CN114921412A - 一种人肿瘤相关巨噬细胞表型特征分析方法 - Google Patents
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Abstract
本发明公开了一种人肿瘤相关巨噬细胞表型特征分析方法，包括以下步骤：S1、细胞培养：首先运用佛波酯(PMA)诱导人单核细胞株THP‑1细胞贴壁分化成巨噬细胞；S2、细胞取样：获取巨噬细胞单细胞悬浮液，并对巨噬细胞采用荧光标记；S3、细胞检测：运用流式细胞分析仪检测巨噬细胞(PMAonly型)表面分子CD14的表达水平；S4、刺激极化：运用人白细胞介素4(IL‑4)和脂多糖(LPS)分别刺激巨噬细胞极化成为M2和M1型巨噬细胞。本发明能快速制备充足的巨噬细胞样本数量，并且能有效进行标记，从而方便进行观察，还能快速将巨噬细胞刺激极化，便于针对进行进行试验，能很好的对M1型和M2型巨噬细胞进行对比，了解两种类别巨噬细胞的表型特征以及其区别。
Description
技术领域
本发明涉及巨噬细胞表型分析技术领域，尤其涉及一种人肿瘤相关巨噬细胞表型特征分析方法。
背景技术
巨噬细胞是由血液中的单核细胞穿出血管后分化而成的。单核细胞进入结缔组织后，体积增大，内质网和线粒体增生，溶酶体增多，吞噬功能增强。巨噬细胞的寿命因所在组织器官而异，一般可存活数月或更久。巨噬细胞不仅存在于血液中，还分布全身。根据所处的位置不同，名字和形状也各不相同。单核细胞随血液循环至全身，奔向炎症部位。肺巨噬细胞在肺部活动，肝巨噬细胞在肝脏中活动，神经胶质细胞则在脑部活动。
在微环境中，组织蛋白酶蛋白酶活性在大多数巨噬细胞中被诱导巨噬细胞至少可以被分为两种类型：M1型，即经典活化的巨噬细胞和M2型，即替代性活化的巨噬细胞。不同活化表型的巨噬细胞所起的作用完全不同：M1型巨噬细胞表现为:高递呈抗原的能力；高分泌白细胞介素12(IL-12)和白细胞介素23(IL-23)及其随后诱导的I型免疫应答；高产生毒性中间产物、活性氧中间产物(ROI)的能力。因此，M1型巨噬细胞被认为具有杀伤细菌和肿瘤细胞并可以分泌多种促炎性细胞因子的能力。而M2型巨噬细胞表现为:低的递呈抗原的能力；能够分泌IL-1受体拮抗剂(IL-1RA)、IL-10和多种趋化因子，高表达精氨酸酶(Arg)-1、巨噬细胞甘露糖受体(MMR)、清道夫受体(scavengerreceptor)等，产生很少的NO和IL-12，主要参与细胞生长、血管生成、免疫抑制、组织修复和间质形成等过程；
综上所述，为了更好的对巨噬细胞的特性进行研究，如何控制巨噬细胞的产生以及如何促进巨噬细胞进行快速分化是本领域的重点问题，为此，我们提出了一种人肿瘤相关巨噬细胞表型特征分析方法来解决上述问题。
发明内容
本发明的目的是为了解决如何控制巨噬细胞的产生以及如何促进巨噬细胞进行快速分化这一本领域的重点问题，而提出的一种人肿瘤相关巨噬细胞表型特征分析方法。
为了实现上述目的，本发明采用了如下技术方案：
一种人肿瘤相关巨噬细胞表型特征分析方法，包括以下步骤：
S1、细胞培养：首先运用佛波酯(PMA)诱导人单核细胞株THP-1细胞贴壁分化成巨噬细胞；
S2、细胞取样：获取巨噬细胞单细胞悬浮液，并对巨噬细胞采用荧光标记；
S3、细胞检测：运用流式细胞分析仪检测巨噬细胞(PMAonly型)表面分子CD14的表达水平；
S4、刺激极化：运用人白细胞介素4(IL-4)和脂多糖(LPS)分别刺激巨噬细胞极化成为M2和M1型巨噬细胞；
S5、对比检测：通过半定量RT-PCR法和ELISA实验分析法对多组巨噬细胞的IL-6mRNA和TNF-α蛋白的表达情况。
优选地，为了有效促进巨噬细胞的产生，为试验提供充足样本，所述佛波酯(PMA)为肿瘤促进剂，所述佛波酯(PMA)为用于体内外试验的佛波酯PKC激活剂，所述PMA结合PKC并激活其功能，能在细胞和组织中都呈现出极其广的抑制谱；所述PMA可以抑制Fas诱导的细胞凋亡，又可以诱导HL-60细胞的凋亡。
