CN114414541A - 一种应用3d细胞成像分析***检测t细胞杀伤效力的方法 - Google Patents
一种应用3d细胞成像分析***检测t细胞杀伤效力的方法 Download PDFInfo
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Abstract
本发明公开了一种应用3D细胞成像分析***检测T细胞杀伤效力的方法,包括以下步骤:构建三维肿瘤球体;将靶细胞,效应细胞与TCE抗体共培养并进行染色标记;应用高内涵成像分析***进行图像采集;应用软件分析***对所得到的图像进行单一或叠加分析。本发明方法稳定性好,准确度高,直观可视,且能更接近体内生理环境评估T细胞杀伤效力。
Description
技术领域
本发明涉及一种检测T细胞杀伤效力的方法,具体涉及应用3D细胞成像分析***检测T细胞对肿瘤细胞杀伤效力的方法。
背景技术
目前,肿瘤免疫治疗已经成为继手术治疗、放射治疗和化疗药物治疗之后的第四大肿瘤治疗疗法,也是目前最热门的肿瘤治疗手段。肿瘤免疫治疗就是利用技术手段将免疫***重新激活,使其能够识别癌细胞并将其清除。在肿瘤免疫治疗领域中,抗体药物的研发一直是热门的方向。靶向CD3抗原的双特异性抗体是一类能够将T细胞与肿瘤细胞在空间上拉近,利用T细胞对肿瘤细胞的杀伤性作用治疗血液瘤和实体肿瘤的抗体疗法,这类抗体被称为T细胞衔接器(T cell-engaging bsAb,TCE双抗)。TCE双抗药物的作用机理是通过同时结合T细胞表面抗原和肿瘤细胞表面抗原形成免疫突触,从而直接激活,使T细胞增殖,进而释放细胞毒素或细胞因子来杀伤肿瘤细胞。
细胞杀伤实验,即细胞毒性检测,是用来评价TCE双抗介导的T细胞对肿瘤细胞杀伤效力的一类实验。常用的细胞毒性检测方法有51Cr释放实验、乳酸脱氢酶(LDH)释放法、BATDA法和CytoTox-Glo法等,经典的检测方法是51Cr释放法,但由于放射性同位素不利于安全操作及废物处置,对环境和人体健康存在巨大威胁,自发释放率高而限制了该方法的应用。LDH和CytoTox-Glo方法操作简便,但是存在半衰期短,灵敏度不高,且无法区分靶细胞和效应细胞释放的缺点。BATDA法需要对靶细胞进行标记,操作繁琐且自发释放率高,不适合较长时间的细胞杀伤实验。
当前细胞毒性检测的方法是在肿瘤细胞二维平面培养的环境中进行,这种平面培养、生长方式与机体内立体环境差别很大,导致细胞形态、分化、细胞与基质间的相互作用以及细胞与细胞间的相互作用与体内生理条件下细胞的行为存在明显差异。与传统的细胞培养不同,三维(3D)细胞培养重现了细胞的体内环境。可以形成氧气、营养物质、代谢物和可溶信号的梯度,为细胞提供更加接近体内生存条件的微环境,能够更好地模拟生理状态,从而获得与体内实验更加一致的实验结果。
发明内容
本发明的目的在于,针对传统检测方法操作繁琐,稳定性不高,不能准确反映体内真实的生理状态等缺点,本发明通过构建3D肿瘤球体,使用高内涵成像分析***,建立了一种稳定的、准确度高直观可视化且更接近体内生理环境的评估T细胞杀伤效力的方法。
为实现上述目的,本发明采用以下技术方案,
一种应用3D细胞成像分析***检测T细胞杀伤效力的方法,包括以下步骤:
1.应用3D细胞培养技术构建三维肿瘤球体;
2.将靶细胞,效应细胞与TCE抗体共培养,使用荧光标记A对靶细胞进行染色标记,使用荧光标记B对效应细胞进行染色标记,使用荧光标记C对共培养后产生的死细胞进行染色标记;
3.应用高内涵成像分析***,分别使用荧光标记A对应的激发光通道、荧光标记B对应的激发光通道和荧光标记C对应的激发光通道对同一视野进行图像采集;
4.应用软件分析***对所得到的图像进行单一或叠加分析,根据肿瘤球体体积的大小计算出T细胞对靶细胞的杀伤效力。
