CN109486766A - 一种泪腺干细胞、泪腺干细胞的培养体系和培养方法 - Google Patents
一种泪腺干细胞、泪腺干细胞的培养体系和培养方法 Download PDFInfo
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Abstract
本发明涉及一种泪腺干细胞、泪腺干细胞的培养体系和培养方法,涉及细胞工程领域。本发明的泪腺干细胞的培养体系以DMEM/F12为基础培养基,添加以下组分构成培养液:细胞培养添加剂、非必需氨基酸、L‑丙氨酰‑L‑谷氨酰胺、丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路配体、成纤维细胞生长因子、Wnt信号通路配体和ROCK信号通路抑制剂的组合物,并以基质胶作为立体培养的支架。本发明的培养方法为,将小鼠泪腺组织消化成单细胞接种到基质胶中,待其凝固后,加入上述泪腺干细胞培养液中进行原代培养。采用本发明的方法培养的小鼠泪腺干细胞,数量较多,够稳定持续传代,并且具备恢复泪腺分泌功能的作用,可有效治疗干眼症。
Description
技术领域
本发明涉及细胞工程领域,特别是涉及一种三维立体培养体系及采用此培养体系培养的小鼠泪腺干细胞(LGSC)。
背景技术
泪腺是眼部重要器官,主要由分泌腺泡和分泌管组成。泪腺受到交感神经和副交感神经调节,其中大部分是胆碱能神经,少部分是肾上腺素能神经。交感神经源自颈上神经节,副交感神经则与翼腭神经节和睫状神经节相连接,并且泪腺的某些感觉神经与三叉神经相连。泪腺中有多种乙酰胆碱受体,包括毒蕈硷受体、I型和II型血管活性肠肽受体及去甲肾上腺素受体α1、β。神经调节下泪腺能分泌溶菌酶,乳铁传递蛋白,脂质运载蛋白,免疫球蛋白A等。
泪腺分泌眼泪,形成泪膜,使眼睛保持湿润,以保护眼睛。一旦泪腺病变或受到损伤,眼泪分泌不足,人就会患上干眼症(Dry eye Disease,DED)。人的衰老、自身免疫***的紊乱、眼部的放疗、以及雄性激素水平的降低都会引起泪腺功能失调。干眼症患者所分泌形成的泪膜比正常泪膜渗透压高,从而引起角膜炎症,导致角膜细胞受损、形成溃疡,最终导致患者视力下降,严重影响其生活质量。
对于干眼症患者,目前的药物治疗方案主要有点滴等渗或低渗的眼药水,抑制因干眼症引起的眼结膜和角膜的炎症发生,阻塞泪孔增加泪膜厚度等。但这些治疗只能缓解干眼症的症状,使眼睛尽可能地保持湿润,避免角膜细胞受到损伤。而且,长时间使用眼药水和抗炎症药物,也会杀伤角膜细胞并且引发更难治愈的炎症反应。因此,药物治疗并不能从根本上解决泪腺分泌功能失调的问题,不能彻底治愈老化或者病变的泪腺。
为了更有效更彻底地治疗干眼症,研究者们开展了一系列研究,旨在恢复泪腺的分泌功能。要完全恢复泪腺的分泌功能,就需要获得新的健康细胞或者器官进行移植治疗。为了得到用于移植的泪腺细胞,研究者便开始尝试体外分离培养泪腺干细胞。经过近三十年的努力,泪腺干细胞的体外培养有了突破性的研究,然而,现有的泪腺干细胞体外培养方法分选祖细胞的效率非常低、数量少,并且不能持续传代培养,大大限制了其临床应用。因此,需要更加优良的分离方法和培养体系来分离和培养泪腺干细胞,为临床应用提供良好的技术平台。
发明内容
基于此,有必要针对现有的泪腺干细胞培养体系和培养方法分选祖细胞效率低、数量少的问题,提供一种泪腺干细胞的培养体系和培养方法,以获得较多数量的泪腺干细胞,并且该泪腺干细胞能够进行稳定的传代培养。
