CN115386551A - 小鼠骨髓造血干/祖细胞体外衰老细胞模型的应用 - Google Patents

小鼠骨髓造血干/祖细胞体外衰老细胞模型的应用 Download PDF

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CN115386551A
CN115386551A CN202210836566.2A CN202210836566A CN115386551A CN 115386551 A CN115386551 A CN 115386551A CN 202210836566 A CN202210836566 A CN 202210836566A CN 115386551 A CN115386551 A CN 115386551A
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张丽娜
胡海燕
顾伟梁
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Shanghai University of Traditional Chinese Medicine
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Abstract

本发明涉及生物技术以及医药领域，具体小鼠骨髓造血干/祖细胞的体外衰老细胞模型的应用。小鼠骨髓造血干/祖细胞体外衰老细胞模型可用于建立骨髓增生异常综合征病理模型，筛选治疗骨髓增生异常综合征的药物。经本发明研究发现，HSPC衰老在MDS发病机制中有重要作用。HSPC衰老的变化与MDS在定性上相似。

Description

小鼠骨髓造血干/祖细胞体外衰老细胞模型的应用
技术领域
本发明涉及生物技术以及医药领域，涉及小鼠骨髓造血干/祖细胞的体外衰老细胞模型的应用。
背景技术
虽然血液是确定的自我更新组织，但造血依然难逃衰老。造血衰老在人群中表现为恶性髓系血液病的增加(如：骨髓增生异常综合征、白血病等)、机体衰老、免疫功能障碍等。
因此，开发延缓造血衰老的药物，对防治相关疾病有着重要的理论意义和应用价值。哺乳动物的血液系统由多种类型的成熟血细胞组成，如：红细胞、T细胞、B细胞等。但这些血细胞大多是短命的，需要依靠造血祖细胞(hematopoietic progenitor cells，HPCs)和最终由造血干细胞(hematopoietic stem cells，HSCs)来替代。以往研究也表明，造血干/祖细胞衰老是机体发生造血衰老的首要因素。
造血干/祖细胞衰老模型是造血衰老研究的重要平台。迄今为止，文献所报道的体外造血干/祖细胞衰老模型主要包括如下几种：衰老动物的衰老造血干/祖细胞模型，白消安体外诱导的衰老造血干/祖细胞模型，辐射导致的衰老造血干/祖细胞模型，t-BHP体外诱导的衰老造血干/祖细胞模型等。但是，这些方法都不同程度地存在着或操作步骤繁琐或致老效果不佳等局限。
发明人在前期工作中，建立了一种可以快速简便地大量获得衰老HSPC的细胞模型(专利申请号：201711157564.6，公开号CN107858330A)公开了一种小鼠骨髓造血干/祖细胞体外衰老细胞模型的建立方法，可以快速获得大量衰老的体外造血干细胞/祖细胞。此模型是一个可以快速简便地大量获得衰老HSPCs的细胞模型，甚至可以说是一个在一定程度上加速加剧体内HSPCs衰老的体外HSPCs衰老模型。
经过研究提示，在各种血液性疾病中，包括骨髓增生异常综合征(myelodysplastic syndromes，MDS)、获得性再生障碍性贫血(AA)、纯红细胞再生障碍(PRCA)、获得性无巨核细胞血小板减少症(AATP)、阵发性睡眠性血红蛋白尿(PNH)等，与造血衰老最相关的疾病可能是MDS。
MDS是一组后天获得性、异质性、髓系克隆性的血液疾病，其基本病变是HSC/HSPC发育异常，导致无效造血及高风险向恶性血液病如急性髓系白血病(AML)转化。MDS呈现高度的异质性，临床表现差异很大，总体预后较差，其致病机制复杂，至今未能明确。临床资料显示，MDS发病年龄呈上升趋势，中间年龄77岁。但是，MDS的发病机制复杂，目前尚未明确。
如果能够找到一种细胞模型，用来建立骨髓增生异常综合征病理模型，可用来研究病症机制，开发筛选相关的药物。
发明内容
本发明旨在提供一种MDS的细胞病理模型，将小鼠骨髓造血干/祖细胞(HSPC)体外衰老细胞模型可用于建立骨髓增生异常综合征病理模型。
CFU-Mix和SA-β-Gal两个指标用于评价HSPC衰老，发现：无论在MDS病人组HSPC中还是在小鼠HSPC衰老模型HSPC中，SA-gal阳性率明显升高，CFU-Mix集落数明显减少，与衰老密切相关的P21 mRNA表达显著升高，“aging”是RNA-seq GO分析中显著富集到的Term，“Cellular Senescence”是RNA-seq KEGG分析中显著富集到的Pathway。因此，这种小鼠造血干祖细胞衰老细胞模型能够用于建立骨髓增生异常综合征病理模型，并且能够用于筛选治疗骨髓增生异常综合征的药物。因此，该模型可以用作MDS病理模型。
本发明技术方案：
小鼠骨髓造血干/祖细胞体外衰老细胞模型可用于建立骨髓增生异常综合征病理模型。
小鼠骨髓造血干/祖细胞体外衰老细胞模型可用于筛选治疗骨髓增生异常综合征的药物。
优选的，所述的小鼠骨髓造血干/祖细胞体外衰老细胞模型的建立方法如CN107858330A，包括以下步骤：
小鼠骨髓细胞经过lineage阴性富集和CD117阳性筛选，分离获取得到小鼠骨髓干/祖细胞，其中以造血祖细胞为主；所述的lineage阴性富集为CD5、CD45R/B220、CD11b、Anti-Gr-1、7-4和Ter-119阴性富集；用加入IL3、IL6和SCF的干细胞培养基培养5～10天。
所述的干细胞培养基中，IL3的含量为8～20ng/mL，IL6的含量为8～20ng/mL，SCF的含量为25～60ng/mL。
优选的，所述的干细胞培养基中，IL3的含量为9～12ng/mL，IL6的含量为9～12ng/mL，SCF的含量为27～35ng/mL。
培养过程中，每2～3天更换干细胞培养基。所述的干细胞培养基为无血清培养基。
所述的干细胞培养基中还含有链霉素和青霉素。
临床资料显示，MDS发病年龄呈上升趋势，中间年龄77岁。但是，MDS的发病机制复杂，目前尚未明确。我们明确了HSPC衰老在MDS发病机制中的作用，并在本研究中结合MDS患者的HSPC进行了研究，结果显示，MDS患者的hspc SA-gal阳性率明显升高，CFU-Mix集落数量明显减少。与衰老密切相关的P21 mRNA表达在MDS组和小鼠造血干祖细胞衰老模型组中均显著升高。RNA-seq数据显示衰老是GO分析中差异显著的“Term”之一，而细胞衰老途径是KEGG分析中差异显著的“Pathway”之一。这与小鼠造血干/祖细胞体外衰老模型的表现相同。