优选地，为了快速将分化的巨噬细胞进行分类，方便针对性试验，所述流式细胞分析仪可提供单个细胞的高度特异性信息。
优选地，为了能有效进行标记，方便快速进行观察，所述荧光标记采用台盼蓝。
优选地，为了了解巨噬细胞IL-6mRNA的表达情况，所述半定量RT-PCR法为通过mRNA反转录成cDNA，再进行PCR扩增，并测定PCR产物的数量，可以推测样品中特异mRNA的相对数量。
优选地，为了了解巨噬细胞TNF-α蛋白的表达情况，所述ELISA实验分析法是将一定浓度的抗原和抗体通过物理吸附的方法固定于聚苯乙烯微孔板表面，加入待检标本，通过酶标物显色的深浅间接反映被检抗原或者抗体的存在以及抗原或者抗体的存量。
与现有技术相比，本发明的有益效果是：
1、能快速刺激生产巨噬细胞，从而便于为试验提供充足的样本，并且方便快速取样以及进行荧光标记，保证观察的精准度，同时能精准的检查每个样本表面分子CD14的表达水平并进行记录，便于进行对比；
2、能快速对巨噬细胞进行刺激极化，从而使其分成M2和M1型巨噬细胞，便于有针对性的进行试验，方便进行记录，并且能很好的进行对比，了解不同类别巨噬细胞的表型特征；
综上所述，本发明能快速制备充足的巨噬细胞样本数量，并且能有效进行标记，从而方便进行观察，还能快速将巨噬细胞刺激极化，便于针对进行进行试验，能很好的对M1型和M2型巨噬细胞进行对比，了解两种类别巨噬细胞的表型特征以及其区别。
附图说明
图1为本发明提出的一种人肿瘤相关巨噬细胞表型特征分析方法的流程图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
参照图1，一种人肿瘤相关巨噬细胞表型特征分析方法，包括以下步骤：
S1、细胞培养：首先运用佛波酯(PMA)诱导人单核细胞株THP-1细胞贴壁分化成巨噬细胞，能快速生产巨噬细胞，能为试验提供充足样本，便于试验的开展；
S2、细胞取样：获取巨噬细胞单细胞悬浮液，并对巨噬细胞采用荧光标记，采用台盼蓝荧光标记，其无法被消化，能很好的保留在巨噬细胞内，从而便于进行观测；
S3、细胞检测：运用流式细胞分析仪检测巨噬细胞(PMAonly型)表面分子CD14的表达水平，能精准的对单个巨噬细胞进行分析检测，便于进行记录，方便和后续对比，能很好的了解极化的规律；
S4、刺激极化：运用人白细胞介素4(IL-4)和脂多糖(LPS)分别刺激巨噬细胞极化成为M2和M1型巨噬细胞；
S5、对比检测：通过半定量RT-PCR法和ELISA实验分析法对多组巨噬细胞的IL-6mRNA和TNF-α蛋白的表达情况。
在本发明中，佛波酯(PMA)为肿瘤促进剂，佛波酯(PMA)为用于体内外试验的佛波酯PKC激活剂，PMA结合PKC并激活其功能，能在细胞和组织中都呈现出极其广的抑制谱；PMA可以抑制Fas诱导的细胞凋亡，又可以诱导HL-60细胞的凋亡。
在本发明中，流式细胞分析仪可提供单个细胞的高度特异性信息，流式细胞仪的具有三个主要系统，其为流体学、光学和电子学系统，流体学系统负责将样品从样品管转移到流动池；一旦通过流动池(并通过激光束)，样品将被分选出来(在细胞分选仪中)或移到废液中；光学系统的元件包括激发光源、透镜和滤光片以及用于产生光电流的检测系统；电子系统是对来自检测器的光电流经过数字化、处理和保存，以用于后续分析。
在本发明中，荧光标记采用台盼蓝，能有效保证其不被消化，保证观察时能快速准确的发现巨噬细胞。
在本发明中，半定量RT-PCR法为通过mRNA反转录成cDNA，再进行PCR扩增，并测定PCR产物的数量，可以推测样品中特异mRNA的相对数量，其操作方式简洁，能测定体内目的基因的表达增加减少与否，即通过目的基因跑出来的电泳带与基因的电泳带的相对含量比较，观测目的基因表达增减，另外还要做一个β-actin的内参照对照。
