CN115029301B - 一种化合物小分子在促进胚胎干细胞自我更新中的应用方法 - Google Patents
一种化合物小分子在促进胚胎干细胞自我更新中的应用方法 Download PDFInfo
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Abstract
本发明提供了一种促进胚胎干细胞自我更新中的化合物小分子Danofloxacin的应用方法,其可以维持人与小鼠胚胎干细胞的自我更新以及多能性。本发明涉及的培养基组成成份为小鼠胚胎干细胞基础培养基,内含有10%胎牛血清等,同时添加Danofloxacin+PD0325901小分子组合。与传统的添加白血病抑制因子(LIF)条件相比,本发明的培养效果更加稳定,避免了动物源性因子的使用,并且能够维持干细胞较佳的自我更新与多能性状态。本发明所提出的培养条件,简化了培养体系中细胞因子的种类,为分离、诱导和培养新型多能干细胞提供了线索,为胚胎干细胞多能性调控网络形成丰富的理论奠定基础。
Description
技术领域
本发明涉及一种化合物小分子在促进胚胎干细胞自我更新中的应用方法。
背景技术
胚胎干细胞(Embryonic stem cells,ESCs)是一类分离自囊胚内细胞团(Innercell mass,ICM)的细胞,主要形态特征为:体积小,细胞核大,细胞集落呈鸟巢状生长,细胞间排列紧密,细胞边缘清晰。该种类型的细胞在体外培养时具有两种显著特性1.自我更新(Self-renewal),即能够对细胞的本身进行无限的复制;2.多向分化(Pluripotency),即在特定的条件下具有诱导该细胞能够分化成多种类型细胞的潜能,如造血干细胞、神经细胞、表皮细胞等几乎所有细胞类型。ESCs的这种既能在体外无限增殖又能在特定条件下被诱导生成多种类型的细胞的能力,为再生医学以及组织修复带来了很大希望,同时也为建立疾病模型和探究生物发育的机制提供了强有力的医学工具。小鼠胚胎干细胞(mouse ESCs,mESCs)是早在1981年被建系成功,1998年人胚胎干细胞(human ESCs,hESCs)被建立成功。小鼠与人ESCs拥有众多的相似之处,目前hESCs研究必须遵循国际公认的胚胎研究遵循“14天原则”,且其体外培养的工序繁琐,成本较高;而mESCs体外培养只需要特定的营养物质和特定细胞因子的共同参与来维持mESCs的特性。为了避免研究人胚胎干细胞所带来的伦理道德问题,mESCs常用作在人类疾病研究中的良好模型。该实验以小鼠胚胎干细胞为研究模型,优化胚胎干细胞现有的培养条件,从而筛选出在体外可以维持胚胎干细胞的化合物小分子(无动物血清成分),该培养方式将为更多动物品系的胚胎干细胞系的建立做出贡献。实验前期我们发现在小鼠胚胎干细胞中添加小分子Danofloxacin可以使小鼠和人胚胎干细胞维持自我更新的状态,这为我们分离及培养胚胎干细胞提供了新的思路。
Danofloxacin小分子又名丹诺沙星,达氟沙星,一种具有口服活性第三代氟喹诺酮抗菌剂,属于动物专用药,一般使用其甲磺酸盐即甲磺酸达氟沙星,对鸡大肠杆菌、多杀性巴氏杆菌、败血支原体等均有较强的抗菌活性,对大多数革兰氏阴性和革兰氏阳性细菌,支原体和衣原体物种具有广谱的活性,并通过抑制细菌的DNA回转酶(bacterial DNA-gyrase)发挥抗菌作用。
