在细胞培养中起到抗氧化和抑制细胞凋亡的作用,并对其浓度针予以优化。
发明人根据多年的经验,选择ITS-X为添加剂,利用其中的胰岛素成分存进SC-ECs分化及存活。
根据本发明具体实施方式的非动物源性的内皮细胞分化培养基,所述培养基还添加有6μMCHIR99021和0.2×Pen/Strep。
根据本发明具体实施方式的非动物源性的内皮细胞分化培养基,所述培养基还添加有0.2×Pen/Strep、50 ng/ml FGF2、50 ng/m VEGFA、10 ng/ml EGF和10 μM SB431542。
本发明提供上述非动物源性的内皮细胞分化培养基的应用。
例如,根据本发明具体实施方式的诱导人胚胎干细胞分化内皮细胞的方法,所述方法利用上述培养基作为基础培养基诱导人胚胎干细胞分化内皮细胞。
根据本发明具体实施方式的诱导人胚胎干细胞分化内皮细胞的方法,所述方法包括以下步骤:
1)取传代后的干细胞,使用所述培养基培养,每隔24 h进行一次换液,该步骤持续72 h;
3)72 h后,分化所得细胞即为中胚层干细胞MPCs;
4)包被1块6孔板;
5)进行中胚层干细胞MPCs的消化,在血球计数板中进行计数;弃去离心管中的上清,用所述培养基重悬细胞以使细胞密度为1×105-2.5×105/ml,轻摇离心管使细胞混匀;
6)将5)中的细胞悬液以每孔2 ml加入6孔板中,上下左右移动培养板10次以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养。
7)每隔24 h进行一次换液,步骤6)与步骤7)共计持续72 h;
8)72 h后,分化得到内皮细胞。
优选的,步骤(1)中,所述培养基的组成为:DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C、1×ITS-X、6μMCHIR99021和0.2×Pen/Strep。
优选的,步骤(5)中,所述培养基的组成为:DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C、1×ITS-X、0.2×Pen/Strep、50 ng/ml FGF2、50 ng/m VEGFA、10 ng/ml EGF和10 μM SB431542。
根据本发明具体实施方式的诱导人胚胎干细胞分化内皮细胞的方法,还包括干细胞的复苏的步骤,
干细胞维持培养基配制
总体积 | 终浓度 | 500 ml | 100 ml | 50 ml |
ncEpic Basal Medium | - | 495 ml | 99 ml | 49.5 ml |
ncEpic Supplement (125×) | 1× | 4 ml | 800 μl | 400 μl |
Pen/Strep (100×) | 0.2× | 1 ml | 200 μl | 100 μl |
1)在6孔板中以 1 μg/cm2加入用DMEM/F12稀释后的Vitronectin(VTN),轻摇6孔板使溶液均匀铺于孔中,室温包被培养板至少30 min;
2)从液氮中取出1管冻存好的hPSC细胞系H1;
3)用镊子夹持细胞在37℃水浴锅中迅速摇晃解冻,将解冻细胞转移至50 ml离心管中;
4)用10 ml移液管吸取10 ml DMEM/F12培养基,缓慢滴加至含有解冻细胞的50 ml离心管中,同时持续摇晃离心管使溶液混匀;全部滴加完毕后,在离心机中以300 g离心3min;
5)弃去上清,用4 ml干细胞维持培养基重悬细胞,同时在细胞悬液中添加终浓度为5 μM的Y27632,轻摇离心管使细胞混匀;
6)弃去包被好的培养板中的DMEM/F12,将5)中的细胞悬液以每孔2 ml加入6孔板的2个孔中,上下左右移动培养板10次以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养;
S2. 干细胞的换液
每隔24 h,弃去6孔板中的培养基,并按每孔2 ml更换干细胞培养基。
S3. 干细胞的传代
当细胞密度达到80-90%时,进行干细胞的传代;
1)以S1中的步骤1)操作包被1块6孔板。
2)弃去6孔板中的培养基,按每孔2 ml加入1×PBS洗涤,轻摇6孔板洗去孔中的死细胞,随后弃去PBS;
3)按每孔1 ml加入0.5 mM EDTA,将细胞放回37℃ 5% CO2细胞培养箱中;
4)6 min后取出细胞,轻柔吹打使细胞脱落并形成均匀的细胞悬液,每孔加入1 mlDMEM/F12终止消化,将细胞转移至离心管中。在离心机中以300 g离心3 min;
5)弃去离心管中上清液,并用12 ml干细胞维持培养基重悬细胞,同时在细胞悬液中添加终浓度为5 μM的Y27632,轻摇离心管使细胞混匀;
