CN112391345B - 一种用于促进造血细胞增殖的组合物及其应用 - Google Patents
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Abstract
本发明提供了一种用于促进造血细胞增殖的组合物及其应用，涉及造血细胞增殖技术领域。本发明提供的组合物包括造血细胞因子和铁死亡抑制剂；所述造血细胞因子包括干细胞因子、Flt3配体、血小板生成素和白介素3；所述干细胞因子的浓度为20～100ng/ml，Flt3配体的浓度为10～50ng/ml，血小板生成素的浓度为10～50ng/ml，白介素3的浓度为10～50ng/ml；所述铁死亡抑制剂的摩尔浓度为0.1～10μmol/L。本发明提供的组合物是将造血细胞因子和铁死亡抑制剂联合使用通过上调谷胱甘肽过氧化酶4的表达，抑制活性氧的产生来促进造血干祖细胞的增殖。
Description
技术领域
本发明涉及造血干祖细胞增殖技术领域，具体涉及一种用于促进造血细胞增殖的组合物及其应用。
背景技术
造血干祖细胞是血液系统中的成体干细胞，是一个异质性的群体，具有长期自我更新的能力和分化成各类成熟血细胞的潜能。它是研究历史最长且最为深入的一类成体干细胞，对研究各类干细胞，包括肿瘤干细胞，具有重要指导意义。
造血干细胞移植已广泛用于恶性血液病、非恶性难治性血液病、遗传性疾病和某些实体瘤治疗。但造血干祖细胞来源有限，体外培养造血干细胞的过程中会出现细胞的死亡，其体外扩增仍然是难以突破大的瓶颈问题。
发明内容
有鉴于此，本发明的目的在于提供一种用于促进造血干细胞增殖的组合物及其应用。本发明通过将铁死亡抑制剂和生长因子联合使用可以促进造血干细胞的增殖。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种用于促进造血细胞增殖的组合物，包括造血细胞因子和铁死亡抑制剂；
所述造血细胞因子包括干细胞因子、Flt3配体、血小板生成素和白介素3；
所述干细胞因子的浓度为20～100ng/ml，Flt3配体的浓度为10～50ng/ml，血小板生成素的浓度为10～50ng/ml，白介素3的浓度为10～50ng/ml；
所述铁死亡抑制剂的摩尔浓度为0.1～10μmol/L。
优选地，所述干细胞因子的浓度为25ng/ml，Flt3配体的浓度为12.5ng/ml，血小板生成素的浓度为12.5ng/ml，白介素3的浓度为12.5ng/ml。
优选地，所述铁死亡抑制剂包括Ferrostatin-1、去铁胺、环吡酮胺、丁羟甲苯和Trolox中的一种或几种。
优选地，所述铁死亡抑制剂的摩尔浓度为0.5～1μmol/L。
本发明还提供了上述技术方案所述组合物在促进造血细胞增殖中的应用。
本发明还提供了上述技术方案所述组合物在制备上调谷胱甘肽过氧化酶4制剂中的应用。
本发明还提供了上述技术方案所述组合物在制备抑制活性氧的产生的制剂中的应用。
本发明提供了一种用于促进造血细胞增殖的组合物及其应用。本发明提供的铁细胞抑制剂能够逆转造血细胞因子缺乏导致的细胞死亡。在本中发明，将造血细胞因子和铁死亡抑制剂联合使用能够通过上调谷胱甘肽过氧化酶4的表达，抑制活性氧的产生来促进造血细胞的增殖。
附图说明
图1为Fer-1可逆转erastin诱导的TF-1细胞的铁死亡结果图；其中，条形图从左至右依次为TF-1，TF-1+Fer-1(0.5μmmol/L)；
图2为Erastin下调TF-1细胞GPX4蛋白表达结果图；
图3为rastin诱导TF-1细胞ROS的释放，并可被Fer-1逆转结果图；
图4为逆转GM-CSF缺乏导致的细胞死亡结果图；其中，条形图从左至右依次为GM-CSF(0ng/ml)，GM-CSF(5ng/ml)；
图5为GM-CSF和Fer-1上调GPX4蛋白结果图；
图6为GM-CSF和Fer-1抑制TF-1细胞ROS产生结果图；其中，图6中的条形图从左至右依次为GM-CSF(5ng/ml)，GM-CSF(0ng/ml)；
图7为人CD34+细胞在去生长因子的条件下会发生细胞死亡结果图；其中，图7中a从左至右的条形图依次为DMSO，0.5μmmol/LFer-1，1μmmol/L Fer-1；图7中a的下方条形图从左至右依次为去生长因子的CD34+细胞，右侧为低浓度生长因子培养的CD34+细胞；
图8为人CD34+细胞在去生长因子的条件下会发生细胞，Fer-1可逆转细胞的铁死亡结果图；其中，从左至右的条形图依次为去生长因子的CD34+细胞，低浓度生长因子培养的CD34+细胞；
