CN116593699B - 应用于流式细胞术检测t细胞表面cd39分子的检测方法 - Google Patents
应用于流式细胞术检测t细胞表面cd39分子的检测方法 Download PDFInfo
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Abstract
本发明公开了可应用于流式细胞术检测T细胞表面CD39分子的抗体组合物,该抗体组合物是由均为单克隆抗体的抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体和抗CD39抗体按照1:1:1:1:2的体积比混合的混合物。可为后续临床的治疗提供完整精准的个体信息,从而实现个体化精准化的治疗。
Description
技术领域
本发明涉及流式检测技术领域,具体涉及应用于流式细胞术检测 T 细胞表面CD39 分子的检测方法。
背景技术
免疫系统是机体执行免疫应答及免疫功能的重要系统,具有监视、防御和调控的作用。免疫系统不仅能够识别外来入侵的病原体,同时也能够识别和清除体内发生突变的肿瘤细胞、衰老细胞等其他有害成分。正常情况下发生突变的肿瘤细胞都会被机体的免疫系统识别并且清除,但是肿瘤患者体内的肿瘤细胞由于肿瘤微环境的复杂性,使其逃脱了机体的免疫监视,进而发生发展甚至转移。如何使机体有效识别肿瘤细胞并对其进行清除是肿瘤治疗的核心问题之一。机体的免疫系统由多种成分构成,包括免疫器官、免疫细胞和免疫分子。淋巴细胞作为主要的免疫细胞,在免疫系统发挥功能的过程中起重要作用。淋巴细胞简单的我们可以划分为CD3阳性T细胞,B淋巴细胞和NK细胞,其中CD3阳性T细胞又可以分为CD4阳性T细胞和CD8阳性T细胞。再往下细分我们又可以将CD4阳性T细胞分为辅助性T细胞,记忆型T细胞,调节性T细胞等等。此外巨噬细胞、DC细胞和MDSC细胞也是免疫细胞的重要成员,协助免疫系统发挥功能。肿瘤免疫微环境是一个非常复杂的内环境,不仅包括肿瘤细胞,免疫细胞,内皮细胞等其他细胞,还包括各种细胞因子和基质成分。通过流式细胞术检测肿瘤患者外周血中CD3阳性T细胞,CD4阳性T细胞,CD8阳性T细胞,B淋巴细胞和NK细胞来评估肿瘤患者的一个免疫功能状态,从而为临床提供有用的信息。T淋巴细胞介导的细胞免疫是机体主动的免疫应答,T淋巴细胞具有多种生物学功能,如直接杀伤靶细胞、辅助B细胞产生抗体以及产生细胞因子等。而CD8阳性T细胞是细胞毒性T细胞,能够直接杀伤靶细胞。细胞毒性T细胞作为机体免疫系统杀伤肿瘤细胞的主要执行者,只有这类淋巴细胞基数达到一定数量时,机体的免疫系统才有可能去杀伤肿瘤细胞;而如果这类细胞的数量基数都很低,机体的免疫系统就无法去杀伤肿瘤细胞,从而影响后续的治疗包括一些免疫单抗治疗,如PD-1,CTLA-4等。外周血淋巴细胞亚群虽然不能够准确反应肿瘤原位中淋巴细胞亚群的状态,但是外周血样本取材方便,检测准确的特点,同时同一患者外周血和肿瘤原位淋巴细胞亚群变化具有一致性,使得外周血淋巴细胞亚群分析成为目前最为可行的评估肿瘤患者免疫状态。
肿瘤免疫学研究表明,癌症的发生、发展与机体的免疫状态密切相关。其中T淋巴细胞是细胞免疫的主要应答形式,在机体抗肿瘤免疫中起主导作用,能够识别和清除肿瘤细胞。代谢组学和免疫学一直是医学生物学的前沿领域。已有文献报道在肿瘤免疫微环境中腺苷是一个较为重要的免疫抑制分子,它能够促进调节性T细胞的生成,同时影响免疫微环境中的其他免疫细胞,从而促进肿瘤细胞的逃逸。而CD39作为腺苷代谢途径中的起始分子,在腺苷产生过程中发挥着重要作用。
随着靶向治疗等新疗法的不断出现,使癌症有了可治愈的希望,目前治疗方法除传统的化疗及靶向药物外,免疫治疗已成为新的发展方向。多种免疫检查点抗体药物的临床应用为癌症的临床治疗提供了新的方案,也为肿瘤患者带去了福音。而CD39分子作为腺苷代谢途径中的关键分子,具备了成为一种新型免疫检查点的潜力。
流式细胞术(Flow Cytometry,FCM)是一种能够对单个细胞实现定量分析的检测手段,具有快速、高精度、多参数等优点,是目前最先进的细胞定量分析方法之一。FCM在临床及科研领域都发挥着重要作用,特别在临床体外诊断已有非常广泛和深入的应用,成为最主流的检测手段之一。