一种泪腺干细胞的培养体系,以DMEM/F12为基础培养基,添加以下组分构成培养液:细胞培养添加剂、非必需氨基酸、L-丙氨酰-L-谷氨酰胺、丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路配体、成纤维细胞生长因子、Wnt信号通路配体和ROCK信号通路抑制剂的组合物,并以基质胶作为立体培养的支架。
进一步地,所述细胞培养添加剂为N2和B27的混合物。
所述细胞培养添加剂也可以选用9F,由β-巯基乙醇、2-氨基乙醇、***钠、肝素、转铁蛋白、维生素C、胰岛素、牛血清白蛋白-油酸和成纤维细胞生长因子-2(FGF2)组成。
进一步地,所述丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路(MAPK/ERK信号通路)配体为EGF,所述成纤维细胞生长因子为FGF10,所述Wnt信号通路配体为Wnt3A,所述ROCK信号通路抑制剂为Y-27632。
进一步地,所述细胞培养添加剂中N2浓度为1%,B27浓度为2%;所述非必需氨基酸浓度为1%,所述L-丙氨酰-L-谷氨酰胺浓度为1%。
进一步地,所述细胞培养添加剂9F中各组分浓度为:β-巯基乙醇5-10μM、2-氨基乙醇5-10μM、***钠10-20μM、肝素50-150ng/ml、转铁蛋白2-5ng/ml、维生素C 5-10ng/ml、胰岛素5-10ng/ml、牛血清白蛋白-油酸6.8-9.4ng/ml和成纤维细胞生长因子-2 5-10ng/ml;所述非必需氨基酸1%,所述L-丙氨酰-L-谷氨酰胺1%。
进一步地,所述丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路配体为EGF,所述成纤维细胞生长因子为FGF10,所述Wnt信号通路配体为Wnt3A,所述ROCK信号通路抑制剂为Y-27632。
进一步地,所述细胞培养添加剂中EGF浓度为10-50ng/ml,FGF10浓度为10-100ng/ml,Wnt3A浓度为5-10ng/ml,Y-27632浓度为5-10mM。
进一步地,所述基质胶为Matrigel。Matrigel是具有生物学活性的三维基质,模拟体内细胞基底膜的结构、组成、物理特性和功能,采用其作为基质胶有利于体外培养小鼠泪腺干细胞的培养和分化。
进一步地,非必需氨基酸使用NEAA,其中含有Gly、L-丙氨酸、L-天冬酰胺、L-天冬氨酸、L-谷氨酸、L-脯氨酸、L-丝氨酸等7种非必需氨基酸,能有效改善细胞培养基配比。
一种泪腺干细胞的培养方法,将小鼠泪腺组织消化成单细胞接种到基质胶中,待其凝固后,加入上述泪腺干细胞培养液中,并在37℃、5%~10%CO2环境下进行原代培养。
一种泪腺干细胞,采用上述培养体系和培养方法制备得到。
与现有技术相比,本发明具有以下有益效果:
一、本发明克服了传统的平面培养方法不能有效地培养得到泪腺干细胞的缺陷,构建了三维立体培养的方法进行体外培养小鼠泪腺干细胞,通过此方法培养的小鼠泪腺干细胞数量较多,为临床研究提供充足的细胞材料;
二、本发明利用基质胶进行三维立体培养泪腺干细胞,随着时间推移干细胞会自我复制,并且一部分会进行分化,形成与该器官结构近似或细胞更新过程类似的微器官,即“类器官体”,干细胞在类器官体中与分化的细胞相互作用,信号分子相互影响,形成了一个相对稳定的干细胞增殖微环境,使得干细胞能够持续传代,并保持一定的组织特性和基因型稳定性;
三、采用本发明建立的体外培养体系得到的小鼠泪腺干细胞,移植后的小鼠泪液分泌量显著增加,不仅具有修复损伤的泪腺,还具备恢复泪腺分泌功能的作用,为研究干眼症的干细胞治疗提供了良好基础。
附图说明
图1为LGSCM条件小鼠泪腺干细胞持续传代时不同培养代次的状态变化图,比例尺:100μm;
图2为LGSCM条件小鼠泪腺干细胞持续传代时不同培养代次的细胞总数统计;
