CN112410282A - 一种体外高效诱导高级分支状肺类器官的方法及实验模型和化合物组合 - Google Patents
一种体外高效诱导高级分支状肺类器官的方法及实验模型和化合物组合 Download PDFInfo
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Abstract
本发明提供一种体外高效诱导高级分支状肺类器官的方法及实验模型和化合物组合,涉及生物技术领域,所述方法是将离体的气道上皮组织进行消化处理,然后利用包含表皮生长因子和胰岛素成分的扩增培养基对气道上皮细胞进行扩增培养,利用胰酶消化成单细胞后加入到分化培养基中培养,获得具有分支状高级结构的、且含有分化的纤毛细胞的小鼠肺类器官,所述小鼠肺类器官可以作为实验模型,用于精准医疗、器官移植、药物筛选、药物作用的机制研究的用途,本发明方法不仅可以实现了将气道上皮细胞培养成与体内肺脏形态和细胞结构更为相似的具有分支状高级结构和分化纤毛细胞的肺类器官,而且所培养的肺类器官产量是常规方法的3倍以上。
Description
技术领域
本发明涉及生物技术领域,尤其涉及体外高效诱导高级分支状肺类器官的方法及实验模型和所用培养基组合。
背景技术
肺类器官是应用体外培养技术建立的结构和功能上类似于肺脏的小型组织,具有组织自我更新及可长期培养的特点,在一定程度上模拟体内肺脏生理活动和病理变化,可成为精准医疗、器官移植、药物筛选、药物作用机制研究的理想体外载体。
肺类器官可通过诱导人类多功能干细胞或者小鼠成体干细胞,在附加与肺脏相关的生长因子的3D培养基上获得。2015年,科学家首次从人类干细胞获得了首个肺类器官(参见参考文献1)。2017年,哥伦比亚大学医学中心的Hans-Willem Snoeck教授及其同事利用人多能干细胞生成了肺部的类器官,这是第一个包含分支和肺泡结构的培养物,可以模拟肺部功能(参见参考文献2-3)。在成年小鼠肺组织中,存在三种类型的成体干细胞:大气道区域表达p63和Krt5的基底细胞,肺内支气管区域表达Scgb1a1的,以及肺泡区域表达Sftpc的二型肺泡上皮细胞(参见参考文献4)。在体外培养条件下,肺脏各类成体干细胞都可以获得具有多种分化细胞类型的肺类器官,其中以肺近端气道中的基底细胞培养形成的肺类器官应用最广。目前,培养基底细胞来源的肺类器官常用的培养方法有:①将细胞在Transwell中2D培养,一段时间后改为气体-液体交接面培养条件,模拟体内气道呼吸环境,诱导细胞分化(参见参考文献5。此类培养方法可以形成类似肺气道上皮细胞的假复层结构,但是没有体内气道组织特定的管腔形态和结构。②将分离的基底细胞在不含生长因子的Matril胶的3D条件下培养,通过诱导分化,可以产生球状的肺类器官(参见参考文献6-7),此类培养方法较方法①能形成和肺气道管状组织更相似的机构,但是不具备体内肺脏气道组织的高级分支结构。另外,由于小鼠气道基底细胞较少以及类器官形成对培养环境要求苛刻,目前存在的培养方法都存在类器官产量低的问题。
综上所述,虽然体外培养形成的肺类器官可广泛应用于基础研究和药物测试领域,但是目前培养条件下形成的肺类器官存在产量低,不具有肺脏体内复杂的分支管腔结构等问题,严重限制了肺脏类器官的应用。
参考文献如下:
1.Dye,B.R.,et al.(2015)."In vitro generation of human pluripotentstem cell derived lung organoids."eLife 4.
2.Chen,Y.-W.,et al.(2017)."A three-dimensional model of human lungdevelopment and disease from pluripotent stem cells."Nature Cell Biology 19(5):542-549.
3.H-W·斯诺克Y-W·陈.具有分支结构的肺芽类器官的生成和其用于肺疾病建模的用途.发明专利。申请公布号:CN 110494555 A.
