CN114946762A - 一种气道纤毛上皮细胞缺失的动物模型的构建及应用 - Google Patents
一种气道纤毛上皮细胞缺失的动物模型的构建及应用 Download PDFInfo
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Abstract
本发明提供一种气道纤毛上皮细胞缺失的动物模型的构建及应用。具体的,本发明通过在肺上皮细胞中特异性敲除蛋白质精氨酸甲基转移酶5(Prmt5)基因,成功构建出一种气道纤毛上皮细胞完全缺失的小鼠模型。本发明还提供两种纤毛细胞完全缺失的体外气道类器官培养方案。该动物模型和体外培养方案可广泛应用于机体发育过程、细胞分化、器官再生和移植等相关研究领域,并在肺病的发病机理、药物开发、药物筛选和诊断治疗等方面有广泛的应用前景。
Description
技术领域
本发明涉及动物模型技术领域,具体为,涉及一种Prmt5基因敲除导致的气道纤毛上皮细胞缺失的动物模型的构建方法和气道上皮细胞缺失的体外类器官培养方案,以及所述动物模型和体外培养方案在器官发育、细胞命运和肺病药物筛选和诊断治疗中的应用。
背景技术
纤毛细胞是气道上皮细胞的主要组成类型,其数目占上皮细胞总数的三分之一,在呼吸系统中行使清扫功能,其细胞表面的运动纤毛对气道发挥“黏膜-纤毛”清除作用是必不可少[1-2]。黏膜-纤毛传输系统由纤毛与纤毛表面的黏液层构成,通过纤毛的节律性摆动,推动黏液层运动,把附着于黏液的异物颗粒、细菌和坏死细胞碎屑排出呼吸道,发挥重要的机械防御作用[3-4]。纤毛细胞缺失或功能异常和多种临床肺病密切相关,包括慢性阻塞性肺病、哮喘、特发性肺纤维化、囊性纤维化、支气管扩张和原发性纤毛运动障碍[5-6]。近年来,随着空气污染和病毒,临床肺病人数巨增,给国家医疗和社会造成沉重负担。由于相关动物模型的缺乏,人们对纤毛缺陷相关肺病的致病机制了解甚少,导致其临床预警和诊疗是一个世界性的难题。
蛋白质精氨酸甲基转移酶 5(Prmt5)是最主要的一种II型精氨酸甲基转移酶,可以对称性地甲基化组蛋白或者非组蛋白底物的精氨酸残基,通过表观遗传的方式调控靶基因的表达,已被证明在多个生物学过程中发挥关键作用[7-8]。临床研究方面,Prmt5的表达水平与多种疾病的发生、发展及预后密切相关,Prmt5近年来被认为是一个具有临床潜力抗肿瘤靶点之一[9]。目前基于 Prmt5在生物体内的作用机制研发的小分子药物已进入临床II 期阶段[10]。但是,关于Prmt5与气道纤毛细胞的关系尚不清楚。
参考文献
[1] 张罗, 韩德民, 呼吸道纤毛运动调控机制的研究现状. 中华耳鼻咽喉科杂志 [J], 2004(03): 63-67.
[2] Whitsett, J.A., Airway epithelial differentiation and mucociliaryclearance. Ann Am Thorac Soc [J], 2018.V 15(Suppl 3): S143-S148.
[3] 李健, 蔡映云, 气道粘液纤毛清除系统及其功能障碍. 国外医学(内科学分册) [J], 2003(12): 524-526+543.
[4] Dantas, T.J., Centrosomes and cilia: always at the center of theaction. Commun Biol [J], 2020.V 3(1): 785.
[5] Knowles, M.R., M. Zariwala, and M. Leigh, Primary ciliarydyskinesia. Clin Chest Med [J], 2016.V 37(3): 449-461.
[6] Knowles, M.R., L.A. Daniels, S.D. Davis, et al., Primary ciliarydyskinesia. Recent advances in diagnostics, genetics, and characterization ofclinical disease. Am J Respir Crit Care Med [J], 2013.V 188(8): 913-922.
[7] Kim, H. and Z.A. Ronai, PRMT5 function and targeting in cancer.Cell Stress [J], 2020.V 4(8): 199-215.
[8] Zhu, F. and L. Rui, PRMT5 in gene regulation and hematologicmalignancies. Genes Dis [J], 2019.V 6(3): 247-257.
[9] 沈昊, 张玲, 刘修恒, PRMTs在肿瘤发生、发展中的作用及机制. 中国现代医学杂志 [J], 2020.V 30(01): 45-51.