图3为LGSCM条件小鼠泪腺干细胞随着培养时间增加的状态变化图,比例尺:100μm;
图4为原代培养泪腺干细胞的状态图,a、LGSCM,b、9F-LGSCM,比例尺:100μm;
图5为RT-PCR检测不同代数的泪腺干细胞的干性基因的表达情况图;
图6为单独撤去各生长因子、配体和抑制剂原代培养泪腺干细胞(P0代)第7天的状态图,比例尺:400μm;
图7为单独撤去各生长因子、配体和抑制剂P0代泪腺干细胞培养7天后的大小和细胞总数图,***,P<0.01;
图8为单独撤去各生长因子、配体和抑制剂P2代泪腺干细胞第7天的状态图,比例尺:400μm;
图9为单独撤去各生长因子、配体和抑制剂P2代泪腺干细胞培养7天后的大小和细胞总数,***,P<0.01;
图10为培养14天泪腺干细胞的IF检测图,比例尺:100μm;
图11为成熟小鼠泪腺的IF检测图,比例尺:100μm;
图12为培养超过14天泪腺干细胞的IF检测,比例尺:200μm;
图13为干眼症小鼠治疗前后的眼部症状图;
图14为干眼症小鼠移植泪腺干细胞8周后泪腺切片的IHC检测图,比例尺:100μm;
图15为干眼症小鼠移植泪腺干细胞8周泪液的分泌量,*,P<0.05;***,P<0.01。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
材料说明:
本发明实验中小鼠购自于广东省医学实验动物中心;N2、B27、Glutamax、NEAA均购自于Gibco,EGF、FGF10、Wnt3A均购自Pepro Tech,Y-27632购自于Sigma。
主要溶液以及配方:
(1)细胞培养实验中用到的溶液或试剂
1)PBS Buffer的配置:称取NaCl 8g,KCl 0.2g,Na2HPO4 1.155g,KH2PO4 0.2g,加入约800ml的超纯水,充分溶解后,pH值调至7.4,定容1L,高温高压灭菌后,4℃保存。
2)10×Trypsin-EDTA(TE)的配置:称取1.25g trypsin,1.00g EDTA溶解于250ml的PBS中,过滤除菌,-20℃保存。使用前用加入PBS稀释成1×TE用于消化细胞。
3)从BD公司购买中性酶(Dispase)直接用于原代培养时,消化泪腺组织使用。
4)泪腺干细胞培养液:LGSCM,9F-LGSCM。
5)DMEM/F12(Sigma)培养基组成成分如下表所示:
(2)核酸电泳实验中用到的溶液或试剂
1)50×TAE电泳Buffer(pH8.5):在1L的玻璃瓶中加入242g Tris,18.6gNa2EDTA·2H2O,然后加入约800ml的二级水,充分搅拌溶解,再加入57.1ml的醋酸,充分搅拌,加二级水定容至1L,室温保存。使用时稀释50倍。
2)溴化乙锭(EB):储存液浓度为10mg/ml,工作浓度为0.5μg/ml。
3)1%琼脂糖电泳凝胶:称取1g琼脂糖(agarose),加入100ml 1×TAE buffer,加热煮沸至琼脂糖完全溶解,冷至50℃左右,加入5μl EB储存液,使终浓度为0.5μg/ml,摇匀后灌制凝胶平板。
(3)免疫荧光实验中用到的溶液或试剂
1)4%PFA:20g多聚甲醛溶解于400ml 1×PBS中,65℃加热溶解过夜(约12h)。待底部剩余少量沉淀时,加入40μl 1N NaOH促溶,并定容500ml,4℃保存。
2)30%蔗糖溶液:3g蔗糖粉末溶解于4%PFA中,定容10ml,4℃保存。
3)封闭液:用1×PBS稀释非免疫山羊血清至10%,现配现用保存。
4)一抗:Krt14(Abcam,ab181595),Krt19(Abcam,ab52625),AQP5(Abcam,ab104751),E-cadherin(Abcam,ab11512),mCherry(Abcam,ab167453)
