CN110042053B - 一种单细胞激光弹射基片、方法及应用 - Google Patents
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Abstract
本发明涉及一种单细胞激光弹射基片及液相单细胞弹射方法,单细胞激光弹射基片依次包括基底层和镀膜层,其中镀膜层上偶联有氨基基团或特异性抗体。本发明解决了干燥处理造成细胞活性受损和动物细胞弹射分离的问题,优点是能够最大程度保持分离单细胞的活性,用于进一步培养,可实现对动物细胞的弹射分离;同时,能够结合其他观察、测量手段对单细胞进行定位、判断,以满足对特殊细胞如CTC细胞的选择性分选。
Description
技术领域
本发明涉及单细胞分析及分选技术领域,具体涉及一种单细胞激光弹射基片及利用单细胞激光弹射基片进行液相单细胞弹射的方法。
背景技术
前期发展的基于脉冲激光的单细胞弹射技术作为一种能够在单细胞水平上对微生物细胞进行精确地定向分离手段,能够结合完善的细胞多模态观察和测量方法(如光学成像、拉曼光谱、荧光标记等),同时耦合下游组学测序技术,弥补了FACS及其他一些技术的不足,因此,对于单细胞功能及遗传信息的精确解析具有极大潜力。然而,该技术在使用前,往往需要将细胞悬液滴涂于弹射基片表面进行干燥固定,因此仅适用于有细胞壁的微生物细胞,如细菌、酵母、微藻等;而干燥处理往往造成细胞活性减弱或丧失,对于动物细胞甚至造成细胞破裂,因此,前期技术对细胞活性影响较大,且无法用于对动物细胞的分离获取。
发明内容
针对以上技术问题,本发明提供了一种单细胞激光弹射基片和液相单细胞弹射的方法,解决了干燥处理造成细胞活性受损和动物细胞弹射分离的问题。
本发明提供一种单细胞激光弹射基片,依次包含基底层和镀膜层,其中镀膜层表面经化学修饰偶联有氨基基团或特异性抗体,所述单细胞激光弹射基片用于单细胞弹射分选。
所述基底层材料为光透明材料,包括硅酸盐玻璃、石英玻璃和氟化钙玻璃中的一种或多种。
所述镀膜层材料为透明金属复合氧化物薄膜材料,包括ITO(氧化铟锡)、FTO(氟掺杂氧化锡)、AZO(铝掺杂氧化锌)中的一种或多种。
所述基底的厚度为0.1-2mm,所述镀膜层的厚度为10-500nm。
所述氨基基团或特异性抗体,用于结合单细胞。
所述氨基基团通过静电吸附方式结合单细胞。
所述特异性抗体通过免疫结合方式结合单细胞。
所述特异性抗体能够与单细胞表面所具有的抗原位点特异性结合,特异性抗体种类因单细胞不同而异。
特异性抗体包括单克隆抗体或多克隆抗体。
所述氨基基团或特异性抗体为单分子层。
所述氨基基团或特异性抗体通过共价反应与镀膜层偶联。
所述单细胞为细胞膜完整且活性不丧失的人或动物来源的细胞。
本发明提供一种利用单细胞激光弹射基片进行液相单细胞弹射的方法,包括以下步骤:
1)将细胞悬液滴涂于单细胞激光弹射基片表面并孵育;
2)冲洗除去单细胞激光弹射基片上未被结合的细胞,并使细胞始终处于液相环境中;
3)利用显微镜观察识别结合于单细胞激光弹射基片上的细胞,采用脉冲激光进行单细胞弹射,使单细胞脱离单细胞激光弹射基片表面;
4)使用接收装置接收被分离的单细胞。
所述液相环境为中性等渗溶液,优选地,为PBS溶液、盐溶液、蔗糖溶液或细胞培养液中的一种或多种。
所述孵育的时间为10-30分钟。
所述结合方式包括静电吸附或免疫结合。
所述显微镜包括正置显微镜。
所述观察识别包括收集单细胞拉曼散射信号或荧光信号,获得单细胞拉曼图谱或荧光图像,分析拉曼图谱或荧光图像识别单细胞。
所述脉冲激光波长为激光波长为266nm-1064nm,优选地,为266nm、532nm或1064nm,更优选地,为532nm。
所述脉冲激光的脉宽为1fs-1 μs中的一种,优选地,为1ns。
所述脉冲激光的能量范围为1μJ-100 μJ,优选地,为10μJ。
所述激光的脉冲频率为5-16.6kHz,优选地,为11.0-14.6kHz。
所述接收装置包括离心管或微孔。
本发明提供一种单细胞激光弹射基片在循环肿瘤细胞(CTC)分析中的应用,包括以下步骤:
1)CTC细胞悬液滴涂于单细胞激光弹射基片表面;
2)利用拉曼检测或荧光成像对CTC细胞进行观察识别,并采用激光弹射技术分离获取单个CTC细胞;
3)对分离后的单个CTC细胞进行单细胞转录组或基因组测序,分析其表达谱或基因突变位点。
