CN110499294A - 双色荧光示踪胶质瘤干细胞微环境模型的制备方法 - Google Patents
双色荧光示踪胶质瘤干细胞微环境模型的制备方法 Download PDFInfo
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Abstract
本发明公开了双色荧光示踪胶质瘤干细胞微环境模型的制备方法。该制备方法包括体外培养GSCs;将具有嘌呤霉素抗性的慢病毒转染红色荧光蛋白(RFP)基因于GSCs;将稳定表达RFP的GSCs接种至全身表达绿色荧光蛋白(GFP)balb/c裸小鼠体内,构建双色荧光示踪异种移植瘤模型;将稳定表达RFP的GSCs与全身表达绿色荧光蛋白(GFP)balb/c裸小鼠骨髓细胞体外共培养,构建双色荧光示踪体外细胞共培养模型;荧光共聚焦显微镜下观察两种荧光细胞的相互作用。本发明具有通过两种荧光分别标记胶质瘤干细胞与微环境中间质细胞,使二者在细胞层面上的相互作用,在荧光显微镜下可见,为体内体外肿瘤微环境的研究提供了一种有效的研究方法的效果。
Description
技术领域
本发明涉及医疗生物标记技术领域,特别涉及双色荧光示踪胶质瘤干细胞微环境的制备方法。
背景技术
胶质瘤是颅内发病率最高的恶性肿瘤,约占中枢神经系统原发肿瘤的43%~50%。其中胶质母细胞瘤(glioblastoma,GBM)因常伴有对周围组织的侵袭和破坏,所以预后极差,中位生存期仅为14.6个月。胶质瘤干细胞(Glioblastoma stem cells,GSCs)及其微环境的相互作用是促进胶质瘤进展的重要因素。研究表明GSCs可诱导微环境中的间质细胞发生恶性转化,这可能是胶质母细胞瘤易复发的潜在因素。间质细胞发生恶性转化的可能性机制归纳如下:1)GSCs通过与微环境中间质细胞直接接触,直接诱导间质细胞发生恶变。2)胶质瘤微环境中GSCs可与间质细胞发生相互作用并发生细胞融合,从而使正常间质细胞具有了肿瘤细胞的恶性表型。3)GSCs可向胶质瘤微环境中释放外泌体,间质细胞通过吞噬这些外泌体使自身发生恶性转化。
胶质瘤在微环境研究的难点在于并不能直接观察GSCs与微环境中的间质细胞之间的相互作用。因此,建立双色荧光示踪GSCs微环境模型为分别示踪肿瘤细胞与间质细胞提供了可视化的研究平台,这对GBM发生发展的分子机制和肿瘤微环境等目前热议的研究方向具有重要意义。
发明内容
鉴于在胶质瘤微环境中,观察胶质瘤干细胞与肿瘤微环境中间质细胞之间相互作用存在困难,根据本发明的一个方面,提供了一种双色荧光示踪胶质瘤干细胞微环境的制备方法,此方法分别用两种荧光标记肿瘤干细胞与间质细胞,从而可直接观察二者之间的相互作用。
该双色荧光示踪胶质瘤干细胞微环境的制备方法至少包括以下步骤:
S1.体外培养GSCs
S2.将具有嘌呤霉素抗性的慢病毒转染红色荧光蛋白(RFP)基因于GSCs
S3.将稳定表达RFP的GSCs接种至全身表达绿色荧光蛋白(GFP)balb/c裸小鼠体内,构建双色荧光示踪异种移植瘤模型。
S4.将稳定表达RFP的GSCs与全身表达绿色荧光蛋白(GFP)balb/c裸小鼠骨髓细胞体外共培养,构建双色荧光示踪体外细胞共培养模型。
S5.荧光共聚焦显微镜下观察两种荧光细胞的相互作用。
上述的双色荧光示踪胶质瘤干细胞微环境的制备方法,其有益效果是,通过两种荧光分别标记胶质瘤干细胞与微环境中间质细胞,使二者在细胞层面上的相互作用,在荧光显微镜下可见。为体内体外肿瘤微环境的研究提供了一种有效的研究方法。
在一些实施方式中,S1中,首先配制干细胞培养基,即添加有浓度为20ng/mlbFGF,20ng/ml EGF,1×B27细胞添加剂的DMEM/F12培养液,另需在培养液中加入1×谷氨酸、复合维生素、丙酮酸钠、非必须氨基酸。双抗可根据具体情况选择性添加。