图9为Fer-1上调人骨髓CD34+细胞GPX4蛋白表达结果图。
具体实施方式
本发明提供了一种用于促进造血细胞增殖的组合物，包括造血细胞因子和铁死亡抑制剂；
所述造血细胞因子包括干细胞因子、Flt3配体、血小板生成素和白介素3；
所述干细胞因子的浓度为20～100ng/ml，Flt3配体的浓度为10～50ng/ml，血小板生成素的浓度为10～50ng/ml，白介素3的浓度为10～50ng/ml；
所述铁死亡抑制剂的摩尔浓度为0.1～10μmol/L。
在本发明中，所述干细胞因子的浓度优选为25ng/ml，Flt3配体的浓度优选为12.5ng/ml，血小板生成素的浓度优选为12.5ng/ml，白介素3的浓度优选为12.5ng/ml。
在本发明中，所述铁死亡抑制剂优选包括Ferrostatin-1(Fer-1)、去铁胺、环吡酮胺、丁羟甲苯和Trolox中的一种或几种。本发明对所述铁死亡抑制剂的的来源没有特殊限定，采用常规市售产品即可。在本发明中，所述铁死亡抑制剂的摩尔浓度优选为0.5～1μmol/L。
本发明还提供了上述技术方案所述组合物在制备上调谷胱甘肽过氧化酶4制剂中的应用。在本发明中，所述造血细胞因子和铁死亡抑制剂优选通过上调谷胱甘肽过氧化酶4(GPX4)的表达来促进造血干祖细胞的增殖。
本发明还提供了上述技术方案所述组合物在制备抑制活性氧的产生制剂中的应用。在本发明中，所述造血细胞因子和铁死亡抑制剂是通过抑制活性氧(ROS)的产生来促进造血干祖细胞的增殖。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。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前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本实验所进行的细胞为TF-1细胞，人脐带血CD34+造血干细胞，人骨髓血CD34+造血干细胞，TF-1细胞为本实验室库存，培养条件为RAPI-1640培养基、5ng/ml GM-CSF、10％血清与1％链霉素/青霉素。人脐带血/骨髓血CD34+造血干细胞培养条件为SFEM培养基、100ng/ml SCF、50ng/mlTPO、50ng/ml Flt3、50ng/ml IL-3与1％链霉素/青霉素。
采用cck8增殖实验观测细胞的增殖，在96孔板接种1×105/100ul的TF-1(人红系白血病细胞)细胞悬液(100ul/孔)，使用小分子化合物erastin在0,2.5,5,10,20(μM)作用于TF-1细胞与fer-1(0.5μmol/l)培养的TF-1细胞，24h后每孔加入10ul cck8检测液，培养板放置培养箱孵育3h，在450nm波长下酶标仪测定各孔OD值。在96孔板分别接种1×105/100ul人脐带血/骨髓血CD34+造血干细胞，分成两组实验条件，一组去各种生长因子，一组只加原有培养条件下因子的1/4，在Fer-10，0.5，1μM作用下48h后cck8检测各孔差异。
采用流式进行铁死亡相关脂质活性氧的测定，在六孔板中接种5×105TF-1细胞/孔，分为两组实验，一组分别在erastin10μM，fer-11μM，erastin10μM+fer-11μM三种条件下培养，一组在无GM-CSF，无GM-CSF+fer-11μM，含GM-CSF(5ng/ml)，含GM-CSF(5ng/ml)+fer11μM四种条件下培养，24h后消化细胞，400μl重选细胞悬液加入2ul5μM C1-BODIPY,37℃孵育30分钟，流式进行测定。
采用为westernblot进行铁死亡相关蛋白GPX4表达的测定，六孔板接种5×105TF-1细胞/孔，分为两组实验，一组分别在erastin10μM,fer-11μM，erastin10μM+fer-11μM三种条件下培养，一组在无GM-CSF,无GM-CSF+fer11μM，含GM-CSF(5ng/ml)三种条件下培养，24h后吸去培养基，预冷PBS洗涤两次，加入直接蛋白裂解液(DLB)150ul/孔，100℃水浴锅加热10min是蛋白变性，后期进行电泳电转，孵育GAPDH，GPX4一抗与山羊抗兔二抗，洗涤显影得出结果。十二孔板铺5×105人骨髓血CD34+细胞/孔，在低因子(原有因子培养条件的1/4)条件下，在Fer-10，0.5，1μM作用48h后收取细胞，按照上述操作完成铁死亡相关蛋白GPX4表达的测定。
结果：
(1)Erastin诱导造血细胞铁死亡