4.Barkauskas,C.E.,et al.(2017)."Lung organoids:current uses andfuture promise."Development 144(6):986-997.
5.Eenjes,E.,et al.(2018)."A novel method for expansion anddifferentiation of mouse tracheal epithelial cells in culture."Scientific Reports 8(1).
6.You,Y.and S.L.Brody(2013)."Culture and differentiation of mousetracheal epithelial cells."Methods Mol Biol 945:123-143.
7.Jason R.Rock,et al.(2009).“Basal cells as stem cells of the mousetrachea and human airway epithelium.”Proc Natl Acad Sci U SA 106(31):12771-5.
发明内容
本发明的目的是提供一种培养分支状高级结构肺类器官的方法,本发明方法不仅可以实现了将气道上皮细胞培养成与体内肺脏形态和细胞结构更为相似的具有分支状高级结构和分化纤毛细胞的肺类器官,而且所培养的肺类器官产量是常规方法的三倍以上。
为实现本发明的技术目的,本发明一方面提供一种利用气道上皮细胞培养分支状高级结构肺类器官的方法,包括:
将离体的气道组织进行消化处理,得到气道上皮细胞;
利用包含表皮生长因子和胰岛素的扩增培养基对气道上皮细胞进行扩增培养,获得数量增加且保留功能的气道上皮细胞;
将数量增加且保留功能的气道上皮细胞利用胰酶消化成单细胞后,最后加入到含有生长因子的分化培养基中,获得具有分支状高级结构和分化纤毛细胞的、小鼠肺类器官。
其中,所述消化处理是用终浓度为1.5mg/ml Pronase消化酶的Ham’F-12培养液消化气道上皮细胞,消化条件为25℃,20分钟,继而4℃,12小时。
本申请消化气道上皮细胞使用Pronase消化酶(即链酶蛋白酶)是近年来从灰霉菌中提取出来的酶。此酶消化作用温和,对气道上皮细胞有较好的消化效果,对细胞本身影响不大,消化后可使细胞分散成单细胞而不受伤害,消化后培养类器官效果比常用的胰蛋白酶和胶原酶相比更好。胰蛋白酶消化后常对上皮细胞损伤较大。由于上皮细胞对胶原酶有耐受性,因此胶原酶消化气道组织后可能形成一些上皮细胞团块,不能完全消化成单细胞状态。
本申请扩增培养基含有表皮生长因子(EGF)以及胰岛素(Insulin)。其中EGF可以持续促进DNA的连续合成,从而起到保持气道上皮细胞增殖与分化潜能的作用。胰岛素用来调节细胞对葡萄糖、氨基酸和脂肪酸的摄取、利用和贮存,同时抑制糖原、蛋白质和脂肪的分解,起到维持细胞生长,促进细胞增殖的作用。
本申请分化培养基使用的FGF10和FGF7,用于模拟小鼠体内肺分支发育过程肺间质细胞分泌的FGFs,诱导分支点的形成。DMH1和Noggin抑制BMP信号通路,促进分支发生中分叉的形成。CHIR99021激活Wnt信号通路,促进气道上皮细胞分化以及细胞干性维持。Y29632,A8301可促进气道上皮细胞扩增和分化。
有益效果
1、本发明方法成功将小鼠原代气道上皮细胞诱导成具有高级分支结构以及终末分化的纤毛细胞的肺类器官,具有显著的进步。
2、利用本发明提供的扩增培养基对小鼠原代气道上皮细胞进行培养后,不仅能够使细胞扩增,还能维持近端气道上皮细胞的特性,可见,本申请提供的扩增培养方法成功实现了气道上皮细胞的扩增培养,解决了现有技术中,扩增培养容易出现的上皮细胞分化特性丧失或减弱的技术问题。
3、本发明将小鼠原代气道上皮细胞进行扩增培养后,再诱导成肺类器官,所获得的肺类器官的数量是传统培养方法培养量的3.5倍,对本领域技术人员而言,可以极大的减少小鼠数量,减少试验成本和时间,解决了现有技术中小鼠原代气道上皮细胞数量少导致肺类器官产量低的的问题。
4、利用本发明提供的分化培养基及培养方法,能够使小鼠气道上皮细胞形成含分支状高级结构的肺类器官,能够更好的模拟体内肺脏的生理功能和病理变化,能够用于肺脏体内复杂的分支管腔结构的机理或药物筛选研究。
