CN110672500B - 一种非肝素抗凝血液样本的Th检测方法 - Google Patents
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Abstract
本发明公开了一种非肝素抗凝血液样本的Th检测方法，使用淋巴细胞分离液对非肝素抗凝的样本进行处理，获得外周血单个核细胞(PBMC)，去除抗凝剂对细胞活化和细胞因子分泌的影响，同时在刺激/阻断阶段，加入血清，给细胞提供足够的营养物质；经抗体染色、固定破膜后，可成功用于Th细胞因子的检测，从而扩大了Th检测样本类型的范围。可使研究人员或检测人员充分利用样本进行Th检测，避免了因收集了非肝素抗凝的样本造成样本浪费和重复采集，大大提高了效率。
Description
技术领域
本发明涉及一种非肝素抗凝血液样本的Th检测方法。
背景技术
通常所说的Th1、Th2和Th17是指在各种生理与病理条件下有能力分化为Th1、Th2和Th17的T辅助细胞(严格意义上是Th0细胞)。静息状态(即未受任何刺激，如人的正常生理状态)下，Th0分化为Th1、Th2和Th17的能力非常弱，所以外周血中仅含有极少量的Th1、Th2和Th17细胞，这时所能检测到的IFN-γ、IL-4和IL-17A也微乎其微。而当Th细胞受到外界因素(如刺激素、病原体等)刺激，其中Th0即会向Th1、Th2或Th17分化，具体分化趋向取决于细胞因子的种类。此时，检测到的IFN-γ、IL-4或IL-17A也较多。实验中检测的Th1、Th2和Th17实际上是检测Th细胞对刺激素刺激的反应能力。
通常选用的刺激素为Phorbol 12-Myristate 13-Acetate(PMA,佛波酯)和Ionomycin(离子霉素)。其中PMA为PKC(蛋白激酶C)的激活物，PKC则可激活下游众多的蛋白激酶的磷酸化，形成级联反应，导致许多蛋白的表达，进而引起T细胞的活化。在细胞内，PKC可被DAG(二脂酰甘油)和Ca2+的共同作用而激活，因此在Ionomycin(Ca2+的转运剂，可将细胞器内的Ca2+转运至胞浆内)的参与下，T细胞内PKC可被进一步激活。可见，PMA与Ionomycin协同活化T细胞。
活化的T细胞可分泌多种细胞因子至细胞外，而流式细胞仪仅能检测细胞内的抗原，所以应将细胞因子阻断在胞内。细胞因子在高尔基体中合成，某些蛋白通过内质网运输以可溶性形式分泌至细胞外。破坏高尔基体即可切断细胞因子的转运途径，阻断其分泌。通常选用的阻断剂为Brefeldin A(BFA,布雷非德菌素A)和/或Monensin(莫能霉素)。
传统的Th检测方法只能受限于肝素抗凝的全血，而其他类型的抗凝样本(如EDTA抗凝血、柠檬酸钠抗凝血)等则会影响Th细胞的活化和细胞因子的分泌，无法检测到细胞因子，使可测的样本类型受到了限制。对研究人员和检测人员来说，就会造成样本浪费或需重新采集的问题，大大影响效率。
发明内容
针对现有技术的不足，本发明提供了一种非肝素抗凝血液样本的Th检测方法，旨在解决非肝素抗凝血液样本在Th检测时，会影响Th细胞的活化和细胞因子的分泌，无法检测到细胞因子，使可测的样本类型受到了限制，造成样本浪费或需重新采集的问题。
为实现上述目的，本发明提供如下技术方案：一种非肝素抗凝血液样本的Th检测方法，包括以下步骤：
步骤一：用淋巴细胞分离液分离外周血单个核细胞(PBMCs)，用含10％胎牛血清的培养基重悬沉淀，使细胞浓度为1×107/ml，取250μl PBMCs至流式管中，加入1μl PMA/Ionomycin Mixture(250×)和1μl BFA/Monensin Mixture(250×)，以只含PBMCs的样本作为对照，混匀，37℃孵育4-6小时，每隔1-2小时取出震荡混匀；
步骤二：从样本管和对照管中取100μl细胞悬液至新的流式管中，加入相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤三：每管加入100μl FIX&PERM MediumA，震荡混匀，室温避光孵育15分钟；
步骤四：用蒸馏水将10×Flow Cytometry Staining Buffer稀释为1×，每管加入2ml预冷1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清；
步骤五：每管加入100μl FIX&PERM Medium B和相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤六：每管加入2ml 1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清。