[10] Fedoriw, A., S.R. Rajapurkar, S. O'Brien, et al., Anti-tumoractivity of the type I PRMT inhibitor, GSK3368715, synergizes with PRMT5inhibition through MTAP loss. Cancer Cell [J], 2019.V 36(1): 100-114 e125。
发明内容
1.本发明旨在解决现有纤毛细胞缺陷动物模型缺乏的问题,旨在提供一种气道纤毛细胞完全缺失的动物模型。
2.为解决上述技术问题,本发明提供Prmt5基因在建立纤毛细胞缺失小鼠模型中的应用。
3.本发明还提供一种纤毛缺失动物模型的构建方法,其包括如下步骤:1. 通过Prmt5 flox/flox 基因型小鼠和Shh-Cre基因型小鼠杂交,获得Prmt5 flox/+ 基因型小鼠。2.Prmt5 flox/ ;Shh-Cre基因型小鼠和Prmt5 flox/flox 小鼠杂交,获得Prmt5 flox/flox;Shh-Cre 基因型小鼠,即为所述纤毛细胞缺失动物模型。
4.本发明还提供一种纤毛细胞缺失的体外3D类器官培养方案和培养条件。具体为,分离纤毛细胞缺失小鼠的大气道上皮祖细胞,将单细胞在50% Matrigel条件下3D培养,所形成的类器官模拟动物体内纤毛细胞完全缺失的现象。
5.本发明还提供一种纤毛细胞缺失的体外2D类器官培养方案和培养条件。具体为,分离纤毛细胞缺失小鼠的大气道上皮祖细胞,将细胞在细胞培养小室内培养,在气-液交界面培养条件下,细胞分化形成气道假复层上皮结构类器官,所形成的类器官纤毛细胞完全缺失,完美模拟动物体内纤毛细胞缺失的现象。
6.本发明还提供上述方法获得的动物模型和培养方案在纤毛缺陷肺病发病机制和药物筛选等研究中的应用。
有益效果:
本发明与现有技术相比,有益效果在于:
1.通过细胞特异性基因敲除的方法构建气道纤毛缺失动物模型,不造成肺组织内其它细胞类型的改变,更不会造成其他器官的毒性。而且此方法构建的动物模型,稳定可靠的重复纤毛细胞缺失的现象,应用范围广泛。
2.本发明提供2种体外类器官培养的方法,在体外模拟动物模型体内纤毛细胞缺失的现象,具有可以批量培养、时间周期短和不用牺牲动物等优点,应用潜力大。
附图说明:
图 1为纤毛细胞缺失的动物模型构建方法模式图;A为通过Prmt5 flox/flox 基因型小鼠和Shh-Cre基因型小鼠杂交,获得Prmt5 flox/+ 基因型小鼠;B为Prmt5 flox/+ ;Shh-Cre基因型雄性小鼠和Prmt5 flox/flox 雌性小鼠杂交,获得Prmt5 flox/flox ;Shh-Cre基因型小鼠。
图2为Prmt5基因在肺上皮细胞中特异性敲低验证结果图;图2A为蛋白质免疫印迹法检测对照组和Prmt5敲除组小鼠肺脏中Prmt5蛋白表达情况;图2B为图2A中Prmt5蛋白表达的定量统计(**P<0.01表示,学生t检验);图2C为用Prmt5和E-cadherin抗体对对照组和Prmt5敲除组小鼠肺组织免疫荧光染色代表图,E-cadherin标记肺上皮细胞,Dapi标记细胞核,箭头指示Prmt5的表达,比例尺为20 μm。
图 3为来自对照组和Prmt5敲除组小鼠大气道内侧细胞扫描电镜分析结果代表照片,比例尺为10 μm。
图4A为纤毛细胞的分化发育过程模式图;图4B为用c-Myb和Sox2抗体对E13.5时期对照组和Prmt5敲除组小鼠大气道免疫荧光共染色代表图,箭头指示c-Myb表达的纤毛祖细胞;图4C为用Foxj1和Sox2抗体对E15.5时期大气道免疫荧光共染色代表图,箭头指示Foxj1表达的上皮细胞;图4D为用ac-tubulin、CC10和Sox2抗体对E18.5时期对照组和Prmt5敲除组小鼠大气道免疫荧光共染色代表图,箭头指示对照组中的ac-tubulin表达的纤毛细胞。图4A-C中Dapi标记细胞核,比例尺为20 μm。
图5为纤毛细胞缺失的3D体外类器官培养过程示意图。
图6A为对照组和Prmt5敲除组小鼠大气道上皮祖细胞在体外3D条件下培养形成的类器官,比例尺为50 μm;图6B为利用Sox2、Krt5、ac-tubulin(ac-tub)和p63抗体染色对照组和Prmt5敲除组类器官免疫荧光染色代表图,箭头指示对照组中的ac-tubulin表达的纤毛细胞,Dapi标记细胞核,比例尺为20 μm。
图7为纤毛细胞缺失的2D体外类器官培养过程示意图。