6)弃去包被好的培养板中的DMEM/F12,将5)中的细胞悬液以每孔2 ml加入6孔板中,上下左右移动培养板10次以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养。
本发明的有益效果:
本发明针对分化SC-ECs所需的必须成分展开研究,发现仅需以DMEM/F12 、1×GlutaMAX 、维生素C和ITS-X作为基础培养基即能分化生成SC-ECs,达到无异种成分分化的目的,并且分化效率高。
本发明采用DMEM/F12并额外添加1×GlutaMAX,1×ITS-X和100 μg/ml维生素C作为基础培养基,用Wnt通路抑制剂CHIR99021作用3天,再用50 ng/ml FGF2,50 ng/mlVEGFA,10 ng/ml EGF和10 μM SB431542作用3天,能有效诱导hPSCs向ECs分化。在该分化策略中,从干细胞培养至内皮细胞分化均处在无异种成分的培养基及基质中,批次之间差异较小,所得到的细胞成分明确,具有临床应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1显示本发明的培养基与LaSR培养基分化内皮细胞流式分析结果;
图2显示免疫细胞化学染色结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1在无动物源性且化学成分确定环境下进行干细胞培养
1.1 干细胞的复苏
表 1干细胞维持培养基配制
1)在6孔板中以 1 μg/cm2加入用DMEM/F12稀释后的Vitronectin(VTN),轻摇6孔板,使溶液均匀铺于孔中,室温包被培养板至少30 min。
2)从液氮中取出1管冻存好的hPSC细胞系H1。
3)用镊子夹持细胞在37℃水浴锅中迅速摇晃解冻,将解冻细胞转移至50 ml离心管中。
4)用10 ml移液管吸取10 ml DMEM/F12培养基,缓慢滴加至含有解冻细胞的50 ml离心管中,同时持续摇晃离心管使溶液混匀。全部滴加完毕后,在离心机中以300 g离心3min。
5)弃去上清,用4 ml干细胞维持培养基重悬细胞,同时在细胞悬液中添加终浓度为5 μM的Y27632,轻摇离心管使细胞混匀。
6)弃去包被好的培养板中的DMEM/F12,将5)中的细胞悬液以每孔2 ml加入6孔板的2个孔中,上下左右移动培养板10次,以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养。
1.2 干细胞的换液
每隔24 h,弃去6孔板中的培养基,并按每孔2 ml更换干细胞培养基。
1.3 干细胞的传代
当细胞密度达到80-90%时,进行干细胞的传代。
1)以1.1中的步骤1)操作包被1块6孔板。
2)弃去6孔板中的培养基,按每孔2 ml加入1×PBS洗涤,轻摇6孔板洗去孔中的死细胞,随后弃去PBS。
3)按每孔1 ml加入0.5 mM EDTA,将细胞放回37℃ 5% CO2细胞培养箱中。
4)6 min后取出细胞,轻柔吹打使细胞脱落并形成均匀的细胞悬液,每孔加入1 mlDMEM/F12终止消化,将细胞转移至离心管中,在离心机中以300 g离心3 min。
5)弃去离心管中上清液,并用12 ml干细胞维持培养基重悬细胞,同时在细胞悬液中添加终浓度为5 μM的Y27632,轻摇离心管使细胞混匀。
6)弃去包被好的培养板中的DMEM/F12,将5)中的细胞悬液以每孔2 ml加入6孔板中,上下左右移动培养板10次以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养。
实施例2确定本发明基础培养基的必要成分
2.1 内皮细胞的分化
表 2阶段1内皮细胞分化培养基配制
总体积 | 终浓度 | 500 ml | 100 ml | 50 ml |
DMEM/F12 | - | 481-489 ml | 96.4-98 ml | 48.2-49 ml |
GlutaMAX (100×) | 1× | 5 ml | 1 ml | 500 μl |
Vc (25 mg/ml) | 0-400 μg/ml | 0-8 ml | 0-1.6 ml | 0-0.8 ml |
ITS-X (100×) | 1× | 5 ml | 1 ml | 500 μl |
Pen/Strep (100×) | 0.2× | 1 ml | 200 μl | 100 μl |
CHIR99021 (20 mM) | 6 μM | 150 μl | 30 μl | 15 μl |
表 3阶段2内皮细胞分化培养基配制