步骤七：每管加入500μl 1×Flow Cytometry Staining Buffer重悬，上机检测；或者加入500μl 1-4％多聚甲醛重悬，2-8℃避光，于24小时内检测。
进一步的，对于人血液样本，所述步骤一中采用人淋巴细胞分离液，步骤二中流式抗体为5μl Anti-Human CD3,FITC和5μl Anti-Human CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μl Anti-Human IFN-γ,PE和5μl Anti-Human IL-4,APC。
进一步的，对于小鼠血液样本，所述步骤一中采用小鼠淋巴细胞分离液，步骤二中流式抗体为5μl Anti-Mouse CD3ε,FITC和5μl Anti-Mouse CD4,PerCP-Cy5.5，步骤五中流式抗体为加入5μlAnti-Mouse IFN-γ,PE。
进一步的，对于大鼠血液样本，所述步骤一中采用大鼠淋巴细胞分离液，步骤二中流式抗体为5μl Anti-Rat CD3,FITC和5μl Anti-Rat CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μlAnti-Rat IFN-γ,efluor660。
本发明的有益效果：使用淋巴细胞分离液对非肝素抗凝的样本进行处理，获得外周血单个核细胞(PBMC)，去除抗凝剂对细胞活化和细胞因子分泌的影响，同时在刺激/阻断阶段，加入血清，给细胞提供足够的营养物质；经抗体染色、固定破膜后，可成功用于Th细胞因子的检测，从而扩大了Th检测样本类型的范围。
可使研究人员或检测人员充分利用样本进行Th检测，避免了因收集了非肝素抗凝的样本造成样本浪费和重复采集，大大提高了效率。
附图说明
图1为本发明实施例1的Th检测结果图：
图1.1为人EDTA抗凝全血的Th检测结果：
A0：未刺激的人EDTA抗凝血来源的PBMCs染色IFN-γ；
A1：PMA/Ionomycin刺激和BFA/Monensin阻断的人EDTA抗凝血来源的PBMCs染色IFN-γ。
图1.2为小鼠EDTA抗凝全血的Th检测结果：
A0：未刺激的小鼠EDTA抗凝血来源的PBMCs染色IFN-γ；
A1：PMA/Ionomycin刺激和BFA/Monensin阻断的小鼠EDTA抗凝血来源的PBMCs染色IFN-γ。
图1.3为大鼠EDTA抗凝全血的Th检测结果：
A0：未刺激的大鼠EDTA抗凝血来源的PBMCs染色IFN-γ；
A1：PMA/Ionomycin刺激和BFA/Monensin阻断的大鼠EDTA抗凝血来源的PBMCs染色IFN-γ。
具体实施方式
实施例1
一种非肝素抗凝血液样本的Th检测方法，包括以下步骤：
步骤一：用淋巴细胞分离液分离外周血单个核细胞(PBMCs)，用含10％胎牛血清的培养基重悬沉淀，使细胞浓度为1×107/ml，取250μl PBMCs至流式管中，加入1μl PMA/Ionomycin Mixture(250×)和1μl BFA/Monensin Mixture(250×)，以只含PBMCs的样本作为对照，混匀，37℃孵育4-6小时，每隔1-2小时取出震荡混匀；
步骤二：从样本管和对照管中取100μl细胞悬液至新的流式管中，加入相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤三：每管加入100μl FIX&PERM MediumA，震荡混匀，室温避光孵育15分钟；
步骤四：用蒸馏水将10×Flow Cytometry Staining Buffer稀释为1×，每管加入2ml预冷1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清；
步骤五：每管加入100μl FIX&PERM Medium B和相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤六：每管加入2ml 1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清。
步骤七：每管加入500μl 1×Flow Cytometry Staining Buffer重悬，上机检测；或者加入500μl 1-4％多聚甲醛重悬，2-8℃避光，于24小时内检测。