图8A为2D气-液交界面培养所形成的类器官免疫荧光染色代表图,箭头指示对照组中的ac-tubulin和Foxj1表达的纤毛细胞,Dapi标记细胞核,比例尺为20 μm;图 8B为qRT-PCR分析2D培养条件下对照组和Prmt5敲除组类器官各种细胞类型标记基因相对表达量,显示Prmt5敲除组形成的类器官表达棒状细胞标记基因CC10,但是不表达纤毛细胞标记基因ac-tubulin和Foxj1,数据表示为平均±SD、ns、不显著、***P<0.001表示,学生t检验。
具体实施方式
为了使本发明的技术方案和实施方式更加清楚明白,以下结合附图及实施例,对本发明的上述内容做进一步详细说明。此处所描述的具体实施例仅用以解释本发明,不应将此理解为本发明上述主题的范围仅限于以下的实例。
需要说明的是,本实施例中所用小鼠均为C57BL/6遗传背景,饲养于安徽大学动物暂养室中;PBS溶液成分:Na2HPO4·12H2O购自Meilun Bio公司,货号为MB0445;KH2PO4购自阿拉丁公司,货号为B1912077;NaCl购自上海生工公司,货号为H305BA0006。AdvancedDMEM/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;PrimocinTM 购于Invivogen,货号:ant-pm-1;Insulin购于Sigma公司,货号为I6634;CHIR99021购于Tocris公司,货号为4423;DMH-1购于Tocris公司,货号为4126;Y27632购于Tocris公司,货号为1254;视黄酸购于Sigma公司,货号为R2625-1g;Transferrin购于Sigma公司,货号为T1147-100 mg;
本发明中所称的英文缩写所代表的含义分别表示:Prmt5:蛋白质精氨酸甲基转移酶5;Shh:音猬因子基因,在肺早期上皮细胞中特异性表达;Cre:Cre重组酶,通过催化特定DNA位点特异性重组实现基因敲除;Shh-Cre小鼠:肺上皮细胞特异性敲除鼠;rpm:转速每分钟;FBS:胎牛血清;PFA:多聚甲醛;PBS:磷酸盐缓冲液;2D:表示二维培养;3D:表示三维培养。Matril gel:Matril 凝胶;EGF:表皮生长因子;Transferrin:转铁蛋白;Insulin:胰岛素;Sox2:原癌基因的一种;Dapi:一种与DNA强力结合的荧光染料;p63:抑癌基因p53家族的一种;Krt5:角蛋白5; ac-tubulin:乙酰化微管蛋白;c-Myb:一种转录因子,在气道纤毛祖细胞核内特异表达;Foxj1:气道纤毛细胞核内特异表达基因;CC10:气道棒状细胞特异表达的10kD分子量蛋白。
1.本发明实施例提供Prmt5基因在建立纤毛缺失小鼠模型中的应用。
2.本发明实施例还提供一种纤毛细胞缺失动物模型的构建方法,其包括如下步骤:将Prmt5 flox/flox 小鼠和Shh-Cre小鼠杂交,获得Prmt5 flox/+ ;Shh-Cre基因型小鼠。再将获得的Prmt5 flox/+ ;Shh-Cre小鼠和Prmt5 flox/flox 小鼠杂交,获得所述纤毛细胞缺失动物模型即Prmt5 flox/flox ;Shh-Cre。
3.本发明实施例还提供一种纤毛细胞缺失的3D类器官培养方案,其包括如下步骤:分离Prmt5 flox/flox ;Shh-Cre小鼠气道上皮祖细胞,将细胞在3D条件下培养18天后,获得所述纤毛细胞缺失类器官模型
4.具体地,所述上皮祖细胞为E13.5天,所述3D培养为50% Matrigel培养。
5.优选地,所述体外3D类器官培养所用培养液为:Advanced DMEM/F-12 89%,青链霉素混合液 1%,GlutaMAX 4 mM,NaHCO3 3.6 mM,HEPES 15 mM,PrimocinTM 100 μg/ml,FBS5%,EGF 25 ng/ml,Transferrin 5 μg/ml,视黄酸 50 nM,DMH-1 10 μg/ml,CHIR99021 5 μM,Y276325 mM。
6.本发明实施例还提供一种纤毛细胞缺失的2D类器官培养方案,其包括如下步骤:分离Prmt5 flox/flox ;Shh-Cre小鼠气道上皮祖细胞,将细胞在2D条件下培养14天后,获得所述纤毛细胞缺失类器官模型。
7.优选地,所述2D培养条件为,气道上皮祖细胞先浸没培养5-7天后,再气-液交界面培养10-14天;所述体外2D培养基为:Ham’s F12 45%,DMEM 45%,FBS 10%,青链霉素混合液 1%,Gluta MAX 4 mM,NaHCO3 3.6 mM,HEPES 15 mM,EGF 50 ng/ml,Insulin 20 ng/ml。
8.本发明还提供上述方法获得的动物模型和培养方法在纤毛缺陷相关肺病发病机制、药物筛选、诊断治疗等方面的应用。
实施例1.
