CN114441413A - 一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法 - Google Patents
一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法 Download PDFInfo
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Abstract
本发明公开了一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法。该方法依次包含Nano‑tip微电极的制备、人多能干细胞的复苏与培养、多巴胺能神经元的定向分化和单囊泡检测。本发明中将基于Nano‑tip微电极的单细胞分析技术与iPSC技术诱导的人源性帕金森病疾病模型相结合,构建了单个神经元中单囊泡分析的方法,为帕金森疾病的病理学研究提供一种新的化学视角,为PD和其他神经退行性疾病的新药发现提供了新的平台和途径。
Description
技术领域
本发明属于分析检测技术领域,具体涉及一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法。
背景技术
神经系统是人体内起主导作用的功能调节系统,是产生认知、学习、记忆、情绪、心理活动及控制运动的重要物质基础。神经元是构成神经系统的基本单位,神经元细胞间的信号传递是通过突触囊泡中的神经递质(如多巴胺,乙酰胆碱,谷氨酸,多肽类等)完成的。单个囊泡中神经递质的含量对于信号传导有着重要的影响,与多种神经疾病的发生发展有关。但由于囊泡极其微小(纳米级),目前仍缺乏高灵敏度高时空分辨率的神经递质分析方法,对于这一过程的调控机制了解甚少。因此,单细胞水平内对囊泡储存情况进行检测,是近些年来神经科学领域面临的一个非常重大的问题。
帕金森病(Parkinson's disease,PD)是一种常见的严重危害人类健康的神经退行性疾病,其最主要的病理改变是中脑黑质多巴胺(dopamine,DA)能神经元的变性死亡,由此而引起纹状体多巴胺含量显著性减少。目前,导致这一病理改变的原因是否与单囊泡中神经递质储存有关有待确证。因此,建立高灵敏度的神经递质分析方法,对神经元内单囊泡储存情况,从单细胞水平揭示神经元囊泡储存情况与帕金森病发病直接的关系具有重要意义。
单细胞的分析可以从单细胞水平上反映细胞活动的规律,能够更深层次地从分子水平反映生物分子在活细胞中的功能和作用,揭示重要的生物过程和信号传导机制的本质和规律,为探究疾病早期的起因、发展和治疗提供更可靠的科学依据。单细胞的分析技术由于发挥关键作用的生物分子含量极低而面临着巨大的挑战。电化学技术因具有灵敏度高、可实现实时监测等优点而被应用于生物系统中单细胞生物过程中重要生物分子的实时定量分析,其中微电极相对于宏观尺寸电极具有更小的尺寸、更强的电流密度,更高的信噪比,更快的传质速度等,非常适合从单细胞水平对生物过程进行快速灵敏地监测。基于微电极的检测方法为神经元之间信号传递规律的研究提供有力的技术支持。
人类诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)是近些年来发展最迅速的领域,2012年山中伸弥获得诺贝尔奖之后,iPSCs被公认为最重要的科技之一,并受到广泛的关注。以病人体细胞重新编程而来的iPSCs在特定条件下,可定向分化为病人身体任意一种细胞。其最大优势在于它取材于人类体细胞,从而建立病人特异性iPSC从而构建疾病的模型。病人iPSC来源的特定谱系细胞已经广泛用于构建疾病模型、药物筛选等研究。hiPSCs直接诱导定向分化为特种神经元技术的出现,为建立应用分化得到多巴胺能神经元的帕金森病病模型提供了可能。
综上所述,本发明拟基于nano-tip微电极建立一种监测iPSC技术诱导分化的多巴胺能神经元中单囊泡储存情况的测定方法。同时分析正常组,帕金森病模型组和药物干预组的多巴胺能神经元囊泡储存情况的变化,揭示帕金森病可能的发病机理同时筛选潜在的治疗药物。本发明不仅为单细胞水平的单囊泡储存情况提供新思路、新方法,而且对神经科学领域疾病的发病机制研究及药物筛选等研究具有重要理论意义和广泛实用价值。
发明内容
本发明公开一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法。囊泡作为神经递质储存的场所在细胞信号传导发挥着重要的作用,与多种神经疾病的发生发展有关。基于Nano-tip微电极的单细胞分析技术可以从单细胞水平对囊泡进行分析,揭示重要的生物过程和信号传导机制的本质和规律,为探究疾病早期的起因、发展和治疗提供更可靠的科学依据。人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)可以建立病人特异性iPSC,进而定向诱导分化构建人源性的疾病模型。本发明中将基于Nano-tip微电极的单细胞分析技术与iPSC技术诱导的人源性帕金森病疾病模型相结合,构建了单个神经元中单囊泡分析的方法,为帕金森疾病的病理学研究提供一种新的化学视角,为PD和其他神经退行性疾病的新药发现提供了新的平台和途径。
