CN115161282A - 一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法 - Google Patents
一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法 Download PDFInfo
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Abstract
本发明提供一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,该方法新颖而简单,采用“两步酶解法”可从小鼠中枢神经系统中分离和培养大量内皮细胞与周细胞,该方法易于建立,并在较短时间内提供大量高纯度的内皮细胞和周细胞,进而用于多种下游应用,包括体外和体内实验,转录组学、蛋白质组学、代谢组学、表观基因组学和单细胞分析。
Description
技术领域
本发明涉及领域,特别是一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法。
背景技术
微血管是血液循环系统的最末梢部分,在机体内分布最广,也是功能最为复杂的一部分,多项研究表明微血管系统可以为局部组织提供屏障保护,和发挥半透膜的营养输送作用,同时还具有调控局部微环境代谢和分泌多种细胞因子等极为重要的作用;此外,在很多脏器中,实质细胞均需依托微血管提供的支架作用生长、发育,进而形成立体结构。在中枢神经系统中,由微血管和血管周围的星形细胞突起及由这些突起所支持的神经元以及轴突共同组成的复合体被称为“神经血管单元”。而内皮细胞和周细胞是构成“神经血管单元”的关键细胞。二者在神经轴突与屏障环境的发育、成熟和重塑中起着关键作用。
但是由于缺乏特异性细胞标记物,以及难以提取到足够数量及纯度的细胞,从而限制了对这些重要细胞类型的研究。以往的分离策略通常从简单的组织酶解开始,通过连续的过滤步骤分离微血管碎片进行培养。片段生长策略相对容易做,但往往杂细胞较多,导致细胞种群不纯。
虽然可以通过使用磁珠或流式细胞术来提高细胞纯度,磁珠分选是基于细胞表面抗原能与连接有磁珠的特异性单抗相结合,在外加磁场中,通过抗体与磁珠相连的细胞被吸附而滞留在磁场中,无该种表面抗原的细胞由于不能与连接着磁珠的特异性单抗结合而没有磁性,不在磁场中停留,从而使细胞得以分离。
流式细胞术分选是同样是基于样品细胞表面特有抗原,标记上荧光素偶联抗体,并将其通过高压电场,在激光照射后,收集每个细胞的发射荧光信号,处理分析得到样品细胞表达某抗原分子强弱等情况,进行分选收集。但这种方法的缺点明显,如对实验设备要求高,技术操作难度大,实验步骤复杂,并且常常导致非常低的细胞产量,并且严重影响细胞活力,导致无法进行下一步实验。
发明内容
本发明提出一种新颖而简单的方法,可以从小鼠中枢神经系统中分离和培养大量内皮细胞与周细胞,该方法易于建立并在较短时间内提供大量高纯度的内皮细胞和周细胞,进而用于多种下游应用,包括体外和体内实验,转录组学、蛋白质组学、代谢组学、表观基因组学和单细胞分析。是通过如下技术方案实现的。
一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,采用“两步酶解法”联合提取内皮细胞与周细胞,步骤包括:
利用II型胶原酶充分解离小鼠的脑组织,使得脑组织中微血管段得到充分释放;
向酶解组织中添加20%的BSA,去除因为脑组织消化过程中释放出来的大量脂质或磷脂类碎片,减少其对细胞的毒性作用,并富集微血管段;
采用胶原酶/分散酶联合消化富集后的血管段,使得微血管段内的内皮细胞进一步释放出来;
用含嘌呤霉素的筛选培养基重悬细胞然后种板,利用嘌呤霉素来抑制其他杂细胞的生长,待细胞获得群体生长优势后再更换正常培养基;
同时联合提取周细胞,将分离出的细胞使用不含嘌呤霉素的低糖培养基重悬,48h后更换周细胞专用培养基。
本发明的有益效果是:在短时间内提供大量高纯度的内皮细胞和周细胞,方法简单。
附图说明
图1是本发明实施例中微血管内皮细胞和周细胞光镜图。
图2是本发明实施例中微血管内皮细胞的免疫荧光鉴定图。
图3是本发明实施例中微血管周细胞的免疫荧光鉴定图
具体实施方式
以下结合附图对本发明的实施例进行详细说明,但是本发明可以由权利要求限定和覆盖的多种不同方式实施。
本发明的实施例,一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,采用“两步酶解法”可同时提取微血管内皮细胞与周细胞。