将小分子Danofloxacin加入小鼠胚胎干细胞的培养体系,我们发现Danofloxacin在最适浓度下,能够在不添加传统的细胞因子或者不过表山中申弥四因子OSKM的条件下短暂的对小鼠胚胎干细胞的自我更新状态进行维持。进一步研究发现,仅使用Danofloxacin和PD0325901两种小分子组合可促进小鼠胚胎干细胞的自我更新,因此Danofloxacin作为一种干细胞培养的新因素,有望优化对未来其他哺乳动物或其他类型动物干细胞的体外培养条件的建立。
发明内容
本发明所要解决的问题是通过将小分子库里药物分别加入小鼠胚胎干细胞中,筛选出能够维持胚胎干细胞的小分子,该发明将为维持小鼠胚胎干细胞自我更新提供一种新的应用方法,从而为其他动物的胚胎干细胞的建系以及培养提供新的策略
本发明解决上述技术问题所采用的技术方案是在含有10%FBS基础上,筛选出Danofloxacin的最适培养浓度,通过加入Danofloxacin与PD0325901两种小分子组合来促进小鼠胚胎干细胞的自我更新。
本发明的具体操作,包括以下步骤:
(1)取1.5ml的浓度为0.1%的明胶包被的细胞培养板,将包被好的培养板置于37℃的CO2浓度为5%的细胞培养箱中,孵育30min;
(2)选取生长密度为70-80%六孔板培养的P20代小鼠胚胎干细胞,弃掉上层培养液,使用PBS缓冲液清洗一次,去除残留的培养液;
(3)弃掉PBS,加入浓度为0.25%的胰蛋白酶1ml,使用胰蛋白酶消化细胞2min左右,至细胞边缘浮起,用移液枪均匀的轻轻吹打细胞,吸取细胞悬液,转移至盛有2ml的10%血清高糖培养液的离心管中,轻轻吹打混匀,从而终止消化;
(4)将盛有细胞的离心管置于离心机,1000rpm,离心3min,离心结束后弃去上清,加入2ml含血清高糖的培养液重悬细胞,使用细胞计数仪计数,确保细胞接种的密度;
(5)从培箱中取出明胶包好的培养板,吸掉明胶,加入2ml血清高糖培养液到细胞培养板中;
(6)在两组细胞培养板中分别接种细胞密度为4×105,水平十字晃动培养板,使细胞分布均匀;
(7)其中一组细胞培养板中分别加入1μM、2.5μM、5μM、7.5μM浓度Danofloxacin小分子处理的细胞,水平十字晃动培养板,使其混合均匀;
(8)另一组细胞培养板中添加5μM Danofloxacin+1.5μMPD0325901小分子组合,水平十字晃动,使其混合均匀;
(9)将细胞培养皿置于37℃、5%CO2浓度的细胞培养箱内培养。
细胞自我更新状态检测:
(1)形态观察,使用Leica DMIL倒置显微镜分别观察添加了不同浓度Danofloxacin的小鼠胚胎干细胞的形态状态,显微镜显示不添加外源性小分子的时候细胞发生了分化,而在添加了Danofloxacin的条件下能够促进小鼠胚胎干细胞的自我更新,当小分子Danofloxacin低于5μM浓度时绝大部分干细胞处于分化状态,而高于5μM浓度的时候对细胞是有致死性的,所以选择5μM作为最佳处理浓度。
(2)使用Leica DMIL倒置显微镜分别对阴性对照组(不添加小分子)和阳性对照组(仅添加小分子Danofloxacin)以及实验组(添加Danofloxacin+PD0325901两种小分子)的干细胞形态进行观察,发现对照组中不添加小分子的细胞发生了分化,而阳性对照组与实验组的观察视野中存在胚胎干细胞样克隆且实验组的胚胎干细胞样克隆多于阳性对照组的克隆数。
本发明具有如下优点:
本发明采用在含有10%FBS的DMEM培养基中加入Danofloxacin和PD0325901两种小分子组合来培养小鼠胚胎干细胞,培养在该条件下的胚胎干细胞生长情况优良,并且能够维持自我更新的状态。而且单独使用Danofloxacin能促进人胚胎干细胞在体外维持干性。