本发明的目的是通过以下方式实现的:
一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法,该方法包括以下步骤:
a)Nano-tip微电极的制备:将碳纤维吸入硼硅玻璃毛细管中,通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露,在显微镜下将暴露的碳纤维切至100-150μm,将碳纤维置于丁烷喷枪的火焰边缘蚀刻在3秒以下,碳纤维的tip端出现红点,将电极从火焰离开,电极中的碳纤维形成尖头,将电极放入环氧树脂胶中1-3min,封闭玻璃管与碳纤维之间的空隙,接着浸入丙酮中洗去碳纤维上的胶,随后,放入100℃的烘箱中,过夜,制备完成的电极放入含0.10mM多巴胺的pH 7.4的PBS溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,且电流平台在1.5-3nA的电极即为达到要求的电极;
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出解冻得到细胞悬液,将细胞悬液加入5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内,使用无异源性培养基Essential 8维持其生长,每日换液,直至人多能性干细胞呈现核质比高,形状规则的状态;
c)多巴胺能神经元的定向分化:首先将适量人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μMMH1、0.4μM CHIR、20-500ng/mL SHH的NIM培养基中贴壁培养,隔天换液,第九天时,将人多能干细胞吹下,并离心处理,离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬,最后转移至培养瓶中进行悬浮培养,隔天换液,第13天将培养液替换为含有SAG以及80-120ng/mL FGF8b的NIM培养基,第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基,第34天时,接种至Matrigel包被的小圆玻片上,用含有10ng/mL BDNF、10ng/mL GDNF、5-15ng/mLTGF-β3、1μM cAMP、1μM Compound E的NIM培养基继续培养至第45天;
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,测定时的工作电极为步骤a)制备的Nano-tip微电极,参比电极为Ag/AgCl电极;用微操作器将Nano-tip微电极插入步骤c)得到的多巴胺能神经元,插入同时采用膜片钳放大器以恒电位法记录电流,测定电位为+700-800mV,输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪以5kHz数字化;记录的电化学数据使用Igor Pro 6.22程序进行数据处理;电流的过滤器为1kHz;电流峰大于噪音三倍的峰被保留,不满足条件的峰去除;每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况;根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
优选步骤“a)”中,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻2秒。
优选步骤“a)”中,电极放入环氧树脂胶中时间为1-3min。
优选步骤“c)”中的前八天培养基中含有的SHH的浓度为250-300ng/mL。
优选步骤“c)”中第13-19天培养基中含有的FGF8b的浓度为80-120ng/mL。
优选步骤“c)”中第34天后培养基中含有的TGF-β3的浓度为10ng/mL。
步骤“d)”中以恒电位法记录电流,优选测定电位为+700-780mV。
上述人多能干细胞优选为帕金森病人来源的多能干细胞。
如本发明图4所示,可以测定单囊泡且空间分辨率很高;如图5所示,每个囊泡检测的时间为ms级别且时间分辨率很高,该方法的优势体现明显。
本发明方法依次包含Nano-tip微电极的制备、人多能干细胞的复苏与培养、多巴胺能神经元的定向分化和单囊泡检测。本发明中将基于Nano-tip微电极的单细胞分析技术与iPSC技术诱导的人源性帕金森病疾病模型相结合,构建了单个神经元中单囊泡分析的方法,为帕金森疾病的病理学研究提供一种新的化学视角,为PD和其他神经退行性疾病的新药发现提供了新的平台和途径。
与现有技术比较本发明的有益效果:
1.本发明得益于Nano-tip微电极的高时空分辨率和高灵敏度,实现了单囊泡储存的分析,从单细胞层面为疾病的病理机制研究提供全新的化学视角。
2.本发明基于iPSC技术构建的人源性疾病模型,其保留了病人独特的遗传特征,可用于不同类型的疾病研究。
