CN110373387B - 组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途 - Google Patents
组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途 Download PDFInfo
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Abstract
本发明公开了组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途。本发明可促使每104人多能干细胞分化产生15.7％的CD31+CD43+的造血干祖细胞，比对照组高了近4倍，造血干祖细胞生成的效率也显著高于现有技术中其他小分子促进后产生造血干祖细胞的一般范围，且处理时间短，可操作性和重复性更强，可实现体外高效、快速产生造血干祖细胞。本发明通过分化早期添加已经成药的HDAC抑制剂，这种低成本、高度可控的无血清、无基质高效产生造血干祖细胞的方式为将来大规模生产用于临床治疗的功能性血细胞奠定了基础。
Description
技术领域
本发明涉及生物技术领域，特别涉及组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途。
背景技术
“血荒”(blood supply shortage)是指出现血液偏型或告急的现象。近年来，内地70个大中城市中，“血荒”报道的已经多达40个。血细胞的直接输注是缓解血液病患者和外伤出血等缺血的有效手段，而国内“血荒”以及血制品污染、免疫排斥等问题，导致血液来源不足，为血液病治疗带来了困扰。因此，没有免疫排斥的自体干细胞在体外规模化生产功能性血细胞是我国面临的重大战略需求。
血细胞是由具有自我更新潜能和多向分化能力的造血干祖细胞HSPC不断产生的。迄今为止，尚未建立统一且高效的方法用于体外高效产生功能性血细胞。为了得到足够的高质量和大量的血细胞，优化体外人多能干细胞造血分化条件以促进HSPC的产生是一个重要的研究方向。HSPC在胚胎发育的过程中，由位于主动脉-性腺-中肾区(AGM)的背主动脉腹侧壁的生血内皮(HE)产生。在妊娠中期，HE细胞通过进化上高度保守的内皮-造血细胞转换(EHT)过程产生HSPC。类似的，人多能干细胞体外造血分化主要经历中胚层细胞，生血内皮细胞最终产生造血干祖细胞。已知EHT是HSPC产生的重要过程，这一过程可以被TGFb信号通路调节。(Yzaguirre AD,Bruijn M F T R D,Speck N A.The Role of Runx1 inEmbryonic Blood Cell Formation[J].Advances in Experimental Medicine&Biology.)(Kwan W,Cortes M,Frost I,et al.The Central Nervous System Regulates EmbryonicHSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling[J].Cell Stem Cell,2016:S1934590916301540.)
小分子化合物指相对分子质量小于10000的化合物。相较于基因操作，小分子化合物可通过调节化合物结构、浓度使其具有简单可控可规模化的优势。相较于细胞因子，小分子化合物更为廉价和稳定。目前体外已经实现了在传统扩增HSC的方法中加入某些小分子化合物甚至能完全采用小分子化合物替代血清，细胞因子。[张宇,程涛,高瀛岱.小分子化合物扩增造血干细胞的研究进展[J].中华血液学杂志,2014,35(1):82-85.)]为了在体外建立高效、成分明确的产生造血干祖细胞的体系，邓宏魁团队建立了人多能干细胞体外小分子诱导产生造血干祖细胞的分化模型，但其产生的HSPCs(CD43+CD45+)仅有18％，而其发现的于EHT阶段添加的TGFb信号通路抑制剂SB-431542也仅能对HSPCs的产生有两倍的促进。[Wang C,Tang X,Sun X,et al.TGFβinhibition enhances the generation ofhematopoietic progenitors from human ES cell-derived hemogenic endothelialcells using a stepwise strategy[J].Cell Research,2012,22(1):194-207.]同时，那洁团队建立的人多能干细胞体外分化模型，同样在EHT阶段通过添加胰岛素能够产生27％的HSPCs。[Fuyu D,Rujin H,Fengzhi Z,et al.Biphasic modulation of insulinsignaling enables highly efficient hematopoietic differentiation from humanpluripotent stem cells[J].Stem Cell Research&Therapy,2018,9(1):205.]