附图说明:
图1是本发明实施例1提供的本发明利用消化气道上皮细胞进行体外高效诱导高级分支状肺类器官的方法示意图;
图2是本发明扩增后的气道上皮细胞形态图,标尺:20微米;
图3是本发明扩增后的气道上皮细胞2D扩增后免疫荧光染色图,标尺:20微米;
图4是本发明扩增后的肺类器官的相对获得量统计,**:p<0.01,双尾学生检测;
图5是本发明进行分化培养分支状高级结构肺类器官形成过程图,标尺:50微米;
图6是本发明获得的肺类器官的免疫荧光染色显示,具有分支状高级结构的肺类器官表达近端气道上皮细胞标记基因Sox2,不表达远端气道上皮细胞标记基因Sox9,标尺:50微米;
图7是本发明诱导形成的分支状高级结构肺类器官免疫荧光染色图,肺类器官表达基底细胞标记基因p63和Krt5,同时表达分化的纤毛细胞标记基因ac-tubulin,标尺:20微米;
需要说明的是,上述图2-7均为共聚焦显微镜拍摄的图片。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定,需要说明的是,下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
需要说明的是,本文中所称的英文缩写所代表的含义分别表示:PBS:磷酸缓冲液;FBS:胎牛血清;Fgf-10:成纤维细胞生长因子10;RA:视黄酸;EGF:表皮生长因子;BPE:牛垂体提取物;CT:霍乱霉素;p63:抑癌基因p53家族的一种;Krt5:角蛋白5;Sftpc:肺表面活性物质;Bmp:骨形态发生蛋白;Sox2、Sox9:原癌基因的一种,Clara细胞:克拉拉细胞,终末细支气管上皮中的主要细胞;Insulin:胰岛素;Transwell:可穿透性细胞培养小室;Pronase:链霉蛋白酶;GlutaMAX:谷氨酸添加剂;NaHCO3:碳酸氢钠;HEPES:一种缓冲试剂名称;Primocin:催产素;Transferrin:铁传递蛋白;Matril gel:Matril凝胶;DAPI:与DNA强力结合的荧光染料;Merged:融合;E-cad:E-钙粘附素;2D表示二维,即扩增培养;3D表示三维,即分化培养。
实施例1化合物组合
1、扩增培养基(即2D培养基)
按照表1所示的成分表进行扩增培养基的配置
具体的,在本发明的一个实施例中,所述Ham’s F12和DMEM、青链霉素混合液均购于Life Technologies公司,货号分别是31765-092、11330032和15140-163,FBS购于Biowest公司,货号为S1580-500,GlutaMAX和HEPES购于Gibco公司,货号分别为05966L17和15630-080,NaHCO3购于Fisher公司,货号为S233-500,EGF购于Corning公司,货号为354001,Insulin购于Sigma公司,货号为I6634。当然,本领域技术人员还可以采用其他公司生产的具有相同功效的试剂配置本申请的扩增培养基,本发明不做具体限制。
优选的,发明人经过大量实验发现,EGF的用量还可以选自50-80ng/ml范围内的任一数值与表1中的其他成分浓度进行扩增培养基的配置,Insulin的用量还可以选自20-40μg/ml内的任一数值与表1中的其他成分浓度进行扩增培养基的配置。
2、分化培养基(即3D培养基)
按照表2所示的成分表进行分化培养基的配置
具体的,在本发明的一个实施例中,所述Advanced DMEM/F-12和GlutaMAX、HEPES均购于Gibco公司,货号分别是C11330500BT、05966L17和15630-080;青链霉素混合液购于Life Technologies公司,货号是15140-163;FBS购于Biowest公司,货号为S1580-500;EGF购于Corning公司,货号为354001;Transferrin、Cholera Toxin、Bovine PituitaryExtract、Retinoic acid和Insulin均购于Sigma公司,货号分别是T1147、C8052、P1476、R2625-1g和I6634;DMH-1、CHIR99021和Y27632购于Tocris公司,货号分别是4126、4423和1254;Noggin购于SinoBiological公司,货号为10267。