总体积 | 终浓度 | 500 ml | 100 ml | 50 ml |
DMEM/F12 | - | 480-488 ml | 96.4-98 ml | 48.2-49 ml |
GlutaMAX (100×) | 1× | 5 ml | 1 ml | 500 μl |
Vc (25 mg/ml) | 0-400 μg/ml | 0-8 ml | 0-1.6 ml | 0-0.8 ml |
ITS-X (100×) | 1× | 5 ml | 1 ml | 500 μl |
Pen/Strep (100×) | 0.2× | 1 ml | 200 μl | 100 μl |
FGF2 (100 μg/ml) | 50 ng/ml | 250 μl | 50 μl | 25 μl |
VEGFA (100 μg/ml) | 50 ng/ml | 250 μl | 50 μl | 25 μl |
EGF (250 μg/ml) | 10 ng/ml | 20 μl | 4 μl | 2 μl |
SB431542 (20 mM) | 10 μM | 250 μl | 50 μl | 25 μl |
注:当更换不同细胞系进行分化时,需按照此方案确定针对不同细胞系的最佳Vc浓度。
1)按1.3中方法进行干细胞的传代。
2)传代24 h后,更换培养基为阶段1内皮细胞分化培养基,每隔24 h进行一次换液,该步骤持续72 h。
3)72 h后,分化所得细胞即为中胚层干细胞(Mesoderm Progenitor Cells,MPCs)。
4)以1.1中的步骤1)操作包被1块6孔板。
5)MPCs的消化:按1.3中步骤1)-步骤5)进行,在血球计数板中进行计数。弃去离心管中的上清,用适当体积的阶段2内皮细胞分化培养基重悬细胞,使细胞密度为1×105-2.5×105/ml,轻摇离心管使细胞混匀。
6)弃去包被好的培养板中的DMEM/F12,将5)中的细胞悬液以每孔2 ml加入6孔板中,上下左右移动培养板10次,使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养。
7)每隔24 h进行一次换液,步骤6)与步骤7)共计持续72 h。
8)72 h后,所得细胞中含有一定比例的内皮细胞。
该分化方案重复3次确保分化效率的稳定及数据可信度,以确定以DMEM/F12 + 1×GlutaMAX + 1×ITS-X作为培养基成分时的最佳Vc浓度。
设置以LaSR作为基础培养基的Control组,保证细胞拥有正常的起始状态。
用流式细胞技术评估分化效率
1)按照2.1中方案进行分化后,按1.3中步骤1)-步骤5)消化细胞;
2)在离心机中以300 g离心3 min,弃去上清,用FACS buffer(组成:1×PBS,5 g/LBSA,2 mM EDTA)重悬细胞,重复离心操作。
3)弃去上清,用100 μl FACS buffer重悬细胞,加入1 μl APC Anti-Human CD31与1 μl PE Anti-Human CD144,混匀,4℃避光染色15 min。
4)加入1 ml FACS buffer,在离心机中以300 g离心3 min。
5)弃去上清,用500 μl FACS buffer重悬细胞并用带有滤网的流式上样管过滤。
6)打开Guava easyCyte流式细胞仪,按操作说明清洗仪器。
7)上样后调整FSC-H,SSC-H,Yellow-B和Red-R通道的电压及增益强度,使细胞位于图中。圈出单细胞。
8)分析Yellow-B(即荧光基团PE信号)通道与Red-R(即荧光基团APC信号)通道信号强度。
9)利用软件FlowJo分析,其中Yellow-B与Red-R通道均为阳性的细胞即为CD31+CD144+的内皮细胞。记录双阳性细胞的占比作为内皮分化效率。
表4Vc的浓度对分化效率的影响
*Control组即为以LaSR作为基础培养基的阳性对照组。
如表4所示,随Vc浓度逐渐升高,内皮分化效率随之升高并趋于稳定,当添加浓度为100 μg/ml以上时,分化效率无明显差异,故100 μg/ml为合适的Vc浓度。
探究BSA是否为必要成分
1)按2.1所述进行内皮细胞的分化,设置对照组使用LaSR作为基础培养基,阴性对照为添加同种属非特异IgG进行染色的对照组,实验组使用DMEM/F12 + 1×GlutaMAX +100 μg/ml Vc + 1×ITS-X + 0.2×Pen/Strep作为基础培养基。
2)按2.2所述进行染色及流式细胞分析,本部分使用的抗体为CD144-PE。