3.本发明首次在单细胞水平研究帕金森疾病模型中的单囊泡的多巴胺储存、囊泡数量及在微电极表面的小孔形成动力学情况,可以为帕金森疾病和其他神经退行性疾病的新药发现提供了新的平台和途径。
附图说明
图1为iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法示意图。
图2A为Nano-tip微电极的扫描电镜表征(标尺:20μM)。
图2B为电极在0.1mM的多巴胺溶液中的典型循环伏安图谱。
图3A为多能干细胞向多巴胺能神经元特异性分化的流程图。
图3B为正常对照组与PD患者来源的多巴胺能神经元中中脑标记物FOXA2、OTX2和CORIN(第24天)和多巴胺能神经元标记物TH(第45天)免疫荧光染色体染色。
图3C为FOXA2与OTX2染色结果的阳性率统计。
图4A为实施例5对照组单个神经元中囊泡储存情况的典型图。
图4B为实施例5PD疾病组单个神经元中囊泡储存情况的典型图。
图4C为实施例5对照组(24个神经元,n=872)和PD组(22个神经元,n=993)囊泡内容物的标准化频率分布图。
图4D为实施例5对照组和PD组诱导分化的神经元中每个囊泡的儿茶酚胺分子平均数。每个点代表单个神经元的平均水平。**:p<0.01。
图4E为实施例5对照组和PD组诱导分化的单个神经元中检测到的事件平均数。每个点代表单个神经元的平均水平。
图5A为实施例5从对照组和PD组中测定的获得的单个囊泡储存情况的典型峰。
图5B为实施例5单囊泡典型峰中不同参数示意图。
图5C为实施例5半峰宽(thalf)的比较。每个点代表来自单个神经元的单囊泡的平均水平。**:p<0.01。
图5D为实施例5峰值电流(Imax)比较。每个点代表来自单个神经元的单囊泡的平均水平。**:p<0.01。
具体实施方式
以下通过具体实施例对本发明做进一步解释说明:
药品和试剂来源:
Essential 8Basal medium | Gibco(A15169-01) |
Essential 8Supplement,50x | Gibco(A1517001) |
Vitronectin | Life Technologies(A14700) |
Matrigel | BD Biosciences(356234) |
DMEM/F12 | Gibco(C11330500BT) |
N2 supplement | Gibco公司(17502048) |
非必需氨基酸 | Gibco公司(11140) |
B27supplement | Life Technologies(P4957) |
Fetal bovine serum(FBS) | Life Technologies(10099-141) |
抗荧光淬灭封片剂(Fluromount-G) | SouthernBiotech(0100-01) |
Triton X-100 | Amresco(0694) |
Ethelyne glycol | Sigma(293237-1L) |
NaCl | 上海沪试(10019318) |
葡萄糖 | VETEC(V900116-500G) |
HEPES | Sigma(H3375-25G) |
KCl | 上海沪试(10016318) |
GaCl2 | Sigma(21097-250G) |
MgCl | Sigma(21097-250G) |
OTX2 | R&D(AF1979) |
FOXA2 | SantaCruz(sc-374376) |
NESTIN | SantaCruz(sc-21247) |
PKC-λ | BD(610207) |
CORIN | R&D(MABA2209) |
TH | Pel-Freez(P40101) |
试剂配制:
(1)人多能干细胞培养基配制:将10mL Essential 8TM Flex Supplement加入490ml Essential 8TM Basal Medium中。
(2)神经诱导培养基配制:将5mL N2 Supplement(N2因子)和5ml非必须氨基酸(non-essential amino acids solution,NEAA)加入490mL DMEM/F12基础培养基中。
(3)电分析外液配制:150mM NaCl,5mM KCl,2mM CaCl2,1.2mM MgCl2,5mMglucose,10mM HEPES。调至PH为7.4。
实施例1
a)Nano-tip微电极的制备:首先,将碳纤维吸入硼硅玻璃毛细管中,随后通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露。在显微镜下用锋利的刀片将暴露的碳纤维切至100μm。随后,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻2秒。一旦碳纤维的tip端出现红点,将电极从火焰离开,并在显微镜下检查电极中的碳纤维是否成功形成尖头。成功形成尖头的电极放入环氧树脂胶中1.5min封闭玻璃管与碳纤维之间的空隙。接着浸入丙酮中洗去碳纤维上的胶水。随后,放入100℃的烘箱中过夜。制备完成的电极放入含0.10mM多巴胺的PBS(pH 7.4)溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,并且电流平台在1.5-3nA的电极,可用于后续实验。