所述S1中,干细胞培养过程中,需要进行干细胞传代培养,是指将干细胞悬液1000rpm 5min离心,去除上清,加入2ml Accutase消化液,37℃孵育5min,将干细胞球吹打成单细胞悬液,1000rpm 5min离心去除上清,加入5ml干细胞培养液悬浮细胞,37℃5%CO2细胞培养箱中继续培养。
在一些实施方式中,所述S2中,构建具有嘌呤霉素抗性的,且稳定表达RFP的慢病毒。
所述S2中,将处于对数生长期的干细胞悬液1000rpm 5min离心,去除上清,Accutase消化液在细胞培养箱中孵育5min,移液枪轻柔吹打干细胞球成单细胞悬液。按1*105/L密度接种于12孔板,同时加入PBS浓度梯度稀释的病毒液,混合均匀后放入细胞培养箱中。
所述S2中,慢病毒转染细胞24h左右换液。离心干细胞悬液,去除上清,加入新鲜干细胞培养液重悬干细胞,转染48h后荧光显微镜下观察细胞转染效率。
所述S2中,将根据不同稀释浓度的病毒及细胞生长状态,选择最佳适宜稀释浓度进行转染。
所述S2中,荧光显微镜下观察细胞转染效率大于70-80%时,加入10g/ml的嘌呤霉素,以杀灭为转染细胞,使接近100%的细胞均表达RFP(图1)。
在一些实施方式中,所述S3中,选择4-6周龄的全身表达绿色荧光蛋白(GFP)balb/c雄性裸小鼠(图2),4%水合氯醛以0.1ml/10g腹腔注射麻醉后,75%酒精消毒皮肤。
所述S3中,计数处于对数生长期的稳定表达RFP的GSCs,每只裸小鼠接种107个细胞,分别接种于小鼠颅内、腹腔及皮下(图3)。
所述S3中,致瘤成功后,活体动物成像仪下观察异种移植瘤。
所述S3中,断颈处死荷瘤鼠,切去移植瘤,直接用OCT(optimal cuttingtemperature compound)包埋,-80℃保存。
所述S3中,用冰冻切片机切片OCT包埋的移植瘤组织,并与荧光共聚焦显微镜下观察GSCs-RFP与表达GFP的间质细胞(图4)。
在一些实施方式中,所述S4中,取4-6周龄表达绿色荧光蛋白(GFP)balb/c裸小鼠,麻醉后断颈处死,离断小鼠股骨和胫骨并在75%乙醇中浸泡5~10min,无菌条件下剥离附着的皮肤、筋膜和肌肉。用含1%双抗的PBS缓冲液冲洗后,切开股骨和胫骨干骺端。用1ml注射器吸取10%胎牛血清的DMEM完全培养基,插入干骺端反复冲洗骨腔,直到骨透明为止。用完全培养基于37℃5%CO2细胞培养箱中继续培养骨髓细胞(图5)。
所述S4中,分别计数GSCs-RFP与表达GFP的骨髓细胞,将GSCs与骨髓细胞按1:20的比例,共培养于同一细胞培养皿中。荧光显微镜(图6)及活细胞工作站(图7)下观察两种细胞的相互作用。
如上所述,该双色荧光示踪胶质瘤干细胞微环境模型制备方法,具有以下有益效果:
本方法通过两种荧光分别标记胶质瘤干细胞与微环境中间质细胞,使二者在细胞层面上的相互作用,在荧光显微镜下可见。为体内体外肿瘤微环境的研究提供了一种有效的研究方法。
附图说明
图1为本发明的实施例1、2实施方式中的双色荧光示踪胶质瘤干细胞微环境模型的制备方法的所涉及的稳定表达RFP的胶质瘤干细胞荧光显微镜下白光与荧光照片;
图2为本发明的实施例1、2实施方式中所涉及的本实验室自建的全身表达GFP的裸小鼠活体动物成像仪视图;
图3为本发明实施例1的实施方式的双色荧光示踪胶质瘤干细胞微环境模型的制备方法中的获得的GSCs-RFP分别接种至裸小鼠颅内、腹腔、皮下体内双色荧光示踪异种移植瘤模型视图;
图4为本发明实施例1的实施方式的双色荧光示踪胶质瘤干细胞微环境模型的制备方法中的获得的颅内移植瘤组织冰冻切片荧光共聚焦显微镜视图;
图5为本发明实施例2的实施方式的双色荧光示踪胶质瘤干细胞微环境模型的制备方法中的获得的表达GFP的裸小鼠骨髓细胞荧光显微镜视图;
