CN116875604A - 一种用于pd-l1阳性细胞检测的dna适配体及其应用 - Google Patents
一种用于pd-l1阳性细胞检测的dna适配体及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及一种DNA适配体及利用流式细胞仪高灵敏度检测PD‑L1阳性细胞的方法与应用。本发明提供的核苷酸适配体序列特异性高,对PD‑L1阳性细胞有较强的亲和力,本发明提供的核酸适配体合成简单,成本低,分析过程简单方便,所耗时间短。本发明提供的一种可用于检测PD‑L1阳性细胞的方法,该方法设计的核酸适配体上修饰有荧光基团,既可在荧光显微镜下观察,也可在流式细胞仪上分析,在流式细胞仪分析具有灵敏度高的特点,同时具有定量分析的特点。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种DNA适配体及利用流式细胞仪高灵敏度检测PD-L1阳性细胞的方法与应用。
背景技术
与组织活检相比较,液体活检对于癌症的动态检测和指导用药等方面有着重要意义。PD-L1广泛表达于癌细胞细胞膜表面,其表达含量与癌细胞对免疫抑制剂敏感性有直接关联,其表达量对免疫监测点的治疗及其预后都有着重要的临床意义。PD-L1组织免疫组化分析法是目前较为公认的检测方法。但这种方法的缺点是组织获取困难、有创伤、不能实时检测体内PD-L1表达,因此及时开发一种无创、高效、简便、低成本的分析方法极其具有临床价值。
核酸适配体是指利用SELEX技术从核酸分子文库中得到的寡核苷酸序列,其能与生物靶标等稳定地高特异性地结合。核酸适配体与可和蛋白靶标相结合的抗体相比较:DNA适配体具有以下优势:第一,适配体具有很高的结合特异性和亲和力。第二,没有免疫原性。第三,化学结构稳定,没有毒性,便于存储。第四,适配体序列分子量小,可以在体外低成本大量快速的合成,同时保持高再现性和可靠性。第五,核酸结构灵活多变,可与不同的荧光基团结合,从而得以实现高灵敏度的定量检测。
流式细胞仪是通过测量流体中单个微观颗粒或生物细胞的物理或化学信号,从而实现对单个微粒或细胞进行分析的仪器。流式细胞仪是一种在临床诊断和细胞生物学广泛使用的细胞分析与分选工具。流式细胞仪可以通过采集荧光信号来测定细胞信息,实现实时、简便、无损伤的检测。
将DNA适配体应用于目标细胞检测是本领域近年来新的研究方向。利用DNA适配体可以实现高效快速地检测特定细胞,此外DNA适配体分子量小,合成简单,稳定性好,可低成本快速大量合成,同时DNA序列可修饰不同的荧光基团,为流式细胞仪信号检测奠定了基础。
发明内容
本发明的主要目的是针对现有技术存在的不足,提供一种DNA适配体及基于DNA适配体的PD-L1阳性细胞在流式细胞仪上的检测方法。所述的DNA适配体,可以特异性强、灵敏度高的识别PD-L1阳性细胞,然后利用流式细胞仪实现PD-L1阳性细胞的高灵敏检测。所述检测方法检测时间短,灵敏度高,特异性强,同时涉及的试剂耗材易得、检测成本低,具有很高的临床应用价值。
为实现上述发明目的,本发明包括如下技术内容:
本发明第一方面,提供一种用于PD-L1阳性细胞检测的DNA适配体,所述适配体序列为SEQ ID NO:1:
SEQ ID NO:1(5‘-3’):ACGGGCCACATCAACTCATTGATAGACAATGCGT CCACTGCCCGTGGGGGGTTACCTGGGAAGTCTAACTAAGTCTAAATCTACGACG
进一步地,所述序列SEQ ID NO:1其中5’端修饰有荧光基团FAM(绿色荧光)。