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出,并在在37℃水浴锅内解冻。之后,将细胞悬液加入提前备好5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内。使用无异源性培养基Essential 8(E8)维持其生长,每日换液并观察。状态良好的人多能性干细胞呈现核质比高,形状规则的状态。
c)多巴胺能神经元的定向分化:首先将适量的人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μM MH1、0.4μM CHIR、250ng/mL SHH的NIM培养基中贴壁培养,隔天换液。第九天时,将人多能干细胞吹下,并转移至15mL离心管后进行离心处理。离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬。最后转移至培养瓶中进行悬浮培养,隔天换液。第13天将培养液替换为含有SAG以及100ng/mL FGF8b的NIM培养基。第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基。第34天时,接种至Matrigel包被的小圆玻片上。用含有10ng/mL BDNF、10ng/mL GDNF、10ng/mL TGF-β3、1μMcAMP、1μM Compound E的NIM培养基继续培养至第45天。
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,可以保护内部不受外部电场的干扰。测定时的工作电极为步骤a)制备的Nano-tip微电极,参比电极为Ag/AgCl电极。用微操作器(Sutter MP-225)将Nano-tip微电极插入神经元,插入同时采用膜片钳放大器(Axon 700B)以恒电位法记录电流,测定电位为+780mV。输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪(Axon 1550A)以5kHz数字化。记录的电化学数据使用Igor Pro 6.22程序进行数据处理。电流的过滤器为1kHz。电流峰大于噪音三倍的峰被保留,不满足条件的峰去除。每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况。根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
实施例2
a)Nano-tip微电极的制备:首先,将碳纤维吸入硼硅玻璃毛细管中,随后通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露。在显微镜下用锋利的刀片将暴露的碳纤维切至150μm。随后,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻3秒。一旦碳纤维的tip端出现红点,将电极从火焰离开,并在显微镜下检查电极中的碳纤维是否成功形成尖头。成功形成尖头的电极放入环氧树脂胶中1min封闭玻璃管与碳纤维之间的空隙。接着浸入丙酮中洗去碳纤维上的胶水。随后,放入100℃的烘箱中过夜。制备完成的电极放入含0.10mM多巴胺的PBS(pH 7.4)溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,并且电流平台在1.5-3nA的电极方可用于后续实验。
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出,并在在37℃水浴锅内解冻。之后,将细胞悬液加入提前备好5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内。使用无异源性培养基Essential 8(E8)维持其生长,每日换液并观察。状态良好的人多能性干细胞应当呈现核质比高,形状规则的状态。
c)多巴胺能神经元的定向分化:首先将适量的人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μM MH1、0.4μM CHIR、300ng/mL SHH的NIM培养基中贴壁培养,隔天换液。第九天时,将人多能干细胞吹下,并转移至15mL离心管后进行离心处理。离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬。最后转移至培养瓶中进行悬浮培养,隔天换液。第13天将培养液替换为含有SAG以及80ng/mL FGF8b的NIM培养基。第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基。第34天时,接种至Matrigel包被的小圆玻片上。用含有10ng/mL BDNF、10ng/mL GDNF、5ng/mL TGF-β3、1μMcAMP、1μM Compound E的NIM培养基继续培养至第45天。