利用FCM对人体外周血T细胞表面分子CD39,使用特定的抗体组合物,配合多色流式细胞仪,可以有效检测其表达水平。然而,抗体组合物的正确选择是实现上述目的的关键所在。
发明内容
鉴于现有技术中的上述缺陷或不足,期望提供抗体组合物及其在检测T细胞表面分子CD39表达水平的应用。
根据本申请实施例提供的技术方案,应用于流式细胞术检测 T 细胞表面 CD39分子的检测方法,该抗体组合物是由均为单克隆抗体的抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体和抗CD39抗体按照1:1:1:1:2的体积比混合的混合物。
本发明中,进一步的,各抗体均为荧光素标记的抗体;抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体、抗CD39抗体,抗体的荧光素标记按顺序分别为:FITC、PE、PerCP-Cy5.5、PE-Cy7、APC。
抗体组合物在所述检测T细胞表面CD39分子的过程包括以下步骤:
(1)将待测样本加入到流式管中,使呈单个细胞悬液状态,并保证 细胞量1×106/管-1×107/管;所述待测样本为外周血或组织单细胞悬液;
(2)将步骤(1)处理得到的样本,流式管中加入所述的抗体组合物中的抗体,混匀后室温避光孵育;
(3)向步骤(2)孵育后的流式管中加入1×溶血素,室温避光孵育;
(4)向步骤(3)孵育后的流式管中加入PBS缓冲液洗涤,离心后去上清,用PBS缓冲液重悬细胞;
(5)对步骤(4)重悬细胞进行流式细胞上机检测,样本选择CD45、CD3、CD4、CD8抗体分别组合SSC设门,筛选CD3+CD4+辅助型T细胞和CD3+CD8+杀伤型T细胞,进行CD39分子表达水平分析。
综上所述,本申请的有益效果:使用多种标志组合设门,可以准确检测所有T细胞表面CD39分子的表达水平。CD39作为腺苷代谢途径中的重要分子,可能对人体的免疫系统具有重要影响作用,是一个潜在的可能的治疗靶点。可为后续临床的治疗提供完整精准的个体信息,从而实现个体化精准化的治疗。
附图说明
通过阅读参照以下附图所作的对非限制性实施例所作的详细描述,本申请的其它特征、目的和优点将会变得更明显:
图1为SSC-peak和SSC-int设门,去粘连细胞P1的示意图;
图2是在P1门下CD45/SSC设门,找到Lym细胞门;
图3是在Lym门下CD3/SSC设门,找到CD3+ T细胞门;
图4是在CD3门下,CD4/CD8设门,圈出CD4+T细胞门,CD8+T细胞门;
图5是在CD4门下,CD4/CD39设门,圈出CD4+CD39+门;
图6是在CD8门下,CD8/CD39设门,圈出CD8+CD39+门;
图7是在CD4门下,显示CD39的二维图,其中图(b)的线为对照组,图(a)的线为实验组;
图8是在CD8门下,显示CD39的二维图,其中图(b)的线为对照组,图(a)的线为实验组。
具体实施方式
下面结合附图和实施例对本申请作进一步的详细说明。可以理解的是,此处所描述的具体实施例仅仅用于解释相关发明,而非对该发明的限定。另外还需要说明的是,为了便于描述,附图中仅示出了与发明相关的部分。
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本申请。
腺苷代谢途径是指ATP和其他核苷酸先水解为AMP(磷酸腺苷)并最终生成腺苷的过程。胞外核苷酸的水解由几类核苷酸酶控制,其中包括胞外核苷三磷酸二磷酸水解酶(ENTPDases,基因名称为ENTPD)、外核苷酸焦磷酸磷酸二酯酶(E-NPPs)、NAD糖水解酶、碱性磷酸酶、核苷二磷酸激酶和ecto-F1-Fo ATP合酶。CD39是ENTPD的原型,是第一个被测序和克隆的NTPDase酶。与所有的NTPDase一样,CD39具有5个高度保守的序列结构域,被称为“焦磷酸酶保守区”,它们能够通过磷酸化水解参与活性位点的形成和胞外核苷酸的催化。CD39通过两个跨膜结构域锚定在细胞膜上,这两个跨膜结构域对于维持催化活性和底物特异性至关重要。CD39能够发生功能性修饰,包括蛋白水解和糖基化。而糖基化是赋予CD39催化活性的决定因素。