CN114085813B - 用于评价物质具有免疫调节功能的方法 - Google Patents
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Abstract
本发明公开了一种评价物质具有免疫调节功能的方法，包括：取后天免疫细胞缺陷小鼠的脾脏，制成原代天然免疫细胞悬液；将免疫细胞与OVA培养，获得培养体系；收集T细胞受体转基因小鼠的脾脏和淋巴结单细胞悬液，磁珠分选获得CD4+CD62L+Naive T细胞；将CD4+CD62L+Naive T细胞加入培养体系中，培养过程中，待评价物质以100～500μg/mL的浓度加入培养体系中；收集全部细胞，细胞用佛波醇和离子霉素刺激培养，破膜配合固定后添加荧光抗体，再利用流式细胞仪检测Th1、Th2或Treg的比例，以评估物质的免疫调节功能。该方法能够准确的评估活性物质是否具有免疫调节功能，该方法成本低、周期短。
Description
技术领域
本发明涉及细胞体外模型技术领域，具体涉及一种用于评价物质具有免疫调节功能的方法。
背景技术
天然产物中富含糖类、蛋白质、矿物质等活性物质，被广泛应用于食品制造业和药物研发，研究表明多种天然产物具有调控机体免疫反应的生物功能，表现在免疫调节活性、抗炎症、抗肿瘤、抗过敏、抗病毒等多方面。越来越多学者关注于天然产物调控机体免疫反应的研究。
现阶段常用的评估活性物质免疫调节功能的模型是以体内动物实验或体外巨噬细胞吞噬实验为主。体内动物实验包括小鼠或大鼠的体内实验，长期喂食