如图1所示,使用LaSR作为基础培养基时,内皮细胞分化效率为30.4%,使用本发明的基础培养基,在添加100 μg/ml Vc情况下,内皮细胞分化效率为28.2%。故BSA并非内皮分化体系中的必须成分。
利用免疫细胞化学进一步验证该方案所得SC-ECs的标志基因
1)按最佳分化方案(DMEM/F12 + 1×GlutaMAX + 100 μg/ml Vc + 1×ITS-X +0.2×Pen/Strep作为基础培养基)在12孔板中进行内皮细胞分化;
2) 使用CD31-PE抗体与Human anti-PE Nanobeads(Biolegend,货号480092)按照说明书方法进行内皮细胞纯化,将纯化出的内皮细胞铺入明胶包被的48孔板中。
3)弃去培养基上清,加入200 μl 1×PBS洗涤3次。
4)加入100 μl 4%多聚甲醛室温固定15 min。
5) 弃去多聚甲醛,加入200 μl 1×PBS洗涤3次,加入10 μl 2% BSA室温封闭30min。
6)弃去BSA,在2% BSA中稀释CD31及CD144一抗,加入孔中室温染色30 min。
7)弃去含有一抗的BSA,加入200 μl 1×PBS洗涤3次,按1:500比例在2% BSA中稀释Donkey anti-rabbit Alexa Fluor 488及Donkey anti-mouse Alexa Fluor 568荧光二抗,加入孔中室温避光染色15 min。
8)弃去含有二抗的BSA,用200 μl 1×PBS洗涤3次,加入100 μl 1×4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)室温避光复染10 min。
9)用200 μl 1×PBS洗涤3次后在荧光显微镜下观察。
结果如图2所示,分化所得内皮细胞广泛表达内皮细胞标志基因CD31与CD144,说明分化成功。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (9)
1.一种非动物源性的内皮细胞分化培养基,其特征在于,所述培养基包括以下组分:
DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C和1×ITS-X。
2. 根据权利要求1所述的非动物源性的内皮细胞分化培养基,其特征在于,维生素C的浓度为50-100 μg/ml。
3.根据权利要求1所述的非动物源性的内皮细胞分化培养基,其特征在于,所述培养基还添加有6μMCHIR99021和0.2×Pen/Strep。
4. 根据权利要求1所述的非动物源性的内皮细胞分化培养基,其特征在于,所述培养基还添加有0.2×Pen/Strep、50 ng/ml FGF2、50 ng/m VEGFA、10 ng/ml EGF和10 μMSB431542。
5.权利要求1-4任一项所述的非动物源性的内皮细胞分化培养基的应用。
6.诱导人胚胎干细胞分化内皮细胞的方法,其特征在于,所述方法包括利用权利要求1-4任一项所述的培养基作为基础培养基诱导人胚胎干细胞的步骤。
7.根据权利要求6所述的诱导人胚胎干细胞分化内皮细胞的方法,其特征在于,所述方法包括以下步骤:
1)取传代后的干细胞,使用所述培养基培养,每隔24 h进行一次换液,该步骤持续72h;
3)72 h后,分化所得细胞即为中胚层干细胞MPCs;
4)包被1块6孔板;
5)进行中胚层干细胞MPCs的消化,在血球计数板中进行计数;弃去离心管中的上清,用所述培养基重悬细胞以使细胞密度为1×105-2.5×105/ml,轻摇离心管使细胞混匀;
6)将5)中的细胞悬液以每孔2 ml加入6孔板中,上下左右移动培养板10次以使细胞均匀铺在孔中,将细胞放于37℃ 5% CO2细胞培养箱中培养;
7)每隔24 h进行一次换液,步骤6)与步骤7)共计持续72 h;
8)72 h后,分化得到内皮细胞。
8. 根据权利要求7所述的诱导人胚胎干细胞分化内皮细胞的方法,其特征在于,步骤(1)中,所述培养基的组成为:DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C、1×ITS-X、6μMCHIR99021和0.2×Pen/Strep。
9. 根据权利要求7所述的诱导人胚胎干细胞分化内皮细胞的方法,其特征在于,步骤(5)中,所述培养基的组成为:DMEM/F12、1×GlutaMAX、25-200 μg /ml维生素C、1×ITS-X、0.2×Pen/Strep、50 ng/ml FGF2、50 ng/m VEGFA、10 ng/ml EGF和10 μM SB431542。
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