本发明的有益效果是:能够最大程度保持分离单细胞的活性,用于进一步培养,可实现对人或动物来源的单细胞的弹射分离;同时,能够结合其他观察、测量手段对单细胞进行定位、判断,以满足对特殊细胞如CTC细胞的选择性分选。该方法的建立拓展了单细胞弹射分离技术的应用范围,将为人或动物来源的细胞尤其是特殊细胞的捕获和分选提供新的工具。
应理解,在本发明范围内,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图做简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1A和图1B分别为经过化学修饰和未经化学修饰的单细胞激光弹射基片结合单细胞的显微照片。
图2A和图2B分别为结合有HeLa细胞的单细胞激光弹射基片在液相弹射分离前和液相弹射分离后的显微照片。
图3A和图3B分别为弹射分离前和弹射分离后聚焦于单细胞激光弹射基片的图像;图3C为微孔中接收到所分离的单细胞图像。
图4为酵母单细胞弹射分离后的培养结果;其中图4A、4B为液相弹射获取的单细胞酵母培养结果,图4C为干性弹射获取的单细胞酵母培养结果,其中图4A、4C为实物拍照照片,4B为采用10倍物镜获得的显微图像。
图5为单个HeLa细胞经弹射分离后进行单细胞MDA扩增及PCR验证的结果;其中,N0为PCR反应阴性对照,N为MDA阴性对照,P为MDA阳性对照,#7为无弹射细胞的对照孔,#1-#6、#8及#9为各弹射一个细胞的样品孔。
图6A、6B分别为HER-2抗体修饰的单细胞激光弹射基片结合MCF-7及MCF-7/HER-2细胞的亮场图像和荧光图像(10倍物镜),其中颜色较亮的细胞为HER2蛋白高表达的乳腺癌细胞,颜色较暗的细胞为普通MCF-7细胞;图6C为单个MCF-7/HER-2细胞经液相弹射分离后的对比图像(10倍物镜);图6D为微孔接收装置中所接收到的单细胞图像(40倍物镜)。
具体实施方式
实施例1:单细胞激光弹射基片的制作及单细胞结合
单细胞激光弹射基片依次包括基底层和镀膜层,其中镀膜层表面经化学修饰偶联有氨基基团或特异性抗体。基底层采用光透明材料,包括但不限于硅酸盐玻璃、石英玻璃、氟化钙玻璃中的一种或多种;镀膜层材料为透明金属复合氧化物薄膜材料,包括但不限于ITO、FTO、AZO中的一种或多种,采用喷涂、蒸发、溅射等沉积方式在基底层表面形成镀膜层,镀膜层经氧气等离子体处理后,将单细胞激光弹射基片迅速放入0.5%-5%的氨基为末端的硅烷偶联试剂(如3-氨基丙基-三甲氧基硅烷,APTMS)甲苯溶液中,处理0.5-2个小时,随后取出,经无水乙醇彻底清洗,氮气吹干,置80℃烘箱中干燥处理1-3个小时,使镀膜层表面偶联氨基基团。正常培养的HeLa细胞经离心清洗后重悬于pH7.0-7.4的PBS缓冲溶液中,将一定量的细胞悬液滴涂于上述经处理后的单细胞激光弹射基片表面,在37℃培养箱中孵育10-30分钟;随后,PBS溶液冲洗2-3次以除去未被结合的细胞。由于单细胞激光弹射基片表面带有氨基基团,其在PBS溶液中解离使表面带有正电荷;而细胞在PBS中表面呈负电性,由于静电作用,细胞被吸引并附着于单细胞激光弹射基片表面。
图1A和图1B分别为经过化学修饰和未经化学修饰的单细胞激光弹射基片结合单细胞的显微照片,经过对比可知,未经化学修饰的单细胞激光弹射基片表面无法直接吸附和结合HeLa细胞。
实施例2:动物细胞的液相单细胞弹射分离及获取
按照实施例1中方法将HeLa细胞结合于单细胞激光弹射基片表面,冲洗除去未被结合的多余HeLa细胞,并使HeLa细胞始终处于液相缓冲体系中以保持细胞活性;随后利用显微镜观察测量结合于单细胞激光弹射基片表面的HeLa细胞,并采用脉冲激光弹射方法选择单个HeLa细胞进行弹射分离,可获得如图2所示的结果,从图中画圈位置可看出原有HeLa细胞的位置已成为空白,说明HeLa单细胞已从单细胞激光弹射基片表面分离。
经弹射分离后的单细胞可通过微孔装置接收,微孔装置位于单细胞激光弹射基片下方,经弹射分离后的单细胞从基片表面掉落至微孔底部,图3A和图3B分别为弹射分离前和弹射分离后聚焦于单细胞激光弹射基片的图像,弹射分离后单细胞掉落至微孔底部,造成了图像的脱焦;图3C为聚焦于微孔底部的图像。
实施例3:酵母单细胞液相弹射分离及培养