较佳的,荧光标记A与荧光标记B及荧光标记C对应的激发光波长均不相同。较佳的,荧光标记A包括荧光蛋白或/和细胞染料,荧光蛋白包括绿色荧光蛋白(GFP)或/和红色荧光蛋白(RFP)等,通过转染或慢病毒感染方式使靶细胞表达荧光蛋白。靶细胞也可以通过细胞染料进行标记,如Hoechst 33342(赫斯特),羧基荧光素二醋酸盐琥珀酰亚胺酯,远红染料,钙黄绿素绿色染料等细胞染料中的一种或多种。
较佳的,荧光标记B为活细胞染色试剂,包括羧基荧光素二醋酸盐琥珀酰亚胺酯,远红染料,钙黄绿素绿色染料,钙黄绿素紫色染料,钙黄绿素蓝色染料,钙黄绿素橙红色染料中的一种或多种。
较佳的,荧光标记C为死细胞染料,如PI(碘化丙啶),Caspase 3/7(半胱氨酸蛋白酶),SYTOX Green(绿菁),7-AAD(7-氨基放线菌素D),和Annexin V(膜联蛋白-V)等中的一种或多种。
本发明中所述的靶细胞是指各类肿瘤细胞或工程化改造的各类细胞,包括但不限于LS174T,HPAF-II,T84,SKBR-3,MCF-7,HepG2,ASPC-1,PC-3等。所述T细胞来源于人PBMC(外周血单核细胞),可以直接使用人PBMC,或通过磁珠分选方法获取T细胞组分,也可以是在体外扩增的T细胞。
叠加分析过程中,只显示荧光标记A的为活靶细胞,同时显示荧光标记A和荧光标记C的为死靶细胞;只显示荧光标记B的为活效应细胞,同时显示荧光标记B和荧光标记C的为死效应细胞。使用高内涵分析软件进行3D图像分析,通过使用荧光标记A染色图像分析识别球体,计算肿瘤球体体积,靶细胞特异性杀伤效率=无抗体组肿瘤球体体积-待测抗体处理组肿瘤球体体积/无抗体组肿瘤球体体积*100。
本发明的优点是:
1.本发明提供了一种基于3D肿瘤球体体积的变化评价细胞杀伤效力的方法,荧光标记的靶细胞在特定的培养条件下形成3D肿瘤球体,按照适当的效靶比,加入荧光标记的T细胞作为效应细胞,同时加入一定浓度梯度稀释的TCE抗体共培养,再加入PI对群体中的死细胞进行标记染色。利用高内涵成像***,可清楚直观的观察到相应的靶细胞及死靶细胞,效应细胞及死效应细胞,通过软件分析计算可得到对应的肿瘤球体体积大小,根据肿瘤球体体积相对于对照组的变化比率计算得出细胞杀伤效力。
2.本方法利用3D细胞培养技术结合高内涵成像,可同时得到图像信息和数据处理结果,结果更加直观形象,与传统的2D平面细胞培养技术相比,3D细胞培养能够更好地模拟生理状态,从而获得与体内实验更加一致的实验结果。
附图说明
图1为三荧光通道叠加下,不同浓度的TCE抗体介导的T细胞对肿瘤球体的杀伤效力;
图2为494nm激发光通道下不同浓度的TCE抗体介导的T细胞对肿瘤球体浸润;
图3为535nm激发光通道下不同浓度的TCE抗体作用下的靶细胞死亡数;
图4为不同浓度的TCE抗体介导的T细胞对肿瘤球体的杀伤效力。
具体实施方式
实施例1:靶细胞染色及3D肿瘤球体的构建
使用Hoechst 33342(赫斯特)对靶细胞LS174T进行荧光标记,离心洗涤之后用细胞计数仪计数,调整细胞密度为5×104个/mL,取100ul细胞悬液接种于Prime3DCulture Spheroid plates(S-bio,#MS-9096UZ)每孔中,放置于37℃,5%CO2培养箱中培养48小时,使之形成3D肿瘤球体。
实施例2:效应细胞染色
48小时之后,使用Calcein Green AM(钙黄绿素乙酰氧基甲酯)对T细胞进行荧光染色,离心洗涤之后用细胞计数仪计数,调整细胞密度为5×105个/mL,取50ul细胞悬液分别加入到已形成肿瘤球体的96孔板每孔中。
实施例3:靶细胞、效应细胞及待测抗体共培养
使用EMEM完全培养基配制不同浓度的待测抗体,从357nM开始,3倍梯度稀释至0.1nM,取50ul稀释好的抗体溶液分别加入到已形成肿瘤球体的96孔板每孔中,于37℃,5%CO2培养箱中培养48小时。