目前现有技术中已有的种种尝试产生的造血干祖细胞的数量并不能满足临床需求。为了更加促进HSPCs的体外产生，寻找更加有效的小分子和调控造血发生的时间窗口十分必要。
发明内容
本发明的一个方面，是针对现有技术中利用小分子诱导产生造血干祖细胞的产量低的问题，提供了组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途。
本发明提供的技术方案为：
组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途。
本发明的发明人通过创造性劳动发现，通过在多能干细胞分化早期添加组蛋白去乙酰化酶抑制剂，能够显著提高造血干祖细胞的生成量，具有较高的可操作性与可重复性，为将来利用自体干细胞进行体外产生血细胞应用于生物医学和临床治疗奠定基础。
在本发明中，所述组蛋白去乙酰化酶抑制剂能够促进人或动物来源的多能干细胞分化为造血干祖细胞，所述动物例如，小鼠。但作为优选，在本发明的一个实施方式中，所述多能干细胞为人诱导多能干细胞。
上述人诱导多能干细胞可通过常规iPSC的制备方法获得。
在本发明中，所述组蛋白去乙酰化酶(HDAC)抑制剂是一类重要的抗肿瘤化合物。它能引起细胞周期的阻断和肿瘤细胞选择性凋亡，在体外细胞培养和动物中都被证明具有明显抗肿瘤作用。所述组蛋白去乙酰化酶抑制剂包括但不限于，例如，曲古抑菌霉素A(TSA，如式1所示)、Vorinostat(SAHA，如式2所示)、trapoxin B(如式3所示)、MS-275(如式4所示)、valproic(如式5所示)、romidepsin(FK-228，如式6所示)、Pracinostat(如式7所示)、Resminostate(如式8所示)。作为优选，在本发明的一个实施方式中，所述组蛋白去乙酰化酶抑制剂为选自Vorinostat或其衍生物、Pracinostat或其衍生物、Resminostate或其衍生物中的一种或几种。更优选地，所述组蛋白去乙酰化酶抑制剂为Vorinostat或其衍生物。
上述组蛋白去乙酰化酶抑制剂可以任意合适的比例联合使用，均能实现本发明的目的。
本发明还提供了一种促进多能干细胞分化为造血干祖细胞的方法，即在中胚层诱导阶段加入终浓度为50μM-100μM所述组蛋白去乙酰化酶抑制剂。
上述中胚层诱导阶段一般为进入诱导阶段的第0～2天。
本发明中所述的方法均为非治疗目的的。
作为优选，在本发明的一个实施方式中，上述方法包括以下步骤：
步骤1)将所述多能干细胞消化成单个细胞，在Rho激酶抑制剂存在的条件下37℃，5％CO2培养24小时；
步骤2)将步骤1)得到的多能干细胞更换培养基为含有终浓度为50μM-100μM的所述组蛋白去乙酰化酶抑制剂的培养基，在常规条件下诱导分化2天，所述组蛋白去乙酰化酶抑制剂的终浓度优选为100μM；
步骤3)将步骤2)得到的细胞与培养基混合物更换培养基为含有VEGF和bFGF的培养基继续诱导分化2天，每24小时更换新鲜的该培养基；
步骤4)将步骤3)得到的细胞与培养基混合物更换培养基为含有VEGF、bFGF和TGFb信号通路抑制剂的培养基继续培养4天，每24小时更换新鲜的该培养基；
步骤5)收集CD31+CD45+造血干祖细胞。
上述步骤2)开始为造血分化阶段，即中胚层诱导阶段。
作为优选，在本发明的一个实施方式中，步骤1)中所述的Rho激酶抑制剂为选自Y-27632、Y-230141、Y-39983、GSK429286A、GSK269962、RKI-1447、Thiazovivin、ZINC00881524、KD025、Fasudil、Hydroxyfasudil、Netarsudil或Ripasudil hydrochloridedihydrate中的一种或几种，更优选为Y-27632；
所述Rho激酶抑制剂的终浓度为5μM。
作为优选，在本发明的一个实施方式中，步骤2)所述含有终浓度为50μM-100μM的所述组蛋白去乙酰化酶抑制剂的培养基还包括激活素A和/或骨形态发生蛋白4；