在本发明中，ELISA实验分析法是将一定浓度的抗原和抗体通过物理吸附的方法固定于聚苯乙烯微孔板表面，加入待检标本，通过酶标物显色的深浅间接反映被检抗原或者抗体的存在以及抗原或者抗体的存量，ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记；结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性；在测定时，受检标本(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应；用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开；再加入酶标记的抗原或抗体，也通过反应而结合在固相载体上；此时固相上的酶量与标本中受检物质的量呈一定的比例；加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，故可根据呈色的深浅进行定性或定量分析；由于酶的催化频率很高，故可极大地地放大反应效果，从而使测定方法达到很高的敏感度。
在本发明中，操作时，首先运用佛波酯(PMA)诱导人单核细胞株THP-1细胞贴壁分化成巨噬细胞(PMAonly型)，运用流式细胞分析仪检测巨噬细胞(PMAonly型)表面分子CD14的表达水平，然后运用人白细胞介素4(IL-4)或脂多糖(LPS)分别刺激巨噬细胞极化成为M2或M1型巨噬细胞；半定量RT-PCR法检测不同组巨噬细胞IL-6mRNA的表达水平，ELISA实验分析不同组巨噬细胞TNF-α蛋白的表达情况。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于，包括以下步骤：
S1、细胞培养：首先运用佛波酯(PMA)诱导人单核细胞株THP-1细胞贴壁分化成巨噬细胞；
S2、细胞取样：获取巨噬细胞单细胞悬浮液，并对巨噬细胞采用荧光标记；
S3、细胞检测：运用流式细胞分析仪检测巨噬细胞(PMAonly型)表面分子CD14的表达水平；
S4、刺激极化：运用人白细胞介素4(IL-4)和脂多糖(LPS)分别刺激巨噬细胞极化成为M2和M1型巨噬细胞；
S5、对比检测：通过半定量RT-PCR法和ELISA实验分析法对多组巨噬细胞的IL-6mRNA和TNF-α蛋白的表达情况。
2.根据权利要求1所述的一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于：所述佛波酯(PMA)为肿瘤促进剂，所述佛波酯(PMA)为用于体内外试验的佛波酯PKC激活剂，所述PMA结合PKC并激活其功能，能在细胞和组织中都呈现出极其广的抑制谱；所述PMA可以抑制Fas诱导的细胞凋亡，又可以诱导HL-60细胞的凋亡。
3.根据权利要求1所述的一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于：所述流式细胞分析仪可提供单个细胞的高度特异性信息。
4.根据权利要求1所述的一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于：所述荧光标记采用台盼蓝。
5.根据权利要求1所述的一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于：所述半定量RT-PCR法为通过mRNA反转录成cDNA，再进行PCR扩增，并测定PCR产物的数量，可以推测样品中特异mRNA的相对数量。
6.根据权利要求1所述的一种人肿瘤相关巨噬细胞表型特征分析方法，其特征在于：所述ELISA实验分析法是将一定浓度的抗原和抗体通过物理吸附的方法固定于聚苯乙烯微孔板表面，加入待检标本，通过酶标物显色的深浅间接反映被检抗原或者抗体的存在以及抗原或者抗体的存量。
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