一种通过特异性敲除肺上皮细胞中Prmt5基因构建纤毛细胞缺失的动物模型的建立方法,具体步骤如下:
1.将Prmt5 flox/flox 基因型小鼠与肺上皮细胞特异性表达的Cre工具小鼠Shh-Cre小鼠杂交,获得Prmt5 flox/+ ;Shh-Cre基因型小鼠(图1A)。
2.将10-12周龄Prmt5 flox/+ ;Shh-Cre基因型雄性小鼠,与成年Prmt5 flox/flox 雌性小鼠杂交,在得到的后代胚胎中,理论上有四分之一的概率获得Prmt5 flox/flox;Shh-Cre 基因型小鼠,即Prmt5基因肺上皮细胞特异性敲除小鼠(图1 B)。
3.通过对照组和Prmt5基因敲除小鼠肺组织表达量分析,显示Prm5敲除后Prmt5表达量显著降低(图2A-B),免疫荧光染色分析结果显示:Prmt5只在小鼠肺上皮细胞中特异性缺失,其在肺间质细胞中的表达不受影响(图2 C)。
4.通过扫描电镜分析,在胚胎发育的18.5(E18.5)天,Prmt5敲除小鼠大气道组织中纤毛结构完全缺失(图3)。
5.在气道和肺发育过程中,在胚胎发育的12.5(E12.5)天,所有气道上皮祖细胞表达转录因子Sox2,E13.5天开始表达纤毛祖细胞标记基因c-Myb,而在E14.5天后,纤毛细胞开始表达转录因子Foxj1,成熟的纤毛细胞表达纤毛蛋白ac-tubulin(图4A)。通过对气道发育各时期纤毛细胞标记基因进行免疫荧光染色分析,发现Prmt5在气道上皮细胞特异性敲除后,纤毛前体细胞特异性表达标记基因c-Myb和Foxj1,以及成熟纤毛细胞标记基因ac-tubulin,都在气道组织中完全缺失(图4B-D)。
6.以上多种分析结果说明,通过将Prmt5基因在肺上皮细胞中特异性敲除,成功构建了纤毛上皮细胞缺失的动物模型。
实施例2.
一种纤毛细胞缺失的3D类器官培养方案和培养基组合。首先将E13.5时期Prmt5基因敲除小鼠气道上皮祖细胞分离,继而将分离的祖细胞在3D 50% Matrigel条件下培养,形成纤毛细胞完全缺失的气道类器官(图5)。具体步骤如下:
1.将E13.5时期对照组和Prmt5敲除组小鼠肺大气道分离出来,纵向剖开。
2.用胶原酶在37℃细胞培养箱中消化15分钟。
3.加入 3D 培养基,重悬吹打成单细胞,细胞悬液与 Matrilgel在冰上混合均匀,使其终浓度为50 %。
4.加到细胞培养皿中,37 ℃放置 20 分钟,待 Matrilgel凝固后加入含有生长因子的 3D 小鼠气道上皮细胞培养基,在细胞培养箱中培养18天。。
5.培养过程中每两天换培养基。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。
6.获得对照组和Prmt5敲除组气道类器官,通过显微镜观察分析,Prmt5敲除组形成的类器官和对照组在外观上并无区别,都是中空的球形结构(图6A)。
7.将类器官用PBS配置的4% PFA室温固定20分钟,1000rpm离心3分钟去掉PFA,再用PBS漂洗3次。
8.对类器官进行免疫荧光染色分析,结果显示Prmt5敲除的上皮祖细胞所形成的类器官,完全不表达纤毛细胞特异标记基因蛋白(图6B)。
9.说明以上培养方案成功建立了纤毛细胞完全缺失的3D气道类器官培养模型。
实施例3.