5)二抗:购买于life公司。
实施例1:
一种泪腺干细胞的培养体系,以DMEM/F12为基础培养基,添加以下组分和浓度构成培养液:1%N2,2%B27,1%NEAA,1%Glutamax,10-50ng/ml EGF,10-100ng/ml FGF10,5-10ng/ml Wnt3A,5-10mM Y-27632,并以Matrigel作为立体培养的支架。此处的培养液称为LGSCM。
实施例2:
一种泪腺干细胞的培养体系,以DMEM/F12为基础培养基,添加以下组分和浓度构成培养液:5-10μMβ-巯基乙醇,5-10μM 2-氨基乙醇,10-20nM***钠,50-150ng/ml肝素,2-5ng/ml转铁蛋白,5-10ng/ml维生素C,5-10μg/ml胰岛素,6.8-9.4μg/ml牛血清白蛋白-油酸,5-10ng/ml FGF2,1%NEAA,1%Glutamax,10-50ng/ml EGF,10-100ng/ml FGF10,5-10ng/ml Wnt3A,5-10mM Y-27632,并以Matrigel作为立体培养的支架。此处的培养液称为9F-LGSCM
实施例3:
一种泪腺干细胞的培养方法,包括以下步骤:
a)采用酶消化法分离获得小鼠泪腺单细胞;
b)按照1×104细胞/孔的比例种入Matrigel基质内,加入调配好的泪腺干细胞培养液LGSCM中进行培养,培养环境为37℃、5%~10%CO2,培养7天以后便有球囊状干细胞群体形成,并采用1×104细胞/孔的比例传代。
实验例4:
一种泪腺干细胞的培养方法,包括以下步骤:
a)采用酶消化法分离获得小鼠泪腺单细胞;
b)按照1×104细胞/孔的比例种入Matrigel基质内,加入调配好的泪腺干细胞培养液9F-LGSCM中进行培养,培养环境为37℃、5%~10%CO2,培养7天以后便有球囊状干细胞群体形成,并采用1×104细胞/孔的比例传代。
实施例5:
采用实施例3或实施例4的培养方法得到的泪腺干细胞。
为了支持上述实施例,本发明进行了小鼠泪腺干细胞的原代培养、传代培养,泪腺干细胞培养液成分验证,小鼠泪腺干细胞的鉴定,小鼠泪腺干细胞的治疗干眼症等一系列实验,各实验的具体实验步骤如以下实验例:
实验例1:小鼠泪腺干细胞原代培养
1)处死小鼠,解剖获取小鼠泪腺,1×PBS浸泡清洗1分钟,75%乙醇浸泡15s,立即1×PBS清洗2次;
2)将组织尽可能剪碎,转移至15ml离心管中,加入500μl Dispase 37℃消化1h;
3)用1ml微量移液枪吹打消化后的组织,使其散开,以获得单细胞,使用预冷的1×PBS清洗两次;
4)收集40μm滤膜过滤后的细胞,计数,按照1×104细胞/孔的比例重悬于40μl培养液中与40μl Matrigel混合后,种入24孔板内,置于37℃培养箱中,使Matrigel凝固,20分钟后加入预先配好的干细胞培养液进行培养。
小鼠泪腺干细胞LGSCM条件下,原代和持续传代不同培养代次的状态变化如图1所示;原代培养泪腺干细胞的状态如图4所示,其中a为以LGSCM为培养液,b为以9F-LGSCM为培养液,9F-LGSCM培养的泪腺干细胞团直径略小于LGSCM培养的泪腺干细胞团。
实验例2:泪腺干细胞传代培养
1)24孔板内每孔加入100μl1×PBS和20μlDispase,将Matrigel与细胞混合物捣碎,置于37℃培养箱中消化30分钟,将Matrigel消化成液体形态,
2)使用15ml离心管收集培养的泪腺干细胞,500rpm离心4分钟,收集沉淀;
3)加入1×TE消化细胞,37℃孵育10分钟;