(1)使用Danofloxacin和PD0325901两种小分子组合与传统培养条件,添加白血病抑制因子(LIF)相比,本发明的培养效果更加稳定,避免了动物源性因子的使用,并且能够将维持在较佳的生长状态下,有利于基础和应用研究的开展,为胚胎干细胞多能性调控网络形成丰富的理论奠定基础。
(2)使用Danofloxacin和PD0325901两种小分子组合与传统培养体系相比,本发明使用化学小分子来代替动物源性小因子,简化了细胞培养体系中添加的细胞因子的种类,有利于科研工作者对细胞内分子调控机制以及相关信号通路的更加进一步的研究的进行。
(3)本发明培养条件Danofloxacin和PD0325901两种小分子组合,为分离、诱导和培养新型多能干细胞提供了线索。
附图说明
图1所示为添加不同浓度Danofloxacin进行处理的P21代小鼠胚胎干细胞形态观察,左侧为不添加小分子处理的阴性对照组细胞,在该状态下干细胞发生了分化;右边依次为添加1μM、2.5μM、5μM、7.5μM浓度Danofloxacin小分子处理的细胞,如图所示,添加不同浓度Danofloxacin后显示能够促进小鼠胚胎干细胞自我更新的特性,然而低于5μM浓度时干细胞分化较多,而高于5μM浓度细胞会呈现致死性,所以挑选5μM作为胚胎干细胞处理的最佳浓度。
图2所示为三种培养条件下进行处理的P21代小鼠胚胎干细胞形态观察,其中左侧为不添加小分子处理的阴性对照组细胞,在该状态下干细胞发生了分化;在单加Danofloxacin小分子处理的细胞较左边阴性组相比,出现部分处于自我更新状态的克隆株;加入Danofloxacin和PD0325901两种小分子组合的条件下,绝大部分克隆株处于自我更新的状态。
具体实施方式
实施例
实验中所用的小鼠胚胎干细胞由美国南加州大学提供。
Danofloxacin促进小鼠胚胎干细胞自我更新最佳浓度的筛选,以及Danofloxacin和PD0325901两种小分子组合条件下的小鼠胚胎干细胞的培养与传代:
(1)取1.5ml的浓度为0.1%的明胶包被细胞培养板,将包被好的培养板置于37℃的CO2浓度为5%的细胞培养箱中,孵育30min;
(2)选取生长密度为70-80%的P20代小鼠胚胎干细胞,弃掉上层培养液,使用PBS缓冲液清洗一次,去除残留的培养液;
(3)弃掉PBS,加入浓度为0.25%的胰蛋白酶1ml,使用胰蛋白酶消化细胞2min左右,至细胞边缘浮起,用移液枪均匀的轻轻吹打细胞,吸取细胞悬液,转移至盛有2ml的10%血清高糖培养液的离心管中,轻轻吹打混匀,从而终止消化;
(4)将盛有细胞的离心管置于离心机,1000rpm,离心3min,离心结束后弃去上清,加入2ml含血清高糖的培养液重悬细胞,使用细胞计数仪计数,确保细胞接种的密度;
(5)从培箱中取出明胶包好的培养板,吸掉明胶,加入2ml血清高糖培养液到细胞培养板中;
(6)在两组细胞培养板中分别接种细胞密度为4×105,水平十字晃动培养板,使细胞分布均匀;
(7)其中一组细胞培养板中分别加入1μM、2.5μM、5μM、7.5μM浓度Danofloxacin小分子处理的细胞,水平十字晃动培养板,使其混合均匀;
(8)另一组细胞培养板中添加5μM Danofloxacin+1.5μMPD0325901小分子组合,水平十字晃动,使其混合均匀;
(9)将细胞培养皿置于37℃、5%CO2浓度的细胞培养箱内培养。
细胞自我更新状态检测:
(1)细胞形态观察,使用Leica DMIL倒置显微镜分别对添加不同浓度Danofloxacin进行处理的小鼠胚胎干细胞进行观察选择并最佳培养浓度;