当然,本领域技术人员还可以采用其他公司生产的具有相同功效的试剂配置本申请的扩增培养基,本发明不做具体限制。
优选的,发明人经过大量实验发现,EGF的用量还可以选自25-50ng/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Transferrin的用量还可以选自5-10μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;CholeraToxin的用量还可以选自0.1-0.2μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Bovine Pituitary Extract的用量还可以选自30-100μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Retinoic acid的用量还可以选自50-60nM范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;FGF10的用量还可以选自30-50ng/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;FGF7的用量还可以选自10-20ng/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Insulin的用量还可以选自30-60μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;DMH-1的用量还可以选自10-20μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Noggin的用量还可以选自20-40μg/ml范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;CHIR99021的用量还可以选自5-10μM范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置;Y27632的用量还可以选自5-10μM范围内的任一数值与表2中的其他成分浓度进行扩增培养基的配置。
实施例2
本发明将消化气道上皮组织诱导为具有高级分支结构以及终末分化的纤毛细胞的小鼠肺类器官的过程如图1所示,具体为:
1.消化气道上皮组织的获得
本发明利用出生后8周的C57 BL/6J雌性小鼠作为实验材料,获取气道上皮组织的过程采用解剖学的常规方法,具体步骤如下:
1.1腹腔注射10%水合氯醛麻醉小鼠,用大头针固定小鼠四肢在手术台上,剪开小鼠胸腔和颈部,取出气道组织,放于预冷的含1%青链霉素混合液的1×PBS中。
1.2在体式镜解剖镜下,用眼科手术镊子将气道组织旁边黏连的肌肉以及结缔组织去除,用眼科手术剪将气道组织纵向刨开,用预冷的含1%青链霉素混合液的1×PBS清洗气道组织2-3次。
2、消化处理
将清洗后的气管组织转移到含1.5mg/ml Pronase的消化培养基中,室温消化20分钟,然后4℃消化12小时,然后在超净工作台中,加入5ml含10%FBS的Ham’s-F12培养基到含气道组织的消化液中,终止消化反应。
用镊子将消化液中的气道组织取出,然后将消化液用300目过滤网过滤到15ml试管中,1000rpm离心5min,去上清,获得气道上皮细胞。
3、扩增培养
将获得的上皮细胞用4ml扩增培养基重悬,然后置于I型鼠尾胶原包被的培养皿中培养,培养条件为37℃,5%CO2浓度,培养5小时。
收集非粘附细胞到新的15ml离心管,1000rpm,离心3分钟,弃上清,再将沉淀的细胞重悬在4ml扩增培养基中,置于100mg/mlⅠ型鼠尾胶原包被的培养皿中培养,培养条件为37℃,5%CO2浓度。
上述培养过程中,每2天换扩增培养基,共培养5天。
将培养扩增后细胞进行细胞学观察和免疫荧光染色,细胞形态观察结果如图2所示,免疫荧光染色结果如图3所示,在图2中可以看到,利用本发明扩增后的上皮组织具备典型的肺气道上皮细胞的形态,在图3中同样可以明显看到扩增后的细胞表达多个肺上皮细胞特有的标记基因,可见,利用本发明扩增后的气道上皮细胞还维持肺上皮细胞特性,没有发生功能丧失和减弱。
4、分化培养