对于人血液样本，所述步骤一中采用人淋巴细胞分离液，步骤二中流式抗体为5μlAnti-Human CD3,FITC和5μlAnti-Human CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μlAnti-Human IFN-γ,PE和5μlAnti-Human IL-4,APC。从图1.1可以看出，人EDTA抗凝全血样本按上述方法处理后，与未刺激对照相比，PMA/Ionomycin刺激和BFA/Monensin阻断后，可检测到明显的IFN-γ细胞因子分泌。
对于小鼠血液样本，所述步骤一中采用小鼠淋巴细胞分离液，步骤二中流式抗体为5μl Anti-Mouse CD3ε,FITC和5μl Anti-Mouse CD4,PerCP-Cy5.5，步骤五中流式抗体为加入5μlAnti-Mouse IFN-γ,PE。从图1.2可以看出，小鼠EDTA抗凝全血样本按上述方法处理后，与未刺激对照相比，PMA/Ionomycin刺激和BFA/Monensin阻断后，可检测到明显的IFN-γ细胞因子分泌。
对于大鼠血液样本，所述步骤一中采用大鼠淋巴细胞分离液，步骤二中流式抗体为5μlAnti-Rat CD3,FITC和5μlAnti-Rat CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μlAnti-Rat IFN-γ,efluor660。从图1.3可以看出，大鼠EDTA抗凝全血样本按上述方法处理后，与未刺激对照相比，PMA/Ionomycin刺激和BFA/Monensin阻断后，可检测到明显的IFN-γ细胞因子分泌。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。
Claims (4)
1.一种非肝素抗凝血液样本的Th检测方法，其特征在于，包括以下步骤：
步骤一：用淋巴细胞分离液分离外周血单个核细胞(PBMCs)，用含10％胎牛血清的培养基重悬沉淀，使细胞浓度为1×107/ml，取250μl PBMCs至流式管中，加入1μl PMA/Ionomycin Mixture(250×)和1μl BFA/Monensin Mixture(250×)，以只含PBMCs的样本作为对照，混匀，37℃孵育4-6小时，每隔1-2小时取出震荡混匀；
步骤二：从样本管和对照管中取100μl细胞悬液至新的流式管中，加入相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤三：每管加入100μl FIX&PERM Medium A，震荡混匀，室温避光孵育15分钟；
步骤四：用蒸馏水将10×Flow Cytometry Staining Buffer稀释为1×，每管加入2ml预冷1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清；
步骤五：每管加入100μl FIX&PERM Medium B和相应的流式抗体，震荡混匀，室温避光孵育15分钟；
步骤六：每管加入2ml 1×Flow Cytometry Staining Buffer，300×g离心5分钟，弃上清；
步骤七：每管加入500μl 1×Flow Cytometry Staining Buffer重悬，上机检测；或者加入500μl 1-4％多聚甲醛重悬，2-8℃避光，于24小时内检测。
2.根据权利要求1所述一种非肝素抗凝血液样本的Th检测方法，其特征在于，对于人血液样本，所述步骤一中采用人淋巴细胞分离液，步骤二中流式抗体为5μl Anti-Human CD3,FITC和5μl Anti-Human CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μl Anti-Human IFN-γ,PE和5μl Anti-Human IL-4,APC。
3.根据权利要求1所述一种非肝素抗凝血液样本的Th检测方法，其特征在于，对于小鼠血液样本，所述步骤一中采用小鼠淋巴细胞分离液，步骤二中流式抗体为5μl Anti-MouseCD3ε,FITC和5μl Anti-Mouse CD4,PerCP-Cy5.5，步骤五中流式抗体为加入5μl Anti-Mouse IFN-γ,PE。
4.根据权利要求1所述一种非肝素抗凝血液样本的Th检测方法，其特征在于，对于大鼠血液样本，所述步骤一中采用大鼠淋巴细胞分离液，步骤二中流式抗体为5μl Anti-RatCD3,FITC和5μl Anti-Rat CD8α,PerCP-Cy5.5，步骤五中流式抗体为5μl Anti-Rat IFN-γ,efluor660。
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