其中，所述激活素A的终浓度为50ng/ml，骨形态发生蛋白4的终浓度为40ng/ml。
作为优选，在本发明的一个实施方式中，步骤3)或步骤4)中所述的VEGF的终浓度为40ng/ml，bFGF的终浓度为50ng/ml；
步骤4)中所述TGFb信号通路抑制剂为SB431542，其终浓度为20ng/ml。
本发明的另一个方面，是提供了一种药物制剂，所述药物制剂包含由上述方法制备的造血干祖细胞。
本发明的另一个方面，是提供了一种上述药物制剂在制备治疗再生障碍性贫血、白细胞减少症或骨髓增生异常综合征的药物中的用途。
本发明的有益效果为：
本发明可促使每104人多能干细胞分化产生15.7％的CD31+CD43+的造血干祖细胞，比对照组高了近4倍，造血干祖细胞生成的效率也显著高于现有技术中其他小分子促进后产生造血干祖细胞的一般范围(每104人多能干细胞产生2-4％)，且处理时间短，可操作性和重复性更强，可实现体外高效、快速产生造血干祖细胞。本发明通过分化早期添加已经成药的HDAC抑制剂，这种低成本、高度可控的无血清、无基质高效产生造血干祖细胞的方式为将来大规模生产用于临床治疗的功能性血细胞奠定了基础。
附图说明
图1为人多能干细胞造血分化第8天流式统计图，其中DMSO为未添加HDAC抑制剂的对照组，其他分别为加入HDAC抑制剂Vorinostat、Resminostate和Pracinostat的实验组；
图2为人多能干细胞造血分化第8天免疫荧光染色结果，其中DMSO为未添加HDAC抑制剂的对照组，其他分别为加入HDAC抑制剂Vorinostat、Resminostate和Pracinostat的实验组。
具体实施方式
本发明公开了一种组蛋白去乙酰化酶抑制剂在制备促进多能干细胞分化为造血干祖细胞的产品中的用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。需要特别指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明，并且相关人员明显能在不脱离本发明内容、精神和范围的基础上对本文所述内容进行改动或适当变更与组合，来实现和应用本发明技术。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。
实施例1：人多能干细胞的培养和传代
人多能干细胞(人胚胎干细胞系H1)利用mTeSRTM1(Stem Cell Technologies)培养基培养在覆盖了Mrigel(corning)基质胶的细胞培养板上以维持其自我更新。
克隆传代：对12孔板内生长较大的克隆加入500μl Dispase酶(Stem CellTechnologies)消化5分钟，吸弃酶液加入1ml DMEM/F12(Hyclone)培养基，将克隆吹下后打入15ml离心管，室温，350g离心5min。弃上清，加入mTeSRTM1重悬沉淀，调整密度传入覆盖了Mrigel基质胶的细胞12孔板，晃匀，在37℃、5％CO2孵箱中静置培养。
单细胞传代：对12孔板内生长较大的克隆加入500μl Accutase酶(Gibco)，37℃静置消化3分钟，加入1ml DMEM/F12(Hyclone)稀释后将克隆吹散成单细胞打入含有5mlDMEM/F12的15ml离心管，室温，350g离心5min。弃上清，加入mTeSRTM1重悬沉淀，加入5μM Rho激酶抑制剂Y-27632，以3×104/孔起始密度接种于12孔板，晃匀，在37℃、5％CO2孵箱中静置培养。
实施例2：人多能干细胞造血分化
1)人多能干细胞株消化成单细胞，添加Rho激酶抑制剂Y-27632后，以3.5×104个细胞/孔的密度单细胞传代种于Growth Factor Reduced Mrigel(corning)覆盖的12孔板上，晃匀，在37℃、5％CO2孵箱中静置培养。
2)24小时后吸弃培养基，添加含有40ng/ml ActivinA(Peprotech)和50ng/mlBMP4(Peprotech)和100μmVorinostat的Custom mTeSR1(StemCell Technologies)培养基诱导分化2天。
