CN105067810B - 一种检测原代神经干细胞迁移极化的方法及其应用 - Google Patents
一种检测原代神经干细胞迁移极化的方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种检测原代神经干/祖细胞迁移极化的方法。用PDMS制备带有显微通道的印章,将印章扣在包被多聚赖氨酸的培养皿上,从通道的一边加入含有作用因子和荧光分子的溶液,溶液进入孔道形成间隔相等的平行条带。在包被好荧光条带的培养皿上进行神经干细胞培养,观察条带间隙中神经干细胞贴壁后伪足的方向,计算干细胞前后端面积二者的比率。本发明不仅有助于神经干细胞迁移起始‑极化可视化,且对极化定量分析及机制的探讨有潜在的开发价值。在本发明的基础上,检测不同分子对神经干细胞迁移极化的影响,探索迁移极化的分子机制,通过外源性给予细胞分泌因子,启动体内神经干细胞的迁移等功能,为神经干细胞治疗神经退行性疾病奠定基础。
Description
技术领域
本发明涉及神经科学技术领域,具体地说,是一种检测原代神经干细胞迁移极化的方法及其应用。
背景技术
神经干细胞(Neural stem cell,NSCs)是一种未分化、多潜能、具有自我更新能力的神经前体细胞,可进一步分化为神经元、星形胶质细胞、少突胶质细胞。此外,NSCs经过多次细胞分裂后,仍能稳定地保持自身的特性。Sally Temple于1989年首次发现,在小鼠室管膜下区(subventricular zone,SVZ)存在自我更新能力和多向潜能的细胞。1992年,Reynolds B.A.首次从成年小鼠的中枢神经系统中分离出NSCs,并引起人们的广泛关注。NSCs存在于动物脑内的很多部位,包括哺乳动物胚胎期的大脑皮质、海马、嗅球、纹状体等处,另外,在成年哺乳动物中枢神经系统(Central nervous system,CNS)海马齿状回(dentate gyrus,DG)的颗粒下层中也分离出NSCs。
细胞迁移,是细胞在接收到迁移信号后,通过一系列黏附与去黏附过程,在基质表面产生的整体位移,是一种重要的细胞运动现象。根据迁移的方式可分为无规律的自由迁移(free/random migration)和定向迁移(directional migration)。近年的研究表明,不仅在哺乳动物的胚胎发育过程中,而且在神经系统疾病状态下NSCs都存在有序的定向迁移能力。在胚胎期中,神经上皮细胞不断向大脑皮层迁移,进一步分化为神经元,并形成大脑的基本神经构成。新生动物的海马和侧脑室中的NSCs不断迁移并分化新的神经元,维持海马和嗅球的神经功能;成年动物的NSCs处于相对静息的状态,仅在中枢神经系统损伤、缺血等病理因素的作用下才被激活和迁移到受损部位发挥其功能。NSCs的定向迁移不仅对机体神经系统的发生发育具有至关重要的作用,对于中枢神经系统的损伤修复也有重大的意义。有研究表明,中枢神经系统发生病理改变后,NSCs会特异性地迁移到损伤部位并可分化并替代缺失的细胞,与其它的神经元建立通路,从而使受损脑组织达到解剖和功能上的修复,这无疑为中枢神经疾病的治疗带来了希望的曙光。目前与NSCs迁移的相关因素很多:包括细胞周围环境、细胞间通讯、细胞代谢产物、NSCs自身及邻近或远隔部位其它细胞所分泌的细胞因子、黏附因子、激素和递质等,但定向迁移的具体机制还不完全清楚,因此了解NSCs定向迁移的机制对于利用NSCs治疗神经系统疾病是必须的。
细胞迁移的机制主要涉及到细胞骨架和其结合蛋白以及细胞外基质蛋白的变化,在迁移信号分子的浓度梯度和胞外基质的密度梯度等外界信号的刺激下,细胞内的信号传递分子和细胞骨架调节分子产生不对称的分布和/或激活,导致细胞骨架的形态、分布和细胞器的定位在即将发生迁移的方向上表现出前后不对称型,这一过程被称作迁移细胞的极性化(polarization),细胞由此分化出运动的前段和后部,之后在前段形成迁移所需的特殊结构-伪足,粘着斑(focal adhesion,FA)。在极化过程中涉及多个信号通路和它们下游效应因子的作用,而不同信号通路的作用又必须紧密配合,形成精密的调控网络,保证极化及迁移的每个步骤能正确完成并密切衔接,而Rho家族小GTP酶(small GTPase)在细胞迁移的协调过程中扮演了关键性的角色,尤其Rac1蛋白在细胞迁移过程中发挥了重要作用。
发明内容
本发明的目的是针对现有技术中的不足,提供一种检测原代神经干细胞迁移极化的方法。
本发明的再一的目的是,提供上述方法的用途。
为实现上述目的,本发明采取的技术方案是:
一种检测原代神经干细胞迁移极化的方法,包括如下步骤:
a.在模具上用二甲基硅氧烷制备下表面有平行显微通道的PDMS印章;
b.条带包被,取消毒的玻片放入培养皿中,加入多聚赖氨酸进行包被,将制备好的PDMS印章扣在多聚赖氨酸包被的玻片上,将需要研究的导向因子用FITC-BSA稀释到作用浓度,然后加入到印章的微通道一端,于通道另一端抽吸使溶液充满整个通道,待溶液干后,移除印章,在玻片上形成含有导向因子的平行条带;
c.在包被好荧光条带的玻片上进行神经干细胞培养;
d.使用免疫荧光技术进行染色,观察条带间隙中神经干细胞贴壁后伪足的方向,计算神经干细胞前端与后端的伪足面积比值,判断神经干细胞是否极化。
所述PDMS印章下表面的通道宽为60μm、高为100μm,每个平行的通道之间间隔为90μm。
所述步骤b中多聚赖氨酸浓度为0.1mg/ml。
所述判断神经干细胞是否极化的方法为:当神经干细胞前端与后端的伪足面积比值为0.8-1.2时为未极化;当神经干细胞前端与后端的伪足面积比值≥1.2时发生极化。
所述的导向因子为对神经纤维有导向作用的因子,所述的导向因子为基质细胞衍生因子(SDF-1)、BDNF(脑源性神经营养因子)、MAG(乙酰胆碱)。
为实现上述第二个目的,本发明采取的技术方案是:
如上任一所述的方法在检测神经干细胞迁移极化中的应用。
本发明优点在于:
本发明的方法不仅有助于使神经干/祖细胞迁移起始‐极化可视化,而且对极化定量分析及机制的探讨具有潜在的开发价值。在本发明的基础上,检测不同分子对神经干细胞迁移极化的影响,同时探索迁移极化的具体分子机制,然后通过外源性给予细胞分泌因子,启动体内神经干细胞的迁移等功能,为神经干细胞治疗神经退行性疾病奠定实验基础。
附图说明
附图1为本发明示意图;其中A部分为PDMS印章的制备;B部分为条带包被。
附图2为趋化因子CXCL12介导的神经祖细胞极化共聚焦拍照图片;其中A、B中的条带只含BSA;C、D中条带含BSA和趋化因子CXCL12;虚线为条带的边界;A、C的标尺为50μm;C、D的标尺为20μm。
附图3为极化细胞的定量分析,其中虚线左侧为前端,虚线右侧为后部;标尺为20μm。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明所记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例
一、PDMS印章的制备
原材料及仪器:模具(模具表面刻有间隔相等的平行微细凹槽,凹槽宽为60μm,槽高为100μm,每个凹槽之间的间隔为90μm。),聚二甲基硅氧烷(PDMS)(Momentive RTV 615A液、B液,苏州汶颢芯片科技有限公司),厚质铝箔,培养皿(150mm);真空箱,烤箱,手术刀。
流程:
1、大培养皿中铺铝箔,称好A液、B液(分别为预聚体与偶联剂),充分搅拌,将铝箔的边缘翻上来,放真空箱里抽真空10min(注1:抽真空时,可一直抽;也可一边抽一边放);
2、新培养皿中铺铝箔,其周围压平,在把模具放进铝箔上,将1中抽真空抽好的胶倒入模具的凹槽中(注2:在倒在模具之前,如果还有气泡,可用针头刺破),再抽5min真空,放在85℃1h;
3、将铝箔和模具从培养皿中拿出,用合适的手术刀沿着模具的边缘小心的分开,去掉铝箔,将印章拿出,切割。印章大小:长宽10mm×5mm,其下表面带有间隔相等的平行显微通道,通道宽为60μm、高为100μm,每个平行的通道之间的间隔为90μm。
二、条带包被
实验试剂及用具:多聚赖氨酸(PDL,Sigma公司),带玻片培养皿(35mm+20mm玻片),灭菌水,无水乙醇,肥皂,FITC-BSA(Life Technologies公司),CXCL12(R&D公司),移液器,枪头;真空泵,超净台。
流程:
1、将制备好的PDMS印章置于肥皂水中70℃过夜,用蒸馏水洗净,并用乙醇常温清洗过夜,紫外消毒;带玻片培养皿PDL多聚赖氨酸(0.1mg/ml)4℃过夜,灭菌水清洗三次后晾干。
2、将印章放置到培养皿中的玻片上,检查是否有气泡,将要印章的因子用FITC-BSA稀释到作用浓度,然后加之于印章的一端,在另一端用真空泵抽吸,使溶液充满整个印章孔道,形成间隔相同的平行条带。
3、放置到超净台,过夜晾干。
4、将印章拿掉,进行后续实验(图1)。
三、小鼠原代神经干细胞的获取、培养与鉴定:
动物:孕14d C57小鼠;
实验试剂及器具:PBS,碘酒、乙醇,眼科剪,显微镊,解剖镜,CO2培养箱,干细胞培养基。
1、原代培养
(1)取孕14天C57孕小鼠,引臼脱颈处死,碘酒、乙醇消毒腹部,逐层剖开腹部皮肤、肌肉、腹膜,取出子宫,放入盛有冷PBS的器皿中;