(2)使用LeicaDMIL倒置显微镜分别对阴性对照组(不添加小分子)、阳性对照组(仅添加Danofloxacin小分子)以及实验组(添加Danofloxacin+PD0325901两种小分子的细胞形态进行观察;
(3)碱性磷酸酶(AlkalinePhosphatase,AP)染色检测自我更新状况,具体方法如下:
a、阳性、阴性对照组和实验组接种2×104细胞,培养7天时间后即可进行AP染色;
b、按照碱性磷酸酶染色试剂盒的说明配制BCIP/NBT染色工作液;
c、弃去细胞培养板中的培养液,PBS洗涤3-5次,加入1ml 4%的多聚甲醛,1-2min固定细胞,弃去固定液,再用PBS洗涤3-5次;
d、最后一次洗涤完毕后,去除PBS,加入适量碱性磷酸酶染色工作液,确保能充分覆盖细胞;室温避光孵育30-60min,直至显色完成;
e、去除碱性磷酸酶染色工作液,用PBS洗涤2-3次可终止显色反应;
f、最后加入适量PBS后,将培养板放在Leica DMIL倒置显微镜观察阴性对照组(不添加小分子)、阳性对照组(仅添加Danofloxacin小分子)与实验组(添加Danofloxacin+PD0325901两种小分子)细胞染色情况,判断细胞自我更新状态。
如图2所示,碱性磷酸酶染色后的小鼠胚胎干细胞的染色情况,阴性对照组细胞显示无色,即无碱性磷酸酶活性,干细胞全部处于分化状态;阳性对照组干细胞有少数克隆呈现紫色,即有较弱的碱性磷酸酶活性,干细胞部分处于自我更新状态;而实验组的干细胞呈现出数量极多的紫色克隆株,即有较强的碱性磷酸酶活性,干细胞大部分处于自我更新状态。
Claims (3)
1.一种化合物小分子在促进胚胎干细胞自我更新的应用方法,其特征在于,在含有10%FBS的DMEM培养基中添加小分子5μM Danofloxacin、1.5μMPD0325901小分子组合来维持小鼠胚胎干细胞自我更新的状态。
2.根据权利要求1所述的方法,其特征在于:
(1)取1.5ml的浓度为0.1%的明胶包被细胞培养板,将包被好的培养板置于37℃的CO2浓度为5%的细胞培养箱中,孵育30min;
(2)选取6孔细胞培养板生长密度为70-80%的P20代小鼠胚胎干细胞,弃掉上层培养液,使用PBS缓冲液清洗一次,去除残留的培养液;
(3)弃掉PBS,加入浓度为0.25%的胰蛋白酶1ml,使用胰蛋白酶消化细胞2min左右,至细胞边缘浮起,用移液枪均匀的轻轻吹打细胞,吸取细胞悬液,转移至盛有2ml的10%FBS的DMEM培养基的离心管中,轻轻吹打混匀,从而终止消化;
(4)将盛有细胞的离心管置于离心机,1000rpm,离心3min,离心结束后弃去上清,加入2ml10%FBS的DMEM培养基重悬细胞,使用细胞计数仪计数,确保细胞接种的密度;
(5)从培箱中取出明胶包好的培养板,吸掉明胶,加入2ml含10%FBS的DMEM培养基到细胞培养板中;
(6)在两组细胞培养板中分别接种细胞密度为4×105,水平十字晃动培养板,使细胞分布均匀;
(7)细胞培养板中添加5μM Danofloxacin+1.5μMPD0325901小分子组合,水平十字晃动,使其混合均匀;
(8)将细胞培养皿置于37℃、5%CO2浓度的细胞培养箱内培养。
3.根据权利要求1或2所述的方法,其特征在于,所述10%FBS的DMEM培养基包括以下组分:2mM L-谷氨酰胺,10%FBS,1mM丙酮酸钠,1%MEM非必需氨基酸,100单位的青霉素,100μg的链霉素和0.1mMβ-巯基乙醇。
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