试剂准备:先准备75%酒精(预冷),鼠尾胶(Sigma,C7661),II型胶原酶, DNaseI,胶原酶/分散酶(Collagenase/Dispase,Roche),BSA(需过滤),正常糖 DMEM(Gibco)(配置各种缓冲液,常规加双抗),低糖DMEM(Gibco)(配置细胞培养液),PBS缓冲液,青霉素/链霉素(Penicillin-Streptomycin Solution,双抗)混合液,生长因子肝素钠,血清(FBS)。
手术器械准备:显微器械3套(75%乙醇中浸泡30min),显微镜,6cm培养皿,15ml离心管,50ml离心管,巴氏管,50ml注射器,过滤器,4℃离心机(预冷),恒温培养箱。
在本发明的提取实验开始前先进行试剂配制:
1、1%的II胶原酶储存液:取15ml无菌离心管,称取0.1g II型胶原酶,加入10mlDMEM配制成1%存储液,转移至超净台中,过滤器过滤后-20℃保存。
2、DNase I(1mg/ml)溶液:100mgDNase I中加入10mlDMEM配制成1%存储液,过滤器过滤后-20℃保存。
3、0.1%的II型胶原酶工作液:取50ml无菌离心管,加入5ml上述1%的II 胶原酶储存液,加入已配好的500μl DNase I(1mg/ml)溶液,44ml DMEM,500μl 双抗溶液,颠倒混匀后,可4℃保存。
4、0.1%Collagenase/Dispase(Roche):100mg用DMEM配制,需加双抗液,用时稀释成0.1%。
5、20%BSA:30ml DMEM+6g BSA(可分两个离心管,15ml+3g,充分振荡混匀,使用前过滤筛过滤)。
5、鼠尾胶:按照5μg/cm2的剂量提前包被培养皿,4℃过夜,使用前1 小时拿出晾干。
6、嘌呤霉素:储存液浓度1mg/ml,最终培养液中溶度为2μg/ml。
7、青霉素/链霉素抗生素:储存液浓度为100mg/2ml。
8、肝素:50mg/ml储存溶度,最终溶度为100μg/ml。
9、内皮细胞生长因子:
EGF:50μg/ml储存溶度,最终培养液中溶度为5ng/ml;
bFGF:50μg/ml储存溶度,最终培养液中溶度为10ng/ml。
10、内皮细胞条件性筛选培养液(50ml):50ml培养基体系:40ml低糖 DMEM+10ml血清+EGF 5μl(50μg/ml)+bFGF 10μl(50μg/ml)+肝素100μl+嘌呤霉素50μl+双抗500μl。
11、增殖培养基:50ml培养基体系:40ml低糖DMEM+10ml血清+EGF 5μl(50μg/ml)+bFGF 10μl(50μg/ml)+肝素100μl+双抗500μl。
在本发明实施例中,内皮细胞提取及培养,包括如下步骤:
步骤1、将小鼠脱颈处死,浸泡于预冷的75%的酒精中,约5-6分钟;
步骤2、在超净工作台中,准备三个培养皿,两个各加入适量PBS+500ul 双抗(一个取出的脑组织,另一个用于显微解剖操作),另一个加入50ml正常糖DMEM+500ul双抗(装剥离后的脑组织);
步骤3、用虹膜剪剪开头部皮肤,换另外一把剪刀从颈后部正中间剪开头骨,并向两侧剥离,使用长柄药匙快速取出大脑,尽量保证结构完整性(注意:剪开皮肤与剪开头骨的剪刀需分开,持物钳夹持老鼠头部前端,不要随意更换位置,减少污染),放入加有PBS(DMEM)+双抗(在细胞房里配置,1个培养皿的量约500ul)的培养皿中;
步骤4、显微镜下去除小鼠大脑外层血痂、脑膜、外层血管,从中间切开小鼠大脑,去除内部的白质、嗅球、小脑和髓质,移入加有冷的DMEM培养皿中 (预先配置好的,50ml正常糖DMEM+500ul双抗);
步骤5、将大脑用显微剪或显微手术刀将组织分割成1×1mm大小的小块碎块状,转移至无菌的50ml离心管中(管中加有含0.1%双抗的DMEM,约15ml),将巴氏管插入试管底部,慢吸快吹,反复吹打,直至试管内组织变成乳状悬浮液;
步骤6、离心机预冷4℃,1200rpm转速,离心5min,弃去上清;
将用胶原包被的培养瓶取出,弃去包被液,放置于超净台晾干;
步骤7、加入0.1%II型胶原酶(内含有0.005%DNase I),约10-15ml,放置于恒温(37℃)摇床上(速度210rpm),震荡消化1.5h;
步骤8、离心机温度4℃下,1000rmp离心8min,去上清;
步骤9、直接向离心管中加入约15ml左右上述配制的20%BSA(需提前用网筛过滤),离心机温度4℃下,4400rmp离心20-22min,离心后可见管内分层,管底有少量深红色脑微血管段或内皮细胞;
步骤10、将巴氏管剪去前端扩大开口,轻轻吸去上层乳白色油脂状物及上清液;
步骤11、加入0.1%Collagenase/Dispase(Roche),放置于恒温(37℃)摇床上(速度210rpm),震荡消化40-60min,等量培养基中和;
步骤12、吹打混匀后,离心机温度4℃下,1200rmp离心5min;
步骤13、继续使用完全培养基清洗2次,离心机温度4℃下,1200rmp离心5min,用提前配置好的含嘌呤霉素的低糖培养基重悬,种板。
