CN114486829B - 一种微量外泌体捕集并计数的方法 - Google Patents
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Abstract
本发明属于生命科学技术领域，具体为一种微量外泌体捕集并计数的方法。本发明方法采用注射泵、毛细管和荧光成像系统，包括毛细管内壁捕获外泌体的特异性配基的修饰、微量外泌体的捕集、外泌体的柱上荧光染色及显微镜观察计数。本发明使用毛细管开管柱，样品无需经过复杂的前处理及纯化过程，可大幅减少样品损失，简化实验步骤并缩短检测时间。该方法所需样品体积在1纳升‑100微升之间，单个显微镜视野的荧光成像可完成对1‑10^3个外泌体的精准计数；经过样品稀释或多次、多视野荧光叠加拍摄可扩展计数范围至10^6数量级。该方法还可以扩展应用到对不同来源和不同类型外泌体进行选择性捕集及计数定量。
Description
技术领域
本发明属于生命科学技术领域，具体涉及微量样品体系中外泌体捕集并计数的方法。
背景技术
外泌体是一类由细胞产生并分泌到胞外的、直径在30-150纳米范围内的具有双层膜结构的微囊泡，天然存在于各种体液中，如血液、尿液等。外泌体内含有广泛的蛋白质、脂质和核酸，不仅提供了对外泌体生物发生机制的见解，更是赋予了外泌体各种生物学功能和临床意义。目前的研究已经表明，外泌体可抑制T细胞抗肿瘤免疫能力及诱导产生耐药性，以此促进肿瘤细胞的迁移及侵袭。因此，对各种体液中的微量外泌体进行富集及定量，具有重要的临床指导意义。
目前常用的外泌体定量方法主要包括纳米粒子示踪分析（NTA）和BCA蛋白质定量法，不仅需要根据样本的体液来源进行不同操作的前处理及纯化过程，而且均是基于大量样本计算得出的外泌体流体力学直径和浓度或其总蛋白含量，无法反映外泌体真实的数量，检测限也无法与体内的外泌体含量数量级相当，需要预富集步骤。近年来，已有学者尝试设计新的外泌体定量方法解决前述问题，如Zeng等开了一种用聚二乙炔囊泡与外泌体结合，通过测定溶剂迁移距离定量外泌体的方法；Jeong等制造了一种用于外泌体分析的手持电化学平台，可实现多通路复用的外泌体测定。尽管如此，这些方法均需要通过复杂的前处理去除样本中的细胞或基质，外泌体定量依据是根据其他物理化学参数的变化间接测定，而且均使用了自制的微流控设备，技术难度较高，这也限制了其应用进一步的推广。
基于以上认识，为了解决微量外泌体的直接计数问题，本发明设计了一种微量外泌体的捕集并计数方法，以内壁修饰了捕获外泌体的特异性配基的毛细管富集并捕获外泌体，提高了检测限；同时结合单分子荧光成像的方法，对外泌体含量进行了直接计数。整个方法不需要用的特定的设备，操作简单，利于在生命科学中的应用及推广。
发明内容
本发明的目的在于提供一种操作简单、对微量外泌体进行捕集并直接计数的方法。
本发明提供的微量外泌体捕集并计数的方法，采用注射泵、毛细管和荧光成像系统，包括毛细管内壁捕获外泌体的特异性配基的修饰、微量外泌体的捕集、外泌体的柱上荧光染色及显微镜观察计数，具体步骤如下：
（1）毛细管内壁上捕获外泌体的特异性配基的修饰：使用注射泵依次向透明毛细管内注入乙二胺-生物素、链霉亲和素、生物素-IgG和IgG抗体，在毛细管内壁上构建捕获外泌体的亲和配基；
（2）微量外泌体的捕集：外泌体样品通过注射泵流经修饰了配基的毛细管柱，流速控制在1-50微升/分钟，使外泌体被充分捕获；
（3）外泌体的荧光染色：在捕获好外泌体的毛细管内注入膜荧光染料或带有荧光标签的亲和配基、抗体或适配体，常温-37℃孵育1-60分钟；
（4）外泌体荧光成像及计数：清洗除去未结合到外泌体的荧光染料，使用荧光显微镜拍摄外泌体荧光成像照片，采用目测计数特定成像区域内的外泌体荧光点数，也可通过软件对特定成像区域内荧光点计数，完成目标外泌体的计数并换算成样品浓度或样品中外泌体总量。
通过目测计数或软件辅助荧光点计数，单位体积内外泌体的个数n的计算式为：
单位体积内外泌体浓度c为：
其中，L为毛细管长度，d为毛细管直径，S为成像面积，m为外泌体荧光点数，V为样品体积，NA为阿夫加得罗常数。
本发明中，毛细管内壁修饰特异性配基时，各反应均可在室温下进行。
本发明中，功能化毛细管内壁时，各步反应之间以磷酸缓冲盐溶液（PBS）和超纯水清洗，且修饰完捕获外泌体的特异性配基后，以牛血清白蛋白（BSA）封闭，以减少非特异性吸附带来的干扰。
本发明中，外泌体的荧光计数以同一视野下不同聚焦平面的荧光亮点叠加、不同视野下荧光亮点叠加得到。