4)终止TE,将泪腺干细胞吹打成单细胞,1200rpm离心4分钟,收集细胞,按照1×104细胞/孔的比例重悬于40μl培养液中与40μl Matrigel混合后,种入24孔板内,置于37℃培养箱中,使Matrigel凝固,20分钟后加入预先配好的干细胞培养液LGSCM或9F-LGSCM进行培养。
采用该方法培养的小鼠泪腺干细胞,在干细胞培养液的条件下,稳定传代至40代以上,持续传代培养时间超过1年以上。小鼠泪腺干细胞持续传代不同培养代次的状态变化如图1所示;小鼠泪腺干细胞持续传代不同培养代次培养7天的细胞总数的统计如图2所示;小鼠泪腺干细胞随着培养时间增加的状态变化如图3所示。
结果分析:小鼠泪腺干细胞的培养一般为7天,原代培养时,小鼠泪腺干细胞呈现出出芽分枝状,而传代以后,其形态则保持实心的球囊状,并且随着传代次数的增加,其状态比较稳定,如图1。统计不同代次的细胞总数发现,泪腺干细胞在LGSCM条件下的增殖能力较强,1×104个细胞培养7天以后,就能扩增出4×105个细胞以上。并随着传代次数的增加,泪腺干细胞逐渐适应体外培养的环境,其增殖能力逐渐增强,并最后趋于稳定,如图2。在LGSCM条件下不同培养时间,泪腺干细胞的状态有所变化,形成的球囊细胞团会越来越大,如图3。同理,9F-LGSCM条件也能得到类似的球囊状泪腺干细胞,其性质也与LGSCM条件下的泪腺干细胞类似,如图4。由于在9F-LGSCM条件下,泪腺干细胞的增殖相对缓慢,因此,在接下来的各种验证实验中,我们都使用LGSCM条件下的泪腺干细胞。
实验例3:泪腺干细胞培养液成分验证
为了验证培养基中主要的生长因子、配体以及抑制剂的必要性,利用实验例1和实验例2的培养方法,分别单独撤去各个生长因子、配体以及抑制剂,配制对应的培养液,各自培养泪腺干细胞,然后对比其生长状态以及增殖速度。
分别单独撤去Wnt3A、FGF10、Y-27632、EGF,原代培养泪腺干细胞(P0代)第7天的状态如图6所示;
分别单独撤去Wnt3A、FGF10、Y-27632、EGF,P0代泪腺干细胞培养7天后的大小和细胞总数如图7所示;
分别单独撤去Wnt3A、FGF10,P2代泪腺干细胞第7天的状态如图8所示;
分别单独撤去Wnt3A、FGF10,P2代泪腺干细胞培养7天后的大小和细胞总数如图9所示。
结果分析:撤去LGSCM中各个主要生长因子以后,我们发现撤去EGF,Y-27632,FGF10对泪腺干细胞的原代培养都有显著影响,泪腺干细胞的球囊直径以及细胞总数都显著降低。传代2次以后,Wnt3A的撤去也会有显著影响。由此可推测,我们建立的培养体系中添加的各个生长因子、配体以及抑制剂对泪腺干细胞的生长与增殖都有重要作用。
实验例4:泪腺干细胞各基因表达mRNA水平的检测
i.细胞RNA的提取
1)收集不同代数培养14天和同一代次不同培养天数的细胞,加入1ml Trizol,涡旋振荡至细胞裂解完全;
2)加入0.2ml氯仿,涡旋振荡30秒,室温静置10分钟,直到液体分层,12000rpm 4℃离心15分钟;
3)离心后,能清除观察到离心管中液体分为三层,小心吸取最上层的透明水相,转移至新的1.5ml无RNA酶离心管中;
4)加入等体积的异丙醇,轻轻混匀,室温静置10分钟,12000rpm4℃离心15分钟;
5)弃上清,加入75%乙醇1ml,颠倒清洗,12000rpm 4℃离心5分钟,弃上清;
6)将离心管置于超净工作台中,吹干,待白色沉淀变成半透明即可加入20μl的无RNA酶水溶解沉淀;
ii.RT-PCR逆转录反应(TOYOBO ReverTra Ace试剂盒)
1)在PCR反应管中按顺序加入:
总RNA 2μl(1000ng)
65℃作用5分钟;
2)变性后的RNA冰浴2分钟后,加入:
37℃作用5分钟;
3)置于冰上,加入5×RT Master MixⅡ;
4)反转录程序为37℃15分钟,50℃5分钟,98℃5分钟,4℃冰浴5分钟,完成反转录的cDNA样品置于-20℃保存备用。
iii.PCR程序
1)在总体积为20μl的PCR体系为:
2)不同引物按照各种退火温度、循环数、延伸时间等设置;
3)核酸电泳检测结果。
结果分析:核酸电泳检测结果如图5所示,培养所得的泪腺干细胞表达Epcam、Krt5、Krt14、Pax6、P63、Nestin等成体干细胞的标记基因,并且,通过对不同培养代次的泪腺干细胞检测,发现各干细胞标记基因都能稳定表达。
实验例5:小鼠泪腺干细胞体外分化和小鼠泪腺IF检测
1)将培养所得的小鼠泪腺干细胞,在体外培养14天并加入血清或者降低基质胶的硬度,都会使其出现分化,
2)分化后的泪腺干细胞或者小鼠泪腺4%PFA溶液固定过夜;
3)脱水:70%乙醇2小时,80%乙醇2小时,95%乙醇30分钟,95%乙醇30分钟,无水乙醇30分钟,无水乙醇2小时,无水乙醇+TO(1:1)30分钟,TO 30分钟,TO 2小时,TO+石蜡(1:1)30分钟,石蜡3小时,石蜡2小时。共12桶,16小时;
4)石蜡包埋;
5)石蜡切片,切片厚度为4μm;
6)染色步骤如下:
①石蜡切片60℃10分钟,二甲苯10分钟,无水乙醇3分钟,90%乙醇3分钟,80%乙醇3分钟,70%乙醇3分钟,蒸馏水洗3次每次3分钟;
②抗原修复17分钟;
③1×PBS摇床上洗3次,每次5分钟;
④10%山羊血清封闭30分钟后,加入一抗,4℃孵育过夜;
⑤去掉一抗,用1×PBS清洗3次,每次5分钟;
⑥加入二抗,室温孵育1小时;
⑦去掉二抗,用1×PBS清洗3次,每次5分钟;
⑧加入DAPI染色,室温5分钟;
⑨去掉DAPI,用1×PBS清洗3次,每次5分钟;
⑩使用荧光封片剂封片并拍照。
降低基质胶硬度培养14天泪腺干细胞的IF检测结果和加入血清培养14天泪腺干细胞的IF检测结果分别如图10和图12所示。泪腺组织的IF检测结果如图11所示。图10a和图11a中泪腺干细胞显示红色荧光,图10b和图11b中成熟管腔细胞显示红色荧光。图12中a为腺泡样细胞显示绿色荧光,b为泪腺干细胞显示红色荧光,c为细胞核显示蓝色荧光。
结果分析:经过免疫荧光检测发现,培养14天的泪腺干细胞团中,只有部分细胞表达Krt14,主要集中于外层,说明这些细胞尚未分化,能维持其干细胞特性;而另一部分细胞则表达Krt19,主要集中于内部,说明这部分细胞已经分化成为具有管腔特性的细胞。对比泪腺组织管腔中两类细胞的定位表达,发现两种细胞的分布定位与泪腺管腔中的定位类似。由图12可见,加入血清培养14天的泪腺干细胞也会分化出表达AQP5,得到核质比很低的腺泡样细胞,说明培养所得的泪腺干细胞在体外培养中,具有向管腔和腺泡分化的潜能。
实验例6:泪腺干细胞体内修复损伤泪腺
i.泪腺干细胞移植
1)培养自带红色荧光(td-Tomato)小鼠的泪腺干细胞到第七天;
2)将泪腺干细胞消化成单细胞,用Matrigel和DMEM/F12等量混合的基质重悬,使细胞成为2×106cells/ml的浓度;
3)麻醉泪腺损伤的干眼症模型小鼠以后,解剖暴露出泪腺;
4)左侧注射20000各细胞,右侧注射重悬细胞的基质作为对照;
5)注射后,缝合伤口,饲养8周后,检测修复效果。
ii.检测干细胞移植后的治疗效果
1)麻醉小鼠,拍摄小鼠眼部症状;
2)使用自制酚红棉线测量小鼠泪液分泌的量,将酚红棉线放置于小鼠眼角约10秒,然后测量变色部分的长度;
3)解剖小鼠,取出泪腺,固定,切片后,进行IHC检测。切片及染色方法与实验例5相同。