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,可以保护内部不受外部电场的干扰。测定时的工作电极为制备的Nano-tip微电极,参比电极为Ag/AgCl电极。用微操作器(Sutter MP-225)将Nano-tip微电极插入神经元,插入同时采用膜片钳放大器(Axon 700B)以恒电位法记录电流,测定电位为+750mV。输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪(Axon 1550A)以5kHz数字化。记录的电化学数据使用Igor Pro 6.22程序进行数据处理。电流的过滤器为1kHz。电流峰大于噪音三倍的峰被保留,不满足条件的峰去除。每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况。根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
实施例3
a)Nano-tip微电极的制备:首先,将碳纤维吸入硼硅玻璃毛细管中,随后通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露。在显微镜下用锋利的刀片将暴露的碳纤维切至120μm。随后,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻1.5秒。一旦碳纤维的tip端出现红点,将电极从火焰离开,并在显微镜下检查电极中的碳纤维是否成功形成尖头。成功形成尖头的电极放入环氧树脂胶中1.5min封闭玻璃管与碳纤维之间的空隙。接着浸入丙酮中洗去碳纤维上的胶水。随后,放入100℃的烘箱中过夜。制备完成的电极放入含0.10mM多巴胺的PBS(pH 7.4)溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,并且电流平台在1.5-3nA的电极方可用于后续实验。
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出,并在在37℃水浴锅内解冻。之后,将细胞悬液加入提前备好5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内。使用无异源性培养基Essential 8(E8)维持其生长,每日换液并观察。状态良好的人多能性干细胞应当呈现核质比高,形状规则的状态。
c)多巴胺能神经元的定向分化:首先将适量的人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μM MH1、0.4μM CHIR、250ng/mL SHH的NIM培养基中贴壁培养,隔天换液。第九天时,将人多能干细胞吹下,并转移至15mL离心管后进行离心处理。离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬。最后转移至培养瓶中进行悬浮培养,隔天换液。第13天将培养液替换为含有SAG以及120ng/mL FGF8b的NIM培养基。第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基。第34天时,接种至Matrigel包被的小圆玻片上。用含有10ng/mL BDNF、10ng/mL GDNF、15ng/mL TGF-β3、1μMcAMP、1μM Compound E的NIM培养基继续培养至第45天。
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,可以保护内部不受外部电场的干扰。测定时的工作电极为制备的Nano-tip微电极,参比电极为Ag/AgCl电极。用微操作器(Sutter MP-225)将Nano-tip微电极插入神经元,插入同时采用膜片钳放大器(Axon 700B)以恒电位法记录电流,测定电位为+700mV。输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪(Axon 1550A)以5kHz数字化。记录的电化学数据使用Igor Pro 6.22程序进行数据处理。电流的过滤器为1kHz。电流峰大于噪音三倍的峰被保留,不满足条件的峰去除。每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况。根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
实施例4