通过将用于识别PD-L1细胞的DNA适配体修饰上荧光基团,单链DNA适配体会与PD-L1阳性细胞特异性结合,这种荧光信号可被流式细胞仪识别并实现高灵敏度分析,上述适配体在临床检测PD-L1阳性细胞方面具有潜在应用前景。
本发明第二方面,提供一种基于DNA适配体荧光传感的PD-L1阳性细胞的检测方法,其步骤包括:
(1)细胞培养:将肿瘤细胞置于水浴锅中复苏,于培养基中培养,放置在二氧化碳培养箱中培养;
(2)制备单细胞悬液:将培养后的细胞用PBS清洗,之后用胰酶消化至镜下观察80%细胞脱落,1000rpm转速离心,弃去上清,加入PBS溶液悬浮;
(3)加入DNA适配体探针:在上述细胞悬液中,加入DNA适配体溶液,避光放置30-40min,中间混匀两次;
(4)流式细胞仪分析:将上述溶液离心,弃去上清,PBS清洗,重复两次,最后用PBS溶液悬浮细胞;流式细胞仪上机分析荧光强度与细胞个数的相关性,同时分析峰面积。
进一步地,步骤1中所述的肿瘤细胞选自HCC827、Hela、乳腺癌细胞、肺癌细胞、宫颈癌细胞等;优选为HCC827、Hela、乳腺癌细胞MCF-7。
进一步地,步骤1中所述的DNA适配体稀释浓度为10mmol/L。
进一步地,步骤1中所述的水浴锅的温度为37℃。
进一步地,步骤1中所述的培养基为DMEM高糖培养基,也可以根据培养细胞类型改变。
进一步地,步骤1中所述的培养箱的温度为37℃,培养时间为36-48h。
进一步地,步骤2中所述的清洗使用PBS清洗2-3次。
进一步地,步骤2中所述的胰酶加入量为1-2mL。
进一步地,步骤2中所述的离心条件为1000rpm/min离心3-5min。
进一步地,步骤2中所述的PBS溶液悬浮时的加入量为100ul。
进一步地,步骤3中所述的DNA适配体适配体序列为SEQ ID NO:1。
进一步地,步骤3中所述的悬浮液与DNA适配体溶液的体积比为5:1。
进一步地,步骤3中所述的DNA适配体溶液的浓度为10mmol/L。
进一步地,步骤3中所述的DNA适配体和细胞结合时间为30-40min。
进一步地,步骤3中所述的细胞悬液浓度不高于1×107cells/mL。
进一步地,步骤4中所述的离心条件为1000rpm/min离心3-5min。
进一步地,步骤4中所述的悬浮液加入量为1ml。
与现有技术相比,本发明的优点及有益效果如下:
(1)本发明提供的核苷酸适配体序列特异性高,对PD-L1阳性细胞有较强的亲和力,且有很好的特异性。细胞结合对照实验表明该适配体可特异性结合PD-L1阳性细胞,不与PD-L1阴性细胞结合。
(2)本发明所述的一种可用于检测PD-L1阳性细胞的方法,该方法设计的核酸适配体上修饰有荧光基团,既可在荧光显微镜下观察,也可在流式细胞仪上分析,在流式细胞仪分析具有灵敏度高的特点,同时具有定量分析的特点。
(3)本发明所述的核酸适配体序列一端修饰有荧光基团,可根据需要更改,具有灵活性、广泛性的特点。
(4)本发明提供的核酸适配体合成简单,成本低,分析过程简单方便,所耗时间短。
附图说明
图1. 本发明的核苷酸适配体序列琼脂糖凝胶电泳图。
图2. 本发明的核苷酸适配体序列二级结构分析图。
图3. 核酸适配体和PD-L1阳性细胞特异性结合的荧光共聚焦显微镜分析图。
图4. 核酸适配体结合PD-L1阳性细胞的流式细胞仪分析结果图。
图5. 核酸适配体特异结合不同浓度PD-L1阳性细胞的定量分析图。
具体实施方式