图6为本发明实施例2的实施方式的双色荧光示踪胶质瘤干细胞微环境模型;
图7为本发明实例2的实施方式的双色荧光示踪胶质瘤干细胞微环境模型的制备方法中的细胞体外共培养体系在活细胞工作站的视图。
具体实施方式
下面结合附图对本发明作进一步详细的说明。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所阐述的内容轻易地了解本发明的优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
材料与来源:DMEM培养液(Gibco,C11995500BT),DMEM/F12培养液(Gibco,11330032),Accutase消化液(StemPro,A1110501),EGF生长因子(Gibco,PHG0311),bFGF生长因子(Gibco,PHG0266),B27细胞添加剂(Gibco,17504044),胎牛血清(Gibco,16000-044)。
实施例1:
胶质瘤干细胞微环境双色荧光示踪颅内移植瘤模型。
1.选择4-6周龄的绿色荧光裸小鼠若干只(图2),4%水合氯醛以0.1ml/10g腹腔注射麻醉后,75%酒精消毒头部皮肤。
2.头顶部正中纵向切开0.5cm皮肤,显露颅骨,于前囟前0.1mm、中线右侧2.5mm处,用直径1.0mm的颅钻钻颅至硬膜。
3.计数处于对数生长期的稳定表达RFP的GSCs(图1),每只裸小鼠接种107个细胞。
4.将待接种的细胞1000rpm 5min离心,除去上清,PBS重悬细胞,再次1000rpm5min离心,将细胞沉淀置于BD碧迪静脉留置针套管内,经钻颅孔垂直穿刺进入4.0mm,后退1.0mm后将套管针内芯推进,细胞植入到小鼠颅内右侧尾状核。
5.缓慢拔针,用骨蜡封闭颅骨孔,0号丝线缝合头皮,护理至苏醒,继续饲养于SPF级动物房。
6.动物活体成像仪下观察颅内异种移植瘤(图3)。
7.麻醉后断颈处死荷瘤鼠,小心剥离出完整颅脑组织(图4),OCT包埋组织储存于-80℃冰箱。
8.使用冰冻切片机对长有异种移植瘤的小鼠脑组织行5m冰冻切片,荧光共聚焦显微镜下观察肿瘤与肿瘤微环境间质细胞的作用(图4)。
实施例2:
胶质瘤干细胞微环境双色荧光示踪体外共培养模型。
1.取4-6周龄表达绿色荧光蛋白(GFP)balb/c裸小鼠(图2),麻醉后断颈处死,离断小鼠股骨和胫骨并在75%乙醇中浸泡5~10min。
2.无菌条件下剥离股骨颈股附着的皮肤、筋膜和肌肉。用含1%双抗的PBS缓冲液冲洗后,切开股骨和胫骨干骺端。
3.用1ml注射器吸取10%胎牛血清的DMEM完全培养基,插入干骺端反复冲洗骨腔,直到骨透明为止。完全培养基培养细胞至对数生长期以备用(图5)。
4.分别计数GSCs-RFP(图1)与表达GFP的骨髓细胞(图5),将GSCs与骨髓细胞按1∶20的比例,共培养于同一细胞培养皿中。
5.荧光显微镜下可见同时表达RFP和GFP的细胞(图6C),以及具有癌性表型的GFP+细胞(图6C),利用单细胞克隆技术分别克隆RFP+/GFP+细胞(图6D-I)和恶性表型的GFP+细胞(图6J,K),可进一步研究微环境中间质细胞对胶质瘤进展过程中的重要作用。
6.活细胞工作站下观察肿瘤微环境中两种细胞的相互作用,可见细胞间的微米级(图7B,C)、纳米级(图7A)连接,连接途径包括细胞间邻近连接(图7C)和跨细胞连接(图7D),连接方式包括细胞间一对一(图7A-D)和一对多(图7E)。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法、组合物的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (7)
1.双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,包括以下步骤,