a)Nano-tip微电极的制备:首先,将碳纤维吸入硼硅玻璃毛细管中,随后通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露。在显微镜下用锋利的刀片将暴露的碳纤维切至150μm。随后,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻2秒。一旦碳纤维的tip端出现红点,将电极从火焰离开,并在显微镜下检查电极中的碳纤维是否成功形成尖头。成功形成尖头的电极放入环氧树脂胶中2min封闭玻璃管与碳纤维之间的空隙。接着浸入丙酮中洗去碳纤维上的胶水。随后,放入100℃的烘箱中过夜。制备完成的电极放入含0.10mM多巴胺的PBS(pH 7.4)溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,并且电流平台在1.5-3nA的电极方可用于后续实验。
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出,并在在37℃水浴锅内解冻。之后,将细胞悬液加入提前备好5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内。使用无异源性培养基Essential 8(E8)维持其生长,每日换液并观察。状态良好的人多能性干细胞应当呈现核质比高,形状规则的状态。
c)多巴胺能神经元的定向分化:首先将适量的人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μM MH1、0.4μM CHIR、300ng/mL SHH的NIM培养基中贴壁培养,隔天换液。第九天时,将人多能干细胞吹下,并转移至15mL离心管后进行离心处理。离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬。最后转移至培养瓶中进行悬浮培养,隔天换液。第13天将培养液替换为含有SAG以及80ng/mL FGF8b的NIM培养基。第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基。第34天时,接种至Matrigel包被的小圆玻片上,用含有10ng/mL BDNF、10ng/mL GDNF、8ng/mL TGF-β3、1μMcAMP、1μM Compound E的NIM培养基继续培养至第45天。
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,可以保护内部不受外部电场的干扰。测定时的工作电极为制备的Nano-tip微电极,参比电极为Ag/AgCl电极。用微操作器(Sutter MP-225)将Nano-tip微电极插入神经元,插入同时采用膜片钳放大器(Axon 700B)以恒电位法记录电流,测定电位为+750mV。输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪(Axon 1550A)以5kHz数字化。记录的电化学数据使用Igor Pro 6.22程序进行数据处理。电流的过滤器为1kHz。电流峰大于噪音三倍的峰被保留,不满足条件的峰去除。每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况。根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
实施例5帕金森疾病机制研究试验例:
采用实施例1中的方法诱导正常对照组和帕金森病人来源的多能干细胞分化为多巴胺能神经元,并采用实施例1中的方法检测并计算单个神经元单囊泡的多巴胺分子数量以及单个神经元中的囊泡数量。如图4,分析检测22个正常来源的多巴胺能神经元和24个疾病来源的多巴胺能神经元,结果表明在PD组单个神经中单囊泡的多巴胺分子平均数量为12.08万,远低于对照组的16.82万;对于对照组和PD组的单个神经元中囊泡数量未发生改变。如图5,进一步对代表单个囊泡情况的单峰进行分析,发现PD组thalf值增大,表明其在微电极表面小孔形成的速度变慢,这可以模拟在帕金森疾病模型组的信息交互中存在一定的障碍。
实施例6药物筛选测试试验例:
化合物佛波醇12-十四酸酯-13-乙酸酯(PMA)进行验证,有研究报道该化合物会触发α-syn减少的化合物,可能能够提高DA神经递质水平,并进一步维持神经元存活。在对照组和PD组诱导分化的神经元中,用10μM PMA孵育12h后,按照实施例1中的方法检测并计算单个神经元单囊泡的多巴胺分子数量。PD组检测24个神经元,PD+PMA组检测25个神经元,并对单个神经中单囊泡中的多巴胺数量进行平均,发现加入该化合物作用后,单囊泡的多巴胺分子数量明显增加(由原来12.08万的增加至18.65万),表明与PMA孵育前可有效逆转单个神经元单个囊泡的DA水平。本发明基于Nano-tip微电极和iPSC技术构建的人源性疾病模型相结合构建的研究平台,为PD及其他神经退行性疾病的预防或治疗策略的设计提供了新的方向。
上述实施例中的对照组为人多能干细胞,为人胚胎干细胞系H9(购于美国威斯康星Wicell bank,授权编号为16-W0060)。