下面将以实施例的方式对本申请作进一步的详细描述,以使本领域技术人员能够实践本申请。应当理解,可以采用其他实施方式,并且可以做出适当的改变而不偏离本申请的精神或范围。为了避免对于使本领域技术人员能够实践本申请来说不必要的细节,说明书可能省略了对于本领域技术人员来说已知的某些信息。因此,以下详细描述不应以限制性的意义来理解,且本发明的范围仅由所附权利要求界定。以下的实施例便于更好地理解本申请,但并不用来限制本申请的范围。
本发明所用材料如下:
胎牛血清、DMEM高糖培养基、胰酶、抗生素、PBS购于Hyclone(USA);
本实验所用到的细胞有HCC827、hela等,购于中科院细胞库;
本实验所用到的DNA序列合成于上海生工,合成方法为PAGE法。PAGE法是使用变性聚丙烯酰胺凝胶电泳,对寡核苷酸序列进行分离纯化,是寡核苷酸序列合成常用办法之一,尤其适用于修饰引物的纯化。寡核苷酸序列合成常用方法包括OPC法、HPLC法、PAGE法和HAP法。OPC法、HPLC法和HAP法适用于短链寡核苷酸序列的合成,不适用于该实验用到的长链寡核苷酸序列的合成。另一方面,本实验用到的序列末端修饰有FAM荧光基团。PAGE法合成寡核苷酸序列的优点包括合成纯度高,纯度大于90%,对于长度大于60mer的末端修饰长链寡核苷酸序列纯化效率高,因此本实验选用PAGE法合成DNA序列。
其他所用的材料、试剂等,如无特定说明,均从商业途径获得。
实施例1
培养细胞中PD-L1阳性细胞的实时检测,其特征在于包括如下步骤:
(1)细胞培养:将购买来的人乳腺癌细胞MCF-7(或其他类型细胞),于37℃的加湿二氧化碳培养箱培养。所用培养基为含有10%胎牛血清和10000IU/ml青霉素和1000ug/ml链霉素的高糖DMEM;
(2)细胞悬液:将培养细胞用预热的PBS清洗2-3次,之后使用预热的胰酶消化3-5min,镜下观察细胞自然脱落80%,停止胰酶消化;细胞液于转速1000rpm离心5min,弃掉上清液。预热PBS溶液100ul悬浮细胞,细胞悬液制成;
(3)荧光探针:本实验用到的适配体序列用ddH2O溶解为浓度为10mmol/L,使用琼脂糖凝胶电泳表征,确认寡核苷酸序列长度无误,如图1所示,左边条带为DNA marker, 右边条带为适配体序列。为验证设计的适配体序列没有复杂的二级结构,进而可能影响后续和PD-L1阳性细胞的特异性结合,利用DNAman软件分析了该序列的二级结构,如图2所示,序列二级结构分析表明可与PD-L1蛋白特异结合的有效序列,没有与其他部分互补配对,不影响序列与PD-L1蛋白的结合力。为表征该序列与PD-L1阳性细胞的亲和力高,以图3为例说明,图3所示,以MCF-7细胞为例,图3左图为白光下视野,右图为绿色荧光通道下视野,结果显示荧光探针可以与该细胞特异性结合。于治好的细胞悬液中,加入20ul的10mmol/L带有FAM标记的核酸适配体单链DNA;操作过程轻柔,避免产生气泡,室温避光放置1h;
(4)流式细胞仪上机:将上述溶液上机,于流式细胞仪分析荧光信号。图4为流式细胞仪分析结果,四个小图从左到右的细胞浓度依次为1,10,100,1000 cells/ml。由此图可以看出跟随细胞浓度增高,细胞荧光强度依次加大,峰面积也依次增大。图5分析了不同细胞浓度与流式细胞仪分析结果图中的峰面积的线性相关性,分析结果显示线性拟合率R2=0.99。细胞浓度与流式细胞仪结果图中的峰面积有着很好的线性相关性。可利用建立线性相关性的方法,实现定量分析PD-L1阳性细胞的数目。
实施例2
一种循环肿瘤细胞中PD-L1阳性细胞的流式细胞仪检测
一种循环肿瘤细胞中PD-L1阳性细胞的检测方法,其特征在于包括如下步骤:
(1)采集人外周血:使用EDTA-K2抗凝采血管采集6.0ml血液,并立即轻柔颠倒混匀8次;
(2)将采血管配平,颠倒混匀后,室温离心15分钟,弃掉上清液,加入清洗液补至采血管中液体浓度到6ml,上下颠倒混匀;
(3)取一新的50ml离心管,加入3ml样本密度分离液。将上一步骤的采血管轻柔颠倒混匀后,使用电动移液器将采血管中的液体沿离心管管壁缓慢加入到密度梯度分离液上层,尽量避免将红细胞将入到分离液中,室温350g/min离心6min;
(4)离心后离心管中液体分为三层,上层为黄色液体,中间层为透明或淡红色液体,第三层为红色液体,将第一层和第二层中的液体转移至另一50ml离心管中,轻轻晃动;
(5)将适配体探针提前融化,每离心管加入30ul,加入后涡旋混匀,室温共同避光孵育30-40min,期间上下颠倒2次;
(6)将孵育完的液体在常温转速350g离心3-5min,弃去上清液,然后用PBS清洗2次,常温350g离心3-5min,弃去上清,加入500ul PBS重选细胞,上流式细胞仪分析,定量分析PD-L1阳性细胞数量。进而为临床诊断和治疗提供客观依据。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (10)
1.一种用于PD-L1阳性细胞检测的DNA适配体,其特征在于,所述适配体序列为SEQ IDNO:1:
SEQ ID NO:1(5‘-3’):ACGGGCCACATCAACTCATTGATAGACAATGCGT CCACTGCCCGTGGGGGGTTACCTGGGAAGTCTAACTAAGTCTAAATCTACGACG。
2.根据权利要求1所述的DNA适配体,其特征在于,所述序列SEQ ID NO:1其中5’端修饰有荧光基团FAM。
3.一种基于DNA适配体荧光传感的PD-L1阳性细胞的检测方法,其特征在于,其步骤包括:
(1)细胞培养:将肿瘤细胞置于水浴锅中复苏,于培养基中培养,放置在二氧化碳培养箱中培养;
(2)制备单细胞悬液:将培养后的细胞清洗,之后用胰酶消化至镜下观察80%细胞脱落,离心,弃去上清,加入PBS溶液悬浮;
(3)加入DNA适配体探针:在上述细胞悬液中,加入DNA适配体溶液,避光放置30-40min,中间混匀两次;
(4)流式细胞仪分析:将上述溶液离心,弃去上清,PBS清洗,重复两次,最后用PBS溶液悬浮细胞;流式细胞仪上机分析荧光强度与细胞个数的相关性。
4.根据权利要求3所述的检测方法,其特征在于,步骤1中所述的肿瘤细胞肿瘤细胞选自肺癌细胞、宫颈癌细胞、乳腺癌细胞等;优选为HCC827、Hela、乳腺癌细胞MCF-7细胞。
5.根据权利要求3所述的检测方法,其特征在于,步骤1中所述的培养箱的温度为37℃,培养时间为36-48h。
6.根据权利要求3所述的检测方法,其特征在于,步骤2中所述的胰酶每个培养皿加入量为1mL-2 mL。
7.根据权利要求3所述的检测方法,其特征在于,步骤2中所述的离心条件为1000rpm/min离心3-5min。
8.根据权利要求3所述的检测方法,其特征在于,步骤3中所述的悬浮液与DNA适配体溶液的体积比为5:1;所述的DNA适配体和细胞结合时间为30-40min。
9.根据权利要求3所述的检测方法,其特征在于,步骤3中所述的细胞悬液浓度不高于1×107cells/mL。
10.根据权利要求3所述的检测方法,其特征在于,步骤4中所述的离心条件为1000rpm/min离心3-5min;所述的悬浮液加入量为1ml。
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