(2)仔细剥离胎膜、羊膜,暴露胎鼠,剪开颅骨,取出胎鼠脑组织并转移至另一盛有冷PBS的器皿中;
(3)体视显微镜下剥离脑膜分离胎鼠大脑皮层,仔细剥离血管及脑膜;
(4)将收集到的大脑皮层在冷PBS中漂洗两次,置于盛有冷PBS液的器皿中,用眼科剪将组织剪碎,然后用1ml移液器吹打,直至大块组织消失;
(5)用40μm过滤,1000rpm离心5min,将沉淀细胞重悬于完全培养基中,计数及调整细胞密度,以106个/mL接种25mL培养瓶中(Corning),于5%CO2培养箱,37℃培养。期间倒置显微镜下观察细胞的生长状况。
2、传代培养
当神经球直径大约100μm时收集细胞于离心管中,1000rpm离心5min,加入Accutase细胞消化液,消化10min终止消化,并用1ml移液器轻轻吹打细胞,使之形成单细胞悬液,计数后以5×104/mL接种25mL培养瓶中,于5%CO2培养箱,37℃培养,完成一次传代。此后每隔两天半量换液,4-5天传代1次。期间倒置显微镜下观察细胞的生长状况。
3、神经干细胞鉴定
将部分神经球或消化后的单个细胞接种到多聚赖氨酸(浓度为0.1mg/ml)处理的盖玻片上,继续培养15min或过夜,4%多聚甲醛固定后进行Nestin及SOX2免疫细胞化学检测。
四、免疫荧光染色
实验试剂及器具:PBS,多聚甲醛(PFA),TritonX-100,BSA,封片剂,盖玻片,罗丹明-鬼笔环肽,Dapi。
流程:
1、将传代后的NPCs用完全培养基重悬后,种于包被好条带的培养皿(带玻片)上;
2、10min后用真空泵吸干培养基,迅速加入1ml 4℃的PFA溶液,静置15分钟,细胞固定;
3、吸干液体,加入1ml PBS,静置5分钟,进行清洗。重复清洗三遍;
4、吸干PBS,加入0.5-1ml,0.4%的Triton X-100静置10-15分钟,进行破膜。之后用PBS清洗三次;
5、吸干液体,加入0.5-1ml PBS稀释的罗丹明-鬼笔环肽(1:200),静置1小时;
6、用PBS清洗三遍。加入用PBS配制的Dapi(1:1000)200ul,常温孵育5min;
7、用抗淬灭封片剂封片,常温避光保存;
8、共聚焦拍照,观察条带间隙中神经干(祖细胞)贴壁后片状伪足的方向,并计算极化神经干(祖细胞)前后端面积二者的比率。
五、结果
图2为由趋化因子CXCL12介导的神经祖细胞极化共聚焦拍照图片;A、B中的条带只含BSA;C、D中条带含BSA与趋化因子CXCL12。
图3为极化细胞的定量分析。将极化细胞分成前端和后部,利用免疫荧光强度分别对伪足面积进行测量,根据前端与后部伪足面积的比值用来评估细胞是否极化。从图中可以看到,BSA对照组细胞前端与后部的伪足面积比值为1,细胞没有发生极化;而在SDF-1α(CXCL12)组,神经细胞已经极化,其比值为2.5,与对照组相比,有显著性统计学差异(P<0.0001)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.一种检测原代神经干细胞迁移极化的方法,其特征在于,包括如下步骤:
a.在模具上用二甲基硅氧烷制备下表面有平行显微通道的PDMS印章;
b.条带包被
取消毒的玻片放入培养皿中,加入多聚赖氨酸进行包被,将制备好的PDMS印章扣在多聚赖氨酸包被的玻片上,将需要研究的导向因子用FITC-BSA稀释到作用浓度,然后加入到印章的微通道一端,于通道另一端抽吸使溶液充满整个通道,待溶液干后,移除印章,在玻片上形成含有导向因子的平行条带;
c.在包被好荧光条带的玻片上进行神经干细胞培养;
d.使用免疫荧光技术进行染色,观察条带间隙中神经干细胞贴壁后伪足的方向,计算神经干细胞前端与后端的伪足面积比值,判断神经干细胞是否极化
所述PDMS印章下表面的通道宽为60μm、高为100μm,每个平行的通道之间间隔为90μm;
所述步骤b中多聚赖氨酸浓度为0.1mg/ml;
所述判断神经干细胞是否极化的方法为:当神经干细胞前端与后端的伪足面积比值为0.8-1.2时为未极化;当神经干细胞前端与后端的伪足面积比值>1.2时发生极化。
2.根据权利要求1所述的方法,其特征在于,所述的神经干细胞为C57小鼠神经干细胞。
3.根据权利要求1所述的方法,其特征在于,所述的导向因子为对神经纤维有导向作用的因子。
4.根据权利要求1所述的方法,其特征在于,所述的导向因子为SDF-1、BDNF或乙酰胆碱。
5.权利要求1-4任一所述的方法在检测神经干细胞迁移极化中的应用。
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