一种纤毛细胞缺失的2D类器官培养方案和培养基组合。首先将E13.5时期对照组和Prmt5缺失组小鼠气道上皮细胞分离,继而将分离的气道祖细胞在细胞培养小室内培养,浸没培养5-7天后,创造气体-液体交界面培养条件,继续培养10-14天,细胞分化为含有纤毛细胞和棒状细胞的假复层气道结构类器官(图7)。具体步骤如下:
1.将E13.5时期对照组和Prmt5缺失组小鼠肺大气道分离出来,纵向切开。
2.用胶原酶在37℃细胞培养箱中消化15分钟,分离气道上皮祖细胞。
3.加入 2D类器官培养基,终止胶原酶反应。
4.1000 rpm,离心5分钟,丢弃上清后,将细胞重悬于2D类器官培养基中,吹打成单细胞。
5.将分离的气道上皮祖细胞接种在细胞培养小室内,浸没培养5-7天。每 2 天更换培养基,每次更换培养基总体积的一半。
6.吸出细胞培养小室上层的培养基,为细胞创造气-液交界面培养条件,继续培养10-14天,获得气道类器官。
7.将在细胞培养小室中的类器官用PBS配置的4% PFA室温固定15分钟,吸掉PFA后再用PBS漂洗3次。
8.对细胞培养小室中的类器官进行免疫荧光染色分析,结果显示,在Prmt5敲除小鼠中,完全没有纤毛细胞标记基因的表达(图8A)。而且qRT-PCR分析结果显示,Prmt5敲除后所形成的类器官不再表达纤毛细胞标记基因Foxj1和ac-tubulin,但是表达棒状细胞标记基因CC10。
9.以上分析结果说明以上培养方案成功建立了纤毛细胞完全缺失的2D气道类器官培养模型。
上述实施例仅示例性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修改。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种Prmt5基因在肺上皮细胞中敲除后气道纤毛上皮细胞缺失的动物模型的构建方法,其特征在于,包括如下步骤:
1)将Prmt5 flox/flox 动物和肺上皮细胞特异性表达的Cre动物杂交,获得Prmt5 flox/+ ;Shh; Cre基因型动物;
2) 将Prmt5 flox/+ ;Shh;Cre动物和Prmt5 flox/flox 动物杂交,获的Prmt5基因在肺上皮细胞特异性敲除的纯合子动物;
3) Prmt5基因在肺上皮细胞特异性敲除的纯合子动物,即气道纤毛上皮细胞缺失的动物模型。
2.一种纤毛细胞缺失的3D体外类器官培养方案和培养基。
3.一种纤毛细胞缺失的2D体外类器官培养方案和培养基。
4.如权利要求3所述的3D体外培养方法,其特征在于,所述3D培养条件为:50%Matrigel培养;所述3D培养基成分为:Advanced DMEM/F-12 89%,青链霉素混合液 1%,GlutaMAX 4 mM,NaHCO3 3.6 mM,HEPES 15 mM,PrimocinTM 100 μg/ml,FBS 5%,EGF 25 ng/ml,Transferrin 5 μg/ml,视黄酸 50 nM,DMH-1 10 μg/ml,CHIR99021 5 μM,Y27632 5mM。
5.如权利要求4所述的2D体外培养方法,其特征在于,所述2D培养条件为:细胞在细胞培养小室中气-液交界面培养。
6.所述体外2D培养基为:Ham’s F12 45%,DMEM 45%,FBS 10%,青链霉素混合液 1%,Gluta MAX 4 mM,NaHCO3 3.6 mM,HEPES 15 mM,EGF 50 ng/ml,Insulin 20 ng/ml。
7.Prmt5基因在肺上皮细胞中特异性敲除在构建气道纤毛缺失动物模型中的用途。
8.一种由权利要求1-8任一所述的构建方法构建的纤毛细胞缺失模型。
9.权利要求9所述的Prmt5基因敲除动物模型在制备或筛选治疗和/或预防纤毛缺陷相关肺病的药物中的用途。
10.一种筛选治疗和/或预防纤毛缺陷相关肺病的药物的方法,其特征在于:所述方法包括以下步骤:
1)由权利要求1-8任一所述的构建方法构建获得Prmt5基因敲除的纤毛缺失动物模型;
2)利用步骤1)制备得到的动物模型筛选对纤毛缺陷相关肺病具有治疗和/或预防效果的药物。
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