将步骤3培养的上皮细胞用胰酶消化成单细胞后,在分化培养基中重悬,细胞悬液在冰水混合物中与Matril胶混匀,得到终浓度为50%的混合液。然后将混合液置于培养皿内,37℃静置20分钟,混合液中的Matril胶凝固,再继续而加入分化培养基进行诱导培养。诱导培养3天后,类器官开始产生分支,随后分支进一步生长并增多,培养14天后在共聚焦显微镜下观察,如图5所示,在图5中可以明显看到清晰的多分支状高级结构的类器官。
同时,对形成的分支状高级结构肺类器官进行免疫荧光染色分析,结果如图6和7所示,在图6和7中可以看出,类器官表达近端气道标记基因Sox2,但是不表达远端气道标记基因Sox9,可见培养形成的分支状高级结构肺类器官其细胞来源为近端气道上皮细胞。同时,分支状高级结构肺类器官表达基底细胞标记基因Krt5和p63,以及分化的纤毛细胞标记基因ac-tubulin,说明培养形成的肺类器官含有终末分化的纤毛细胞。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。
Claims (9)
1.一种体外高效诱导高级分支状肺类器官的方法,其特征在于,包括:
将离体的气道上皮组织进行消化处理,得到气道上皮细胞;
利用包含表皮生长因子和胰岛素成分的扩增培养基对气道上皮细胞进行扩增培养,获得数量增加且保留功能的气道上皮细胞;
将数量增加其保留功能的气道上皮细胞利用胰酶消化成单细胞后加入到富含多种生长因子组合的分化培养基中培养,获得具有分支状高级结构且含有分化纤毛细胞的小鼠肺类器官。
2.如权利要求1所述的利用气道上皮细胞培养分支状高级结构肺类器官的方法,其特征在于,所述消化处理是将用终浓度为1.5mg/ml Pronase消化酶的Ham’F-12培养液消化气道上皮细胞,消化条件为25℃,20分钟,4℃,12小时。
3.如权利要求1所述的方法,其特征在于,所述扩增培养基包括Ham’s F12、DMEM、FBS、青链霉素混合液、NaHCO3、HEPES。其中,所述扩增培养基中各成分浓度为:DMEM为50%,Ham’s F12为50%,FBS为10%,青链霉素混合液为1%,GlutaMAX为4mM,NaHCO3为3.6mM,HEPES为15mM,EGF为50-80ng/ml,Insulin为20-40μg/ml。
4.如权利要求1所述的方法,其特征在于,所述扩增培养条件为:在100ng/ml I型鼠尾胶原包被的培养皿上培养,每2天更换培养基,共培养5天。
5.如权利要求1所述的方法,其特征在于,所述富含生长因子组合的分化培养基包含EGF,Transferrin,CT,BPE,Insulin,DMH1,FGF10、FGF7、Noggin、DMH-1,CHIR9902,Y27632和视黄酸。
6.如权利要求1所述的方法,其特征在于,所述分化培养方法是:
将数量增加的气道上皮细胞加入分化培养基中重悬并吹打成单细胞,形成的单细胞悬液与Matril胶在冰上混合均匀,使其终浓度为50%。继而将细胞-Matril胶-分化培养基混合液加入到细胞培养皿中,37℃静置20分钟,待Matril胶凝固后加入分化培养基;
在细胞培养箱中培养,每两天换培养基;
培养液更换方法为:每次用细胞培养基更换一半体积的原有培养液。培养条件为:37℃,5%CO2浓度。
7.一种利用权利要求1-6的方法得到的实验模型,其为具有高级分支结构以及终末分化的纤毛细胞的小鼠肺类器官。
8.一种将利用权利要求1-6所述的方法获得的小鼠肺类器官或权利要求8所述的实验模型用于精准医疗、器官移植、药物筛选、药物作用的机制研究的用途。
9.一种体外高效诱导高级分支状肺类器官的化合物组合,包括扩增培养液和分化培养液;
其中,所述扩增培养液包括:Ham’s F12、DMEM、FBS、青链霉素混合液、NaHCO3、HEPES;
其中,所述分化培养液包括:Advanced DMEM/F-12,青链霉素混合液,GlutaMAX,NaHCO3,HEPES,PrimocinTM,FBS,EGF,Transferrin,Cholera Toxin,Bovine PituitaryExtract,Retinoic acid,Insulin,DMH-1,FGF10,FGF7,Noggin,CHIR99021和Y27632。
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