实施例4:荧光染料PI标记死细胞
共培养48小时之后,取出培养板,每孔加入5ul 40×工作浓度的PI染色液,轻轻吹打混匀。
实施例5:高内涵成像分析
使用Operetta CLS PreciScan功能,先对样本进行低倍镜扫描,通过编辑算法找出目标对象,再对目标对象进行高倍镜拍摄。对同一视野分别摄取350nm激发光通道采集Hoechst 33342荧光,494nm激发光通道采集Calcein Green荧光,535nm激发光通道采集PI荧光,分别得到单一或多荧光叠加成像图片。对同一视野进行多层扫描,采集Z-stack图像集,Z层步阶为5μm,覆盖至少一半的细胞球体积。高内涵成像结果见图1-图3,图1所示为三荧光通道叠加下,不同浓度的TCE抗体介导的T细胞对肿瘤球体的杀伤效力图像。从左至右抗体的浓度从357nM依次三倍递减至0.1nM,显示蓝色荧光的为肿瘤球体,随着抗体浓度的升高,可以观察到肿瘤体积的减小。红色指示死细胞染料PI的成像,对比同型对照抗体,TCE抗体组的红色荧光信号明显增强,表明TCE抗体组发生了靶细胞的特异性杀伤。图2所示为494nm激发光通道下不同浓度的TCE抗体介导的T细胞对肿瘤球体浸润。从左至右抗体的浓度从357nM依次三倍递减至0.1nM,相较同型对照组,可以观察到TCE抗体组的T细胞浸润程度更高。图3所示为535nm激发光通道下不同浓度的TCE抗体作用下的靶细胞死亡数。
使用Harmony 4.9高内涵成像和分析软件分析3D肿瘤球体积,TCE抗体介导的肿瘤特异性杀伤率=无抗体组肿瘤球体体积-待测抗体处理组肿瘤球体体积/无抗体组肿瘤球体体积*100,结果见图4所示,TCE抗体显示出对肿瘤球体的特异性杀伤,且杀伤效力具有浓度依赖性。
Claims (8)
1.一种应用3D细胞成像分析***检测T细胞杀伤效力的方法,其特征在于包括以下步骤:
1).应用3D细胞培养技术构建三维肿瘤球体;
2).将靶细胞,效应细胞与TCE抗体共培养,使用荧光标记A对靶细胞进行染色标记,使用荧光标记B对效应细胞进行染色标记,使用荧光标记C对共培养后产生的死细胞进行染色标记;
3).应用高内涵成像分析***,分别使用荧光标记A对应的激发光通道、荧光标记B对应的激发光通道和荧光标记C对应的激发光通道对同一视野进行图像采集;
4).应用软件分析***对所得到的图像进行单一或叠加分析,根据肿瘤球体体积的大小计算出T细胞对靶细胞的杀伤效力;
所述效应细胞为人T淋巴细胞或人外周血单核细胞;所述荧光标记A、所述荧光标记B和所述荧光标记C对应的激发光波长均不相同。
2.如权利要求1所述的方法,其中所述荧光标记A为荧光蛋白或/和细胞染料。
3.如权利要求2所述的方法,其中所述荧光蛋白为绿色荧光蛋白或/和红色荧光蛋白。
4.如权利要求1-3任一项所述的方法,其中所述荧光标记B为活细胞染色试剂。
5.如权利要求4所述的方法,其中所述荧光标记B为羧基荧光素二醋酸盐琥珀酰亚胺酯、远红染料、钙黄绿素绿色染料、钙黄绿素紫色染料、钙黄绿素蓝色染料和钙黄绿素橙红色染料中的一种或多种。
6.如权利要求1-3任一项所述的方法,其中所述荧光标记C为碘化丙啶、半胱氨酸蛋白酶、绿菁、7-氨基放线菌素D和膜联蛋白-V中的一种或多种。
7.如权利要求1-3任一项所述的方法,其中所述图像采集包含荧光标记A、荧光标记B和荧光标记C通道中的一种或多种。
8.如权利要求1-3任一项所述的方法,其中步骤4)中的分析方法包括以下步骤:通过分析3D肿瘤球体体积,得到靶细胞特异性杀伤效率;靶细胞特异性杀伤效率=无抗体组肿瘤球体体积-待测抗体处理组肿瘤球体体积/无抗体组肿瘤球体体积*100。
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