步骤14、恒温培养箱内培养48h后,更换无嘌呤霉素的低糖培养基。
在本发明实施例中,周细胞提取在步骤12的基础上进行,
步骤15、周细胞提取在上述实验步骤13之前均与内皮细胞提取操作一致,在第13步时将分离出的细胞使用不含嘌呤霉素的低糖培养基重悬,种板。
步骤16、恒温培养箱内培养48h后,更换周细胞专用培养基。
步骤15-16和步骤13-14为并列步骤。
如图1所示,根据上述步骤获得的内皮细胞和周细胞,从光镜下可看出内皮细胞细胞形态单一,呈梭形或扁平型,呈旋涡状排列。周细胞形态单一,扁平而有突起,胞核明显,呈散在分布。
如图2所示,从免疫荧光鉴定结果可以看出内皮细胞表面标志物CD31阳性细胞占比98%以上,周细胞表面标志物PDGFR-β、NG-2均为阴性,结果证明本实验方法能够获得高纯度的内皮细胞,为后续实验提供可能。
如图3所示,从免疫荧光鉴定结果可以看出周细胞表面标志物PDGFR-β、 NG-2阳性细胞占比95%以上,内皮细胞表面标志物CD31阳性细胞极少,结果证明本实验方法能够获得高纯度的周细胞,为后续实验提供可能。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,其特征在于,联合提取内皮细胞与周细胞,步骤包括:
利用II型胶原酶充分解离小鼠的脑组织,使得脑组织中微血管段得到充分释放;
向酶解组织中添加20%的BSA,去除因为脑组织消化过程中释放出来的大量脂质或磷脂类碎片,减少其对细胞的毒性作用,并富集微血管段;
采用胶原酶/分散酶联合消化富集后的血管段,使得微血管段内的内皮细胞进一步释放出来;
用含嘌呤霉素的筛选培养基重悬细胞然后种板,利用嘌呤霉素来抑制其他杂细胞的生长,待细胞获得群体生长优势后再更换正常培养基;
同时联合提取周细胞,将分离出的细胞使用不含嘌呤霉素的低糖培养基重悬,48h后更换周细胞专用培养基。
2.根据权利要求1所述的一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,其特征在于,内皮细胞的方法包括如下步骤:
步骤1、将小鼠脱颈处死,浸泡于预冷的75%的酒精中,约5-6分钟;
步骤2、准备三个培养皿,两个各加入适量PBS+500ul双抗,另一个加入50ml正常糖DMEM+500ul双抗;
步骤3、快速取出小鼠大脑,放入加有PBS(DMEM)+双抗的培养皿中;
步骤4、去除小鼠大脑外层血痂、脑膜、外层血管,从中间切开小鼠大脑,去除内部的白质、嗅球、小脑和髓质,获取脑组织,并移入加有冷的DMEM培养皿中;
步骤5、将大脑用显微剪或显微手术刀将脑组织分割成1×1mm大小的小块碎块状,转移至无菌的50ml离心管中,管中加有含0.1%双抗的DMEM,约15ml,将巴氏管插入试管底部,慢吸快吹,反复吹打,直至试管内组织变成乳状悬浮液;
步骤6、离心管在预冷4℃的离心机中处理,1200rpm转速,离心5min,弃去上清;
步骤7、加入0.1%II型胶原酶,约10-15ml,放置于恒温摇床上,速度210rpm,震荡消化1.5h;
步骤8、试管在离心机中处理、4℃环境下,1000rmp离心8min,去上清;
步骤9、直接向离心管中加入约15ml左右上述配制的20%BSA,4℃环境下,4400rmp离心20-22min,离心后可见管内分层,管底有少量深红色脑微血管段或内皮细胞;
步骤10、将巴氏管剪去前端扩大开口,轻轻吸去上层乳白色油脂状物及上清液;
步骤11、加入0.1%Collagenase/Dispase(Roche),放置于恒温摇床上,速度210rpm,震荡消化40-60min,等量培养基中和;
步骤12、吹打混匀后,置于离心机中,4℃环境下,1200rmp离心5min;
步骤13、继续使用完全培养基清洗2次,离心机4℃环境下,1200rmp离心5min,用提前配置好的含嘌呤霉素的低糖培养基重悬,种板;
步骤14、恒温培养箱内培养48h后,更换无嘌呤霉素的低糖培养基。
3.根据权利要求3所述的一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,其特征在于,步骤6、将用胶原包被的培养瓶取出,弃去包被液,放置于超净台晾干。
4.根据权利要求2所述的一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法,其特征在于,同时对周细胞进行提取,在步骤1-12的基础上,采用下列步骤:
步骤13、将分离出的细胞使用不含嘌呤霉素的低糖培养基重悬,种板;
步骤14、恒温培养箱内培养48h后,更换周细胞专用培养基。
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