因此，小鼠HSPC衰老细胞模型与MDS患者的HSPC细胞衰老机制相同，本发明将小鼠造血干祖细胞衰老模型作为MDS病理模型，可用来探究血液病MDS的造血干祖细胞衰老机制，以期为临床开发防治造血衰老相关血液病MDS的药物提供实验支持。
附图说明
图1为MDS患者的代表性骨髓涂片
图2为正常组HSPC细胞、MDS组HSPC细胞和小鼠造血干祖细胞衰老细胞模型HSPC细胞的SA-β-gal染色结果
图3为正常组HSPC细胞、MDS组HSPC细胞和小鼠造血干祖细胞衰老细胞模型HSPC细胞的的CFU-Mix试验结果
图4为MDS患者RNA-seq中GO分析柱状图所示显著差异基因富集到的25个Term
图5为MDS患者RNA-seq中GO分析气泡图
图6为MDS患者RNA-seq中KEGG分析柱状图所示显著差异基因富集到的25个Pathway
图7为MDS患者RNA-seq中KEGG分析气泡图
图8为MDS患者HSPC中细胞衰老途径(Cellular Senescence pathway)变化
图9为MDS患者与正常人(体内)的RNA-Seq数据(MvsN)，以及小鼠造血干/祖细胞体外细胞衰老模型(Aging Model hspc)与年轻hspc(体外)的RNA-Seq数据(YoungvsD8)联合分析：9A为重叠基因数量，9B为重叠GO Terms数量，9C为重叠KEGG pathway数量
具体实施方式
实施例1
MDS患者的代表性骨髓涂片如图1所示(瑞士染色，10×100×油)，结果显示：1.骨髓有核细胞增生明显；2.粒系增生明显，原始粒细胞占16.0％，中性粒细胞比例增高，发育形态异常，如双分子层肿瘤、空泡、细胞核和细胞质发育不平衡；3.红细胞增生减少，可见核芽接等异常发育形态。MDS患者外周血细胞减少持续4个月以上，不包括感染、免疫等其他可导致血细胞减少和发育异常的疾病。骨髓中粒细胞系和红细胞系异常比例均在10％以上；诊断为MDS(EB-2)。
采用CFU-Mix和SA-β-Gal评价HSPC衰老。研究MDS病人的HSPC，发现MDS病人的HSPC出现了Gal阳性率显著升高及CFU-Mix集落数显著降低的变化，这与通过CN107858330A方法所建立的小鼠造血干/祖细胞衰老模型是一致的。如图2和图3所示。
SA-β-gal是衰老的标志，可在衰老细胞的细胞质中产生蓝色染色。我们的结果显示，MDS组SA-β-gal染色阳性细胞比例显著高于对照组，P<0.01，P<0.05。蓝染细胞(衰老细胞)比非衰老细胞大，多数呈形态学异常，这与小鼠造血干/祖细胞衰老模型(培养8天后)的结果一致。
CFU-Mix试验用于了解不同发育阶段的造血祖细胞的频率和生长特性，被称为菌落形成单位(cfu)的单个祖细胞增殖和分化形成离散的细胞群或菌落包含可识别的后代。每个菌落来自一个单一的祖先。因此，在CFU试验中计数的菌落数量和类型提供了有关原始细胞群中祖细胞的频率和类型及其增殖和分化能力的信息。由图3可见，MDS患者组的细胞相比于正常人HSPC所形成的肉眼明显可见的集落形成，未见集落形成，说明HSPC集落形成能力和多向分化能力显著下降。提示MDS组hspc增殖和分化能力较对照组明显降低，这也与小鼠造血干/祖细胞衰老模型的结果一致。
实施例2RNA组学实验
联合分析了MDS患者Vs正常人的RNA-Seq数据(用MvsN表示)和衰老模型hspc Vs年轻hspc(体外)的RNA-Seq数据(用Youngvs8D表示)。
分析发现，无论在GO数据库或者是在KEGG数据库，衰老途径是正常人HSPC和MDS病人的HSPC差异基因均可富集到的显著差异的途径。
MDS患者的RNA-seq分析显示，衰老(Aging)是GO分析中差异显著的“term”之一(如图4、5)，细胞衰老途径(Cellular Senescence pathway)是KEGG分析中差异显著的"Pathway"之一(如图6、7)。Aging Term和Cellular Senescence pathway中不同表达的Top25基因结果见表1和表2。MDS患者RNA-seq中GO分析柱状图、气泡图如图4和图5，即为显著差异基因富集到的25个Term，Aging位于其中。MDS患者RNA-seq中KEGG分析柱状图、气泡图如图6和图7，即为显著差异基因富集到的25个Pathway，Cellular Senescence pathway位于其中。细胞衰老途径如图8所示。
Tab.1 Top25 differently expressed genes in GO aging Term
表1 GO衰老阶段表达最显著的25个基因
Figure BDA0003748613320000051
Figure BDA0003748613320000061
Figure BDA0003748613320000071
Tab.2 Top25 differently expressed genes in KEGG cellular senescencepathway
表2 KEGG细胞衰老途径表达最显著的25个基因
Figure BDA0003748613320000072
Figure BDA0003748613320000081
我们结合了MDS(M)与正常细胞(体内)(N)的RNA-Seq数据(MvsN)，以及培养了8天的小鼠造血干/祖细胞体外细胞衰老模型(Aging Model hspc)(D8)与年轻hspc(体外)(Young)的RNA-Seq数据(YoungvsD8)，联合分析结果显示，重叠基因数量为1444个(图9A)，重叠Go Terms数量为412个(图9B)，重叠KEGG pathway数量为22个(图9C)。与衰老密切相关的P21 mRNA表达在MDS组和衰老模型组中均显著升高(P<0.05)，见表3和图9。P53信号通路在MDS组和衰老模型组中均为差异基因显著富集到的通路(P<0.05)，见表4。因此，我们在体内和体外的研究一致表明，与衰老密切相关的P21的mRNA表达显著增加。研究表明，p21的上调是诱导细胞衰老的主要反应。P53通过抑制P53下游靶基因p21/CDKN1A的表达来抑制衰老。
Table.3 Comparison of P21 mRNA expression in RNA-seq analysis
表3 RNA-seq分析中P21 mRNA表达的比较
Figure BDA0003748613320000091
Table.4 Comparison of P53 Pathway in RNA-seq analysis
表4 RNA-seq分析的P53途径比较
Figure BDA0003748613320000092