通过cck8细胞增殖实验与流式活性氧检测表明Fer-1可逆转TF-1细胞在erastin诱导下会发生的铁死亡并抑制铁死亡相关脂质活性氧(ROS)的产生，通过westernblot结果显示Fer-1作用于erastin处理后的TF-1细胞，抑制铁死亡相关蛋白GPX4显著上调。
(2)生长因子缺乏导致细胞铁死亡
CCK8细胞增殖实验显示TF-1细胞在缺乏GM-CSF因子的作用下会发生死亡，且细胞在Fer-11μM作用下出现增长，通过流式活性氧检测显示TF-1细胞在缺乏GM-CSF因子作用下会出现相关脂质ROS的释放并在Fer-11μM的作用下脂质ROS释放显著下降，westernblot结果显示Fer-1作用于缺乏GM-CSF因子的TF1细胞，GPX4显著上调。
(3)抑制铁死亡增强造血细胞的扩增作用
通过cck8增殖实验发现人原代CD34+细胞在去生长因子的条件下会发生细胞铁死亡，并且Fer-1在0.5、1μM的作用下可逆转细胞的死亡，在低造血生长因子的作用下，细胞数随Fer-1浓度的增加而显著增长，通过western blot结果显示在人骨髓CD34+细胞去因子的条件下，GPX4随Fer-1浓度增加而显著高表达。
由以上结果可知，通过铁死亡抑制剂Ferrostatin-1结合生长因子可以抑制CD34+造血干细胞在体外扩增的过程中出现的细胞铁死亡，从而促进造血细胞的增殖。
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 (4)
1.一种抑制细胞铁死亡及促进造血细胞增殖的组合物，其特征在于，由造血细胞因子和铁死亡抑制剂组成；
所述造血细胞因子由干细胞因子、Flt3配体、血小板生成素和白介素3组成；
所述干细胞因子的浓度为100ng/ml，Flt3配体的浓度为50ng/ml，血小板生成素的浓度为50ng/ml，白介素3的浓度为50ng/ml；
所述铁死亡抑制剂的摩尔浓度为0.5~1μmol/L；
所述铁死亡抑制剂为Ferrostatin-1。
2.权利要求1所述的组合物在促进造血细胞增殖中的应用，所述应用为非疾病诊断治疗目的。
3.权利要求1所述的组合物在制备上调谷胱甘肽过氧化酶4表达制剂中的应用。
4.权利要求1所述的组合物在制备抑制活性氧的产生的制剂中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861440A (zh) * | 2016-05-17 | 2016-08-17 | 广东万海细胞生物科技有限公司 | 一种动员骨髓造血干细胞的新型制剂 |
| CN108265028A (zh) * | 2016-12-30 | 2018-07-10 | 联亘生物科技（上海）有限公司 | 用于体外扩增造血干细胞的培养体系 |
| CN108456658A (zh) * | 2017-02-22 | 2018-08-28 | 苏州方舟基因药业有限公司 | 促进造血干细胞扩增并抑制其分化的化合物及细胞因子组合物 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861440A (zh) * | 2016-05-17 | 2016-08-17 | 广东万海细胞生物科技有限公司 | 一种动员骨髓造血干细胞的新型制剂 |
| CN108265028A (zh) * | 2016-12-30 | 2018-07-10 | 联亘生物科技（上海）有限公司 | 用于体外扩增造血干细胞的培养体系 |
| CN108456658A (zh) * | 2017-02-22 | 2018-08-28 | 苏州方舟基因药业有限公司 | 促进造血干细胞扩增并抑制其分化的化合物及细胞因子组合物 |
Non-Patent Citations (2)
| Title |
|---|
| Obesity alters the long-term fitness of the hematopoietic stem cell compartment through modulation of Gfi1 expression;Jung-Mi Lee et al.;《J Exp Med》;20180205;第215卷(第2期);第627-644页 * |
| 不同共培养模式下间充质干细胞对造血干细胞增殖的影响;汪姝玥等;《中国组织工程研究》;20180531;第22卷(第13期);第2068-2074页 * |
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