S1,体外培养GSCs;
S2,将具有嘌呤霉素抗性的慢病毒转染红色荧光蛋白(RFP)基因于GSCs;
S3,将稳定表达RFP的GSCs接种至全身表达绿色荧光蛋白(GFP)balb/c裸小鼠体内,构建双色荧光示踪异种移植瘤模型;
S4,将稳定表达RFP的GSCs与全身表达绿色荧光蛋白(GFP)balb/c裸小鼠骨髓细胞体外共培养,构建双色荧光示踪体外细胞共培养模型;
S5,荧光共聚焦显微镜下观察两种荧光细胞的相互作用。
2.根据权利要求1所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S1中,首先配制干细胞培养基,即添加有bFGF、EGF、B27细胞添加剂的DMEM/F12培养液,另需在培养液中加入谷氨酸、复合维生素、丙酮酸钠、非必须氨基酸或双抗中的一种或多种。
3.根据权利要求2所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S1中,干细胞培养过程中,进行干细胞传代培养,即将干细胞悬液离心,去除上清,加入Accutase消化液,孵育,将干细胞球吹打成单细胞悬液,离心去除上清,加入干细胞培养液悬浮细胞,CO2细胞培养箱中继续培养。
4.根据权利要求3所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S2中,将处于对数生长期的干细胞悬液离心,去除上清,Accutase消化液在细胞培养箱中孵育,移液枪轻柔吹打干细胞球成单细胞悬液,接种于孔板,同时加入PBS浓度梯度稀释的病毒液,混合均匀后放入细胞培养箱中;
慢病毒转染细胞24h左右换液,离心干细胞悬液,去除上清,加入新鲜干细胞培养液重悬干细胞,转染48h后荧光显微镜下观察细胞转染效率;
根据不同稀释浓度的病毒及细胞生长状态,选择最佳适宜稀释浓度进行转染;
荧光显微镜下观察细胞转染效率大于70-80%时,加入嘌呤霉素,以杀灭为转染细胞,使接近100%的细胞均表达RFP。
5.根据权利要求4所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S3中,选择4-6周龄的全身表达绿色荧光蛋白(GFP)balb/c雄性裸小鼠,水合氯醛溶液腹腔注射麻醉后,酒精消毒皮肤;
计数处于对数生长期的稳定表达RFP的GSCs,每只裸小鼠接种107个细胞,分别接种于小鼠颅内、腹腔及皮下;
致瘤成功后,活体动物成像仪下观察异种移植瘤。
6.根据权利要求5所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S3中,断颈处死荷瘤鼠,切去移植瘤,直接用OCT(optimalcutting temperaturecompound)包埋,冰冻保存;用冰冻切片机切片OCT包埋的移植瘤组织,并与荧光共聚焦显微镜下观察GSCs-RFP与表达GFP的间质细胞。
7.根据权利要求6所述的双色荧光示踪胶质瘤干细胞微环境模型的制备方法,其特征在于,所述S4中,取4-6周龄表达绿色荧光蛋白(GFP)balb/c裸小鼠,麻醉后断颈处死,离断小鼠股骨和胫骨并在乙醇中浸泡,无菌条件下剥离附着的皮肤、筋膜和肌肉;
用含1%双抗的PBS缓冲液冲洗后,切开股骨和胫骨干骺端;用注射器吸取部分胎牛血清的DMEM完全培养基,插入干骺端反复冲洗骨腔,直到骨透明为止;用完全培养基于CO2细胞培养箱中继续培养骨髓细胞;
分别计数GSCs-RFP与表达GFP的骨髓细胞,将GSCs与骨髓细胞按比例共培养于同一细胞培养皿中;荧光显微镜及活细胞工作站下观察两种细胞的相互作用。
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