CD39的N端胞内结构域能够发生棕榈酰化,使酶与脂筏结合。通过药物来干扰胆固醇水平的实验,无论是消耗或隔离膜胆固醇,对CD39的胞外酶活性都显示出强烈的抑制作用。CD39是一个完整的膜蛋白,在钙离子和镁离子存在的条件下能够磷酸水解ATP或者ADP,生成AMP。人类的CD39分子是一个由510个氨基酸构成的蛋白质,具有7个N连接糖基化位点,11个半胱氨酸残基和2个跨膜区域。结构上来说,CD39具有两个跨膜结构域,胞内结构域由NH2-和COOH-末端片段组成,胞外则是一个大的疏水结构域,其中包括5个高度保守的区域被称为腺苷三磷酸双膦酸酶保守区域(ACRs)1-5号,这些区域对酶的分解代谢活性至关重要。ACR1和ACR4的氨基酸序列中含有一个磷酸结合基序(DXG),该基序被证明在磷酸裂解过程中对于稳定酶与其核苷酸底物之间的相互作用至关重要。CD39在细胞表面定位后具有催化活性,其糖基化对于蛋白质正确折叠、膜靶向和酶活性至关重要。
已有文献报道在肿瘤免疫微环境中腺苷是一个较为重要的免疫抑制分子,它能够促进调节性T细胞的生成,同时影响免疫微环境中的其他免疫细胞,从而促进肿瘤细胞的逃逸。而CD39作为腺苷代谢途径中的起始分子,在腺苷产生过程中发挥着重要作用。CD39作为腺苷代谢途径中主要的限速酶,能够水解ATP/ADP,从而在肿瘤微环境中发挥重要的作用。CD39一直被作为一个免疫抑制分子在多种肿瘤的发生发展中被广泛研究。但是随着研究的不断深入,越来越多的证据表明在不同细胞表面CD39分子具有多种的生物学功能。有研究显示,CD39分子表达于T细胞表面,尤其是肿瘤细胞浸润的CD8+ T细胞表明,从而使临床获益。也有研究报道CD39分子的表达能够鉴别CD8+ T细胞是否耗竭,同时其他研究也证实了CD39和PD-1分子能够共表达于T淋巴细胞表面。而CD39分子能够诱导调节性T细胞转化和生成。已经由研究报道在非小细胞肺癌肿瘤组织中的免疫细胞上CD39分子和PD-1分子共表达。我们的研究也发现,在多种肿瘤中(包括肺癌、宫颈癌和肉瘤等),患者外周血中T细胞同时表达CD39分子和PD-1。通过进一步的机制探索,我们发现在肿瘤患者体内,腺苷代谢途径的抑制作用主要包括两部分,第一部分代谢生成腺苷,从而抑制局部免疫功能;第二部分CD39参与腺苷代谢途径,而这部分表达于T细胞表面的CD39分子在参与腺苷代谢的过程中,在水解ATP/ADP的时候,CD39分子能够通过胞内部分传递某种信号通路从而使T细胞增加PD-1的表达水平同时也降低了共刺激信号CD28的表达水平。因此CD39的表达可能更早于PD-1分子。
随着靶向治疗等新疗法的不断出现,使癌症有了可治愈的希望,目前治疗方法除传统的化疗及靶向药物外,免疫治疗已成为新的发展方向。多种免疫检查点抗体药物的临床应用为癌症的临床治疗提供了新的方案,也为肿瘤患者带去了福音。尤其是PD-1单抗的应用,大大提高了肿瘤患者的生存水平。而CD39分子作为腺苷代谢途径中的关键分子,在肿瘤免疫过程中起到了非常重要的作用,作为PD-1表达的诱导分子,其表达早于PD-1的表达,因此CD39具备了成为一种新型免疫检查点的潜力。本发明通过检测外周血中T细胞CD39分子的表达水平,能够及时为临床提供更全面准确的患者免疫信息。从而实现临床治疗的精准化和个体化。
因此,本方案提供了一种可应用于流式细胞术检测T细胞表面CD39分子的抗体组合物,其中包括:抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体和抗CD39抗体;本方案的抗体组合物可应用于流式细胞术检测T细胞表面CD39分子表达水平。具体应用时 ,使用抗体组合的方案,通过使用CD45、CD3、CD4和CD8抗体分别和SSC组合设置全部T细胞门 ,可以有效设门选出T细胞,其中包括CD3+CD4+的辅助T细胞和CD3+CD8+的杀伤T细胞,通过检测这些T细胞亚群表面CD39分子的表达水平,结合其他相关免疫检查,从而能够有效描绘患者个体化的免疫图谱,从而为后续的精准化的临床治疗方案制定提供全面的信息和数据支持。