Claims (6)

1.小鼠骨髓造血干/祖细胞体外衰老细胞模型在建立骨髓增生异常综合征病理模型方面的应用。
2.小鼠骨髓造血干/祖细胞体外衰老细胞模型在筛选治疗骨髓增生异常综合征的药物方面的应用。
3.根据权利要求1或2所述的应用，其特征在于，所述的小鼠骨髓造血干/祖细胞体外衰老细胞模型的建立方法包括以下步骤：
小鼠骨髓细胞经过lineage阴性富集和CD117阳性筛选，分离获取得到小鼠骨髓干/祖细胞，其中以造血祖细胞为主；所述的lineage阴性富集为CD5、CD45R/B220、CD11b、Anti-Gr-1、7-4和Ter-119阴性富集；
用加入IL3、IL6和SCF的干细胞培养基培养5～10天；
所述的干细胞培养基中，IL3的含量为8～20ng/mL，IL6的含量为8～20ng/mL，SCF的含量为25～60ng/mL。
4.根据权利要求3所述的应用，其特征在于，所述的干细胞培养基中，IL3的含量为9～12ng/mL，IL6的含量为9～12ng/mL，SCF的含量为27～35ng/mL。
5.根据权利要求1或2所述的应用，其特征在于，培养过程中，每2～3天更换干细胞培养基。
6.根据权利要求5所述的应用，其特征在于，所述的干细胞培养基为无血清培养基。
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CN107858330A (zh) * 2017-11-20 2018-03-30 上海中医药大学 一种小鼠骨髓造血干/祖细胞体外衰老细胞模型的建立方法

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TONY A. NAVAS 等: "Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors", BLOOD, vol. 108, no. 13, pages 4170 - 4177 *
YONG WANG, 等: "Inhibition of p38 Mitogen-Activated Protein Kinase Promotes Ex Vivo Hematopoietic Stem Cell Expansion", STEM CELLS AND DEVELOPMENT, vol. 20, no. 7, pages 1143 - 1152, XP055446478, DOI: 10.1089/scd.2010.0413 *
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