PD组为病人来源的人诱导多功能干细胞系,由帕金森病患者血液重编程所得,具体过程如下:外周血单核细胞(Peripheral Blood Mononuclear Cell,PBMC)分离提取:于15mL离心管内分别加入5mL DPBS、5-10mL ficoll-paque(ficoll的量大于血量)备用。利用移液管将血液加入带有DPBS的离心管内混匀。之后将稀释的血液缓慢滴入带有ficoll的离心管内,使血液与ficoll形成稳定的液面分层。室温离心,400g,30min,升9降4。离心结束后,小心吸取白色层液体于15mL离心管内,并加入3-5mL的DPBS进行稀释重悬。再次离心,200g,5min,弃去上清液,加入2mL MNC培养液重悬后计数。
重编程过程:吸取总量为3×105的细胞接种于CD3+包被的12孔板内,培养液为MNC,37℃培养箱内孵育激活,隔天补液。CD3+包被方法:吸取10μL CD3+均匀溶解于DPBS室温孵育2-4小时,使用前利用PBS清洗3遍,血细胞激活5天后收集PBMC并计数。将细胞总数为3×105的细胞置于24孔板的单孔内,并加入仙台病毒进行转染。培养液为MNC。24小时后,将转染后的细胞接种于铺有MEF的六孔板内进行培养,培养液于一周后替换iPSC培养基。重编程过程中添加小分子化合物10μM JQ1和bFGF,即用现加,直至人多能干细胞出现。
Claims (6)
1.一种iPSC技术诱导分化的多巴胺能神经元中单囊泡储存的分析方法,其特征在于该方法包括以下步骤:
a)Nano-tip微电极的制备:将碳纤维吸入硼硅玻璃毛细管中,通过拉针仪,玻璃受热熔断后,变成长短相同的两部分,碳纤维暴露,在显微镜下将暴露的碳纤维切至100-150μm,将碳纤维置于丁烷喷枪的火焰边缘蚀刻在3秒以下,碳纤维的tip端出现红点,将电极从火焰离开,电极中的碳纤维形成尖头,将电极放入环氧树脂胶中1-3min,封闭玻璃管与碳纤维之间的空隙,接着浸入丙酮中洗去碳纤维上的胶,随后,放入100℃的烘箱中,过夜,制备完成的电极放入含0.10mM多巴胺的pH 7.4的PBS溶液中,测定其循环伏安行为,出现近似S型循环伏安曲线,且电流平台在1.5-3nA的电极即为达到要求的电极;
b)人多能干细胞的复苏与培养:快速将冻于液氮罐内的细胞取出解冻得到细胞悬液,将细胞悬液加入5ml DMEM/F12培养基中,高速离心后弃去上清,E8培养基重悬细胞后转移至Vitronectin包被的培养皿内,使用无异源性培养基Essential 8维持其生长,每日换液,直至人多能性干细胞呈现核质比高,形状规则的状态;
c)多巴胺能神经元的定向分化:首先将适量人多能干细胞接种于铺有Matrigel的六孔板内,并在含有2μM SB、2μMMH1、0.4μM CHIR、20-500ng/mL SHH的NIM培养基中贴壁培养,隔天换液,第九天时,将人多能干细胞吹下,并离心处理,离心结束后,弃上清,用含有CHIR、SHH、0.5-2μM SAG的NIM培养液将沉淀重悬,最后转移至培养瓶中进行悬浮培养,隔天换液,第13天将培养液替换为含有SAG以及80-120ng/mL FGF8b的NIM培养基,第19天时,将培养液替换为含有SHH以及FGF8b的NIM培养基,第34天时,接种至Matrigel包被的小圆玻片上,用含有10ng/mL BDNF、10ng/mL GDNF、5-15ng/mLTGF-β3、1μM cAMP、1μM Compound E的NIM培养基继续培养至第45天;
d)单囊泡检测:单囊泡检测是在倒置显微镜下完成的,整个测定过程在法拉第笼里进行,测定时的工作电极为步骤a)制备的Nano-tip微电极,参比电极为Ag/AgCl电极;用微操作器将Nano-tip微电极插入步骤c)得到的多巴胺能神经元,插入同时采用膜片钳放大器以恒电位法记录电流,测定电位为+700-800mV,输出信号采用Axon过滤器在0.3kHz下过滤信号,输出信号通过数字化仪以5kHz数字化;记录的电化学数据使用Igor Pro 6.22程序进行数据处理;电流的过滤器为1kHz;电流峰大于噪音三倍的峰被保留,不满足条件的峰去除;每个神经元测定后得到多个峰,每个峰表明一个囊泡储存情况;根据单个峰,可以得到如下参数:(1)Imax(2)thalf(3)Q,分子量,曲线下面积值。
2.根据权利要求1所述的单囊泡储存的分析方法,其特征在于步骤“a)”中,将碳纤维放置在丁烷喷枪的火焰边缘蚀刻1-3秒。
3.根据权利要求1所述的单囊泡储存的分析方法,其特征在于步骤“a)”中,电极在环氧树脂胶中的时间为2min。
4.根据权利要求1所述的单囊泡储存的分析方法,步骤“c)”中的前八天培养基中含有的SHH的浓度为250-300ng/mL。
5.根据权利要求1所述的单囊泡储存的分析方法,其特征在于步骤“c)”中第34天后培养基中含有的TGF-β3的浓度为10ng/mL。
6.根据权利要求1所述的单囊泡储存的分析方法,其特征在于步骤“d)”中以恒电位法记录电流,测定电位为+700-780mV。
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