采用液相单细胞弹射方法,可最大程度地保护细胞活性,对分离获得的单细胞可进一步进行培养增殖。采用实施例1及实施例2所述的方法,对酵母细胞进行了液相单细胞弹射分离,同时采用微孔接收所分离的单细胞,并在培养液中对所获得的酵母细胞进行培养,12小时后可获得图4所示结果。采用液相弹射所获得的9个单细胞中有7个活性未受损伤,显示了不同程度增殖(图4A、4B)。而采用干性环境下的弹射分离,所获得的单细胞均未能成功培养增殖(图4C)。
实施例4:单细胞扩增测序
采用液相弹射方法所分离获取的单细胞可进一步进行基因组测序。采用实施例1及实施例2所述的方法,对HeLa细胞进行了液相单细胞弹射分离,同时采用微孔接收所分离的单个HeLa细胞;随后对细胞进行裂解并采用MDA方法对其基因组DNA进行扩增,8小时后以MDA产物为模板,分别进行16S rRNA,18S rRNA及SSU rRNA的PCR扩增,对MDA产物及PCR产物进行凝胶电泳成像可获得图5所示结果。其中,N0为PCR反应阴性对照,N为MDA阴性对照,P为MDA阳性对照,#7为无弹射细胞的对照孔,#1-#6、#8及#9为各弹射一个HeLa细胞的结果。
图5显示,采用液相弹射技术,所获得的单细胞均可成功进行MDA扩增,经18S和SSU标记基因确认可认定MDA产物大部分来自于单细胞基因组;同时,经16S标记基因证实,采用本方法可对外源污染进行较好的控制,能够获得较为纯净的单细胞基因组。进一步的,可对单细胞MDA扩增产物进行常规测序以分析其基因组信息。
实施例5:CTC单细胞分选
本发明所提出的单细胞激光弹射基片与液相单细胞弹射方法,可用于癌症患者血液中CTC细胞的富集捕获和单细胞分选,以进一步进行CTC单细胞测序,对其表达谱和突变位点进行检测,从而用于早期诊断、临床用药评价及预后监测等过程。本实施例中,采用人表皮生长因子受体-2(HER2)蛋白高表达的乳腺癌细胞MCF-7/HER-2模拟血液中CTC细胞,以HER2蛋白特异性抗体对弹射基片进行修饰,对MCF-7/HER-2进行特异性捕获;随后采用液相单细胞弹射方法分离单个MCF-7/HER-2细胞做进一步分析。图6A、6B所示为经抗体修饰过的单细胞激光弹射基片对MCF-7及MCF-7/HER-2细胞的捕获效果,图6C为单个MCF-7/HER-2细胞经液相弹射分离后的显微图像,图6D为微孔接收装置中所接收到的单细胞图像。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,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。
Claims (7)
1.一种单细胞激光弹射基片,其特征在于:依次包含基底层和镀膜层,其中镀膜层上偶联有氨基基团或特异性抗体,所述氨基基团或特异性抗体用于结合单细胞;所述特异性抗体包括单克隆抗体或多克隆抗体,所述单细胞为细胞膜完整且活性不丧失的人或动物来源的细胞,所述氨基基团或特异性抗体通过共价反应与镀膜层偶联。
2.根据权利要求1所述的单细胞激光弹射基片,其特征在于:所述基底层材料选自硅酸盐玻璃、石英玻璃和氟化钙玻璃中的一种或多种,所述镀膜层材料选自ITO(氧化铟锡)、FTO(氟掺杂氧化锡)、AZO(铝掺杂氧化锌)中的一种或多种。
3.一种液相单细胞弹射方法,其特征在于:包括以下步骤:
1) 将细胞悬液滴涂于权利要求1所述的单细胞激光弹射基片镀膜层表面并孵育;
2) 冲洗除去未被结合的细胞,并使细胞始终处于液相环境中;
3) 利用显微镜观察识别结合于单细胞激光弹射基片上的细胞,采用脉冲激光进行单细胞弹射使单细胞脱离单细胞激光弹射基片表面;
4) 使用接收装置接收被分离的单细胞。
4.根据权利要求3所述的液相单细胞弹射方法,其特征在于:所述液相环境为中性等渗溶液。
5.根据权利要求3所述的液相单细胞弹射方法,其特征在于:所述孵育的时间为10-30分钟。
6.根据权利要求3所述的液相单细胞弹射方法,其特征在于:所述观察识别包括收集单细胞拉曼散射信号或荧光信号,获得单细胞拉曼图谱或荧光图像,分析拉曼图谱或荧光图像识别单细胞。
7.一种单细胞激光弹射基片在循环肿瘤细胞(CTC)分析中的应用,所述应用不涉及疾病的诊断和治疗,其特征在于:包括以下步骤:
1)CTC细胞悬液滴涂于权利要求1所述的单细胞激光弹射基片表面;
2)利用拉曼检测或荧光成像对CTC细胞进行观察识别,并采用激光弹射技术分离获取单个CTC细胞;
3)对分离后的单个CTC细胞进行单细胞转录组或基因组测序,分析其表达谱或基因突变位点。
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