本发明使用毛细管开管柱，样品无需经过复杂的前处理及纯化过程，可大幅减少样品损失，简化实验步骤并缩短检测时间。该方法所需样品体积在1纳升-100微升之间，单个显微镜视野的荧光成像可完成对1-10^3个外泌体的精准计数；经过样品稀释或多次、多视野荧光叠加拍摄可扩展计数范围至10^6数量级。该方法还可以扩展应用到对不同来源和不同类型外泌体进行选择性捕集及计数定量。
本发明用于微量外泌体的捕集并计数，可以实现对任何样品体系无前处理的情况下的对极低含量的外泌体分子进行绝对计数，可大幅减少样品转移或清洗带来的损失；通过多视野数据叠加，计数范围可达10^6。该方法还可以拓展应用到对不同来源和不同类型外泌体进行选择性捕集及计数。此方法对于各个体液样品中低含量外泌体定量有着重要作用；同时可对肿瘤细胞分泌的含有重要蛋白质标志物的外泌体进行定量以实现对疾病的诊断和评估，有着广阔的应用前景和实际意义。例如本发明的对微量外泌体进行捕集和计数的方法曾对从人乳腺癌细胞系MCF-7细胞培养基色谱分离得到的外泌体馏分中所含的外泌体进行了计数，算得的外泌体数目在2.3×10^5左右，与NTA测试的结果数量级相当，证明了本方法的可靠性。
附图说明
图1为用于微量外泌体捕集装置示意图。
图2为修饰了捕获外泌体的特异性配基的毛细管柱用于捕获外泌体的示意图。
图3为捕获完外泌体的毛细管柱的直接柱上染色及荧光拍摄图。虚线范围内为成像面积。
图中标号：1为注射泵，2为毛细管，3为荧光成像系统，4为细胞，5为抗体，6为外泌体。
具体实施方式
通过实施例对本发明提供的用于微量外泌体的捕集及计数方法在人乳腺癌细胞系MCF-7细胞外泌体的计数应用做出进一步的说明。
1，毛细管内壁用于捕获外泌体的特异性配基的修饰
修饰内壁的毛细管取50厘米长、150微米内径的，通过无死体积的两通管与注射泵相连并固定，设置反应体积为200微升，依次向透明毛细管内注入乙二胺-生物素、链霉亲和素、生物素-IgG和IgG抗体，完成毛细管内壁捕获外泌体的特异性配基的修饰。
2，MCF-7外泌体的捕集
取经色谱柱分离提纯得到的细胞培养基体系的MCF-7细胞外泌体30微升，通过注射泵以3微升/分钟的速度缓慢流经修饰好的毛细管柱，共10分钟，完成外泌体的捕集。
3，外泌体的柱上荧光染色
向捕获完外泌体的毛细管内注入10微摩的膜荧光染料，在37℃下孵育20min，完成外泌体的荧光染色。
4，外泌体荧光成像及计数
清洗未反应的荧光染料，上机使用荧光显微镜拍摄外泌体的荧光照片，光电倍增倍数设定为300，曝光时间100毫秒，计数荧光点数目，完成外泌体的计数。
Claims (4)
1.一种微量外泌体捕集并计数的方法，其特征在于，采用注射泵、毛细管和荧光成像系统，包括毛细管内壁捕获外泌体的特异性配基的修饰、微量外泌体的捕集、外泌体的柱上荧光染色及显微镜观察计数，具体步骤如下：
（1）毛细管内壁上捕获外泌体的特异性配基的修饰：使用注射泵依次向透明毛细管内注入乙二胺-生物素、链霉亲和素、生物素-IgG和IgG抗体，在毛细管内壁上构建捕获外泌体的亲和配基；
其中，毛细管内壁上修饰捕获外泌体的特异性配基，各步反应之间以磷酸缓冲盐溶液和超纯水清洗，且修饰完捕获外泌体的特异性配基后，以牛血清白蛋白封闭；
（2）微量外泌体的捕集：外泌体样品通过注射泵注入修饰了配基的毛细管柱，流速控制在1-50微升/分钟，使外泌体被充分捕获；
（3）外泌体的荧光染色：在捕获好外泌体的毛细管内注入膜荧光染料或带有荧光标记的亲和配基、抗体或适配体，常温-37℃孵育1-60分钟；
（4）外泌体荧光成像及计数：清洗除去未结合到外泌体上的荧光染料，使用荧光显微镜拍摄外泌体荧光成像照片，采用目测计数特定成像区域内的外泌体荧光点数，或者通过软件对成像区域内荧光点计数，完成目标外泌体的计数并换算成样品中外泌体浓度或样品中外泌体总量。
2.根据权利要求1所述的微量外泌体捕集并计数的方法，其特征在于，所述毛细管内壁上修饰捕获外泌体的特异性配基，反应在室温下进行。
3.根据权利要求1所述的微量外泌体捕集并计数的方法，其特征在于，外泌体的荧光计数以同一视野下不同聚焦平面的荧光亮点叠加、不同视野下荧光亮点叠加得到。
4.根据权利要求1所述的微量外泌体捕集并计数的方法，其特征在于，所述用目测计数或软件辅助荧光点计数，单位体积内外泌体的个数n的计算式为：
单位体积内外泌体浓度c为：
其中，L为毛细管长度，d为毛细管直径，S为成像面积，m为外泌体荧光点数，V为样品体积，NA为阿伏伽德罗常数。
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