3)吸弃培养基，添加含有40ng/ml VEGF(Peprotech)和50ng/ml bFGF(Peprotech)的CustommTeSR1继续诱导分化2天，每24h更换新鲜培养基。
4)吸弃培养基，添加含有40ng/ml VEGF、50ng/mlbFGF和20μM SB 431542(STEMGENT)的Custom mTeSR1继续培养3天，每24h更换新鲜培养基。
实验例1：造血干祖细胞流式检测
1)在培养了8天的细胞中加入最后500μl Accutase酶，37℃静置消化5分钟，利用1ml DMEM/F12重悬后打入于1.5ml EP管，室温，350g离心5min。
2)弃上清，在避光条件下，每组样品用100μl 0.2％BSA重悬后加0.5μl anti-CD31-APC和anti-CD43-PE，在水平摇床上避光孵育30min后，上机(FACS Canto II；BDBiosciences)检测。
3)以IgG为阴性对照圈门，进行流式结果分析。
4)结果如图1所示，与DMSO对照组相比，早期添加HDAC抑制剂有效提高了CD31+CD43+的造血干祖细胞的比例。
实验例2：造血干祖细胞免疫荧光检测
1)在培养了8天的细胞中加入PBS清洗三次，洗去漂浮的死细胞。
2)加入1ml 4％PFA室温固定20min。
3)用PBS清洗三次，加入1ml 0.2％Triton X-100室温通透20min。
4)用PBS清洗三次，加入1ml 0.5％BSA于4℃封闭1小时。
5)用PBS清洗三次，加入PE-mouse anti-human CD43抗体(用0.5％BSA封闭液稀释，终浓度2ug/ml)，4℃过夜孵育。
6)用PBS清洗三次，加入DAPI(1:1000)孵育10min，利用双转盘显微成像。
7)结果如图2所示，与DMSO对照组相比，早期添加HDAC抑制剂显著促进了卵石样CD31+CD43+造血干祖细胞的产生。
本实施例中所有数据以x±s表示，组间比较采用SPSS10.0医学统计软件进行单因素方差分析和t检验，P＜0.05为差异显著，P＜0.0l为差异非常显著。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种促进多能干细胞分化为造血干祖细胞的方法，其特征在于，在中胚层诱导阶段加入终浓度为50μM-100μM的组蛋白去乙酰化酶抑制剂；
所述多能干细胞为人胚胎干细胞系H1；
所述组蛋白去乙酰化酶抑制剂为Vorinostat。
2.根据权利要求1所述的方法，其特征在于，所述促进多能干细胞分化为造血干祖细胞的方法为，包括以下步骤：
步骤1)将所述多能干细胞消化成单个细胞，在Rho激酶抑制剂存在的条件下37℃，5％CO2培养24小时；
步骤2)将步骤1)得到的多能干细胞更换培养基为含有终浓度为50μM-100μM的所述组蛋白去乙酰化酶抑制剂的培养基，在常规条件下诱导分化2天；
步骤3)将步骤2)得到的细胞与培养基混合物更换培养基为含有VEGF和bFGF的培养基继续诱导分化2天，每24小时更换新鲜的该培养基；
步骤4)将步骤3)得到的细胞与培养基混合物更换培养基为含有VEGF、bFGF和TGFb信号通路抑制剂的培养基继续培养至第3天，每24小时更换新鲜的该培养基；
步骤5)收集CD31+CD45+造血干祖细胞；
步骤1)中所述的Rho激酶抑制剂为Y-27632。
3.根据权利要求2所述的方法，其特征在于，步骤2)所述组蛋白去乙酰化酶抑制剂的终浓度为100μM。
4.根据权利要求2所述的方法，其特征在于，步骤1)中所述Rho激酶抑制剂的终浓度为5μM。
5.根据权利要求2所述的方法，其特征在于，步骤3)或步骤4)中所述的VEGF的终浓度为40ng/ml，bFGF的终浓度为50ng/ml；
步骤4)中所述TGFb信号通路抑制剂为SB431542，其终浓度为20ng/ml。
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