干眼症小鼠治疗前后的眼部症状对比如图13所示,干眼症小鼠移植泪腺干细胞8周后泪腺切片的IHC检测如图14所示,干眼症小鼠移植泪腺干细胞8周泪液的分泌量如图15所示。
结果分析:注射泪腺干细胞以后,小鼠的眼部干眼症症状的到抑制和缓解(图13左侧),而注射基质一侧小鼠眼部明显严重溃烂(图13右侧);解剖小鼠后泪腺切片中能检测到来源于移植的泪腺干细胞,并分化成为泪腺的腺泡细胞和管腔细胞(图14左侧),而对照组(图14右侧)未见分化新生腺泡和管腔细胞,说明泪腺干细胞具备在小鼠体内分化的能力,能够修复损伤的泪腺。对比正常(Normal)、病变治疗后(LGSC)、对照组(Vehicle)小鼠泪液的分泌量(图15),泪腺干细胞移植以后的小鼠泪液分泌量略低于正常水平,但相对于未移植干细胞的组别其泪液分泌量显著增加,说明泪腺干细胞移植不仅可以修复损伤的泪腺,还具备恢复其分泌功能的作用。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种泪腺干细胞的培养体系,其特征在于,以DMEM/F12为基础培养基,添加以下组分构成培养液:细胞培养添加剂、非必需氨基酸、L-丙氨酰-L-谷氨酰胺、丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路配体、成纤维细胞生长因子、Wnt信号通路配体和ROCK信号通路抑制剂的组合物,并以基质胶作为立体培养的支架。
2.根据权利要求1所述的泪腺干细胞的培养体系,其特征在于,所述细胞培养添加剂为N2和B27的混合物。
3.根据权利要求1所述的泪腺干细胞的培养体系,其特征在于,所述细胞培养添加剂为9F,所述9F为β-巯基乙醇、2-氨基乙醇、***钠、肝素、转铁蛋白、维生素C、胰岛素、牛血清白蛋白-油酸和成纤维细胞生长因子-2的混合物。
4.根据权利要求2所述的泪腺干细胞的培养体系,其特征在于,所述细胞培养添加剂中N2体积浓度为0.5%-1%,B27体积浓度为1%-2%;所述非必需氨基酸体积浓度为1%,所述L-丙氨酰-L-谷氨酰胺体积浓度为1%。
5.根据权利要求3所述的泪腺干细胞的培养体系,其特征在于,所述细胞培养添加剂9F中各组分浓度为:β-巯基乙醇5-10μM、2-氨基乙醇5-10μM、***钠10-20μM、肝素50-150ng/ml、转铁蛋白2-5ng/ml、维生素C 5-10ng/ml、胰岛素5-10ng/ml、牛血清白蛋白-油酸6.8-9.4ng/ml和成纤维细胞生长因子-2 5-10ng/ml;所述非必需氨基酸1%,所述L-丙氨酰-L-谷氨酰胺1%。
6.根据权利要求1-5任一所述的泪腺干细胞培养体系,其特征在于,所述丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路配体为EGF,所述成纤维细胞生长因子为FGF10,所述Wnt信号通路配体为Wnt3A,所述ROCK信号通路抑制剂为Y-27632。
7.根据权利要求6所述的泪腺干细胞的培养体系,其特征在于,所述细胞培养添加剂中EGF浓度为10-50ng/ml,FGF10浓度为10-100ng/ml,Wnt3A浓度为5-10ng/ml,Y-27632浓度为5-10mM。
8.根据权利要求1所述的泪腺干细胞的培养体系,其特征在于,所述基质胶为Matrigel。
9.一种泪腺干细胞的培养方法,其特征在于,将小鼠泪腺组织消化成单细胞接种到基质胶中,待其凝固后,加入如权利要求1-8任一项所述泪腺干细胞培养液中,在37℃、5%~10%CO2环境下进行培养。
10.一种采用权利要求9的方法培养得到的泪腺干细胞。
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