本方案的抗体组合物中,各抗体均为荧光素标记的抗体。优选地,抗体组合中抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体、抗CD39抗体的荧光素标记按顺序分别为:FITC、PE、PerCP-Cy5 .5、PE-Cy7、APC。本方案中通过对不同抗体搭配特定的荧光素,可以使得本发明的抗体组合物应用于流式细胞术检测T细胞表面CD39分子表达水平,各通道的所有荧光素都能达到优异的染色效果。
本方案的抗体组合物中,各抗体组分均可商购获得。 各抗体应符合相关行业标准要求。
本方案的抗体组合物中,抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体和抗CD39抗体按照1:1:1:1:2的体积比混合(在效价基本相当的情况下)的混合物。
本方案的抗体组合物可应用于试剂盒中,试剂盒中包括:红细胞裂解液、缓冲液、与流式细胞仪配套使用的流式管中的一种或多种。这些试剂和耗材均可商购获得。各试剂材料可分别容置于不同的容器中。
该抗体组合物在试剂盒中的使用的实施例如下:
一、试剂的配制
本实施例使用的抗体组合为,
CD45-FITC、CD3-PE、CD4-PerCP-Cy5 .5、CD8-PE-Cy7、CD39-APC,取以上五种单克隆抗体试剂按体积比1:1:1:1:2混合装于第一容器中;
任选再配红细胞裂解液装于第二容器中,PBS缓冲液装于第三容器中,红细胞裂解液、PBS缓冲液为可商购获得,其中细胞裂解液和PBS缓 冲液为 Beckman Coulter公司产品。
二、标本的处理
按照细胞计数结果,将肝素或EDTA抗凝的骨髓或外周血样本,加入到流式管中,保证加入的细胞量约为2×106个,再按表1向流式管中加入15μl不同荧光素标记的五种胞膜单克隆抗体试剂,与细胞悬液充分混匀后常温避光孵育15分钟,加入0.5ml 1×溶血素,避光孵育10分钟裂解红细胞,1500rpm离心5分钟,最后加入3ml PBS缓冲液洗涤,离心后去上清,用0 .5ml PBS缓冲液重悬细胞,即为处理好的标本,可供上机检测。
三、标本的检测
将标本按照上述“标本的处理”的方法进行标本处理,在 Beckman Coulter公司2激光8色 DxFlex流式细胞仪上机检测,优选获取每管10万细胞(建议至少1万)后,使用kaluza软件分析数据。
其中,流式细胞上机检测时按照以下方式设门:
①固定设门:依次进行去除黏连细胞门、淋巴细胞门;②多标志组合设门:从淋巴细胞开始进行,依次圈出CD3+、CD4+和CD8+的T细胞门,在此基础上再看各T细胞群上CD39分子的表达水平。
固定设门:由去黏连细胞门、淋巴细胞门组成。呈串联关系。
去黏连细胞门:通过SSC-面积(Area,A)/高度(Height,H)可以去除粘连细胞,原理是细胞是球形,A与H成正相关。(见图1)。
淋巴细胞门 :通过CD45/SSC将各群血细胞进行大致区分,原理是根据造血细胞CD45 表达荧光强度不同(成熟淋巴细胞>单核细胞>粒细胞>有核红细胞),SSC大小差异(粒细 胞>单核细胞>成熟淋巴细胞>有核红细胞)(见图2)。
本发明的标志组合,可以精确锁定T细胞。 根据淋巴细胞门,可圈出CD3阳性T细胞(见图3),再依次圈出CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞(见图4)。
在CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞门下,分别查看其表面分子CD39的表达水平。本发明中提供了CD39表达的散点图和二维图,其中图5和图7为CD3+CD4+辅助T细胞的CD39分子表达水平,散点图上可圈出CD3+CD4+CD39+部分,二维图图7中图(b)的线为对照组,图(a)的线为实验室组。同样图6和图8为CD3+CD8+杀伤T细胞的CD39分子表达水平,散点图上可圈出CD3+CD8+CD39+部分,二维图图8中图(b)的线为对照组,图(a)的线为实验室组。
本实施例提供正常一例标本作为示例。图1-图8为标本,除了常规P1(图1)、CD45/SSC设门(图2)以外,分别使用胞浆CD3/SSC设门(图3)、CD4/CD8设门(图4)。图5-图8为CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞表面CD39分子表达水平的散点和二维示例图。
具体而言,图1:正常标本,SSC-A/H设置P1为去 黏连细胞门 ,得到P1内为单个细胞。图2:CD45/SSC设置血细胞门,得到淋巴细胞门(lym)。图3:使用CD3/SSC设置T细胞门(CD3+T细胞),得到T细胞群。图4:在CD3+T细胞门下利用抗体组合CD4/CD8分别显示CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞。图5:在CD3+CD4+辅助T细胞门下,利用抗体组合CD4/CD39,圈出CD3+CD4+CD39+辅助T细胞,从而检测CD3+CD4+辅助T细胞表面CD39分子的表达水平。图6:在CD3+CD8+杀伤T细胞门下,利用抗体组合CD8/CD39,圈出CD3+CD8+CD39+杀伤T细胞,从而检测CD3+CD8+杀伤T细胞表面CD39分子的表达水平。图7:在CD3+CD4+辅助T细胞门下,二维图查看CD3+CD4+辅助T细胞表面CD39分子的表达水平,其中图(b)线为对照组,图(a)线为实验组。图8:在CD3+CD8+杀伤T细胞门下,二维图查看CD3+CD8+杀伤T细胞表面CD39分子的表达水平,其中图(b)线为对照组,图(a)线为实验组。
以上描述仅为本申请的较佳实施例以及对所运用技术原理等方案的说明。同时,本申请中所涉及的发明范围,并不限于上述技术特征的特定组合而成的技术方案,同时也应涵盖在不脱离所述发明构思的情况下,由上述技术特征或其等同特征进行任意组合而形成的其它技术方案。例如上述特征与本申请中公开的(但不限于)具有类似功能的技术特征进行互相替换而形成的技术方案。
Claims (1)
1.应用于流式细胞术检测 T 细胞表面 CD39 分子的检测方法,其特征是,由均为单克隆抗体的抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体和抗CD39抗体按照1:1:1:1:2的体积比混合组成的组合物,各抗体均为荧光素标记的抗体;抗CD45抗体、抗CD3抗体、抗CD4抗体、抗CD8抗体、抗CD39抗体,抗体的荧光素标记按顺序分别为:FITC、PE、PerCP-Cy5.5、PE-Cy7、APC;
然后按照细胞计数结果,将肝素或EDTA抗凝的骨髓或外周血样本,加入到流式管中,保证加入的细胞量约为2×106个,再按上述组合物的体积比向流式管中加入15μl不同荧光素标记的五种胞膜单克隆抗体试剂,与细胞悬液充分混匀后常温避光孵育15分钟,加入0.5ml1×溶血素,避光孵育10分钟裂解红细胞,1500rpm离心5分钟,最后加入3ml PBS缓冲液洗涤,离心后去上清,用0 .5ml PBS缓冲液重悬细胞,即为处理好的标本,然后上机检测;
所述检测方法如下:
A1.通过所述组合物精确锁定T细胞,根据淋巴细胞门,圈出CD3阳性T细胞,再依次圈出CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞;
A2.在实现步骤A1后,分别查看T细胞表面分子CD39的表达水平;
A3.通过P1、CD45/SSC设门及使用胞浆CD3/SSC设门、CD4/CD8设门,从而检测出CD3+CD4+辅助T细胞以及CD3+CD8+杀伤T细胞表面CD39分子表达水平的散点;
A4.通过以上A1-A3步骤,得到以下结果;
a).在CD3+CD4+辅助T细胞门下,利用抗体组合CD4/CD39,圈出CD3+CD4+CD39+辅助T细胞,从而检测CD3+CD4+辅助T细胞表面CD39分子的表达水平;
b).在CD3+CD8+杀伤T细胞门下,利用抗体组合CD8/CD39,圈出CD3+CD8+CD39+杀伤T细胞,从而检测CD3+CD8+杀伤T细胞表面CD39分子的表达水平;
c).在CD3+CD4+辅助T细胞门下,二维图查看CD3+CD4+辅助T细胞表面CD39分子的表达水平;
d).在CD3+CD8+杀伤T细胞门下,二维图查看CD3+CD8+杀伤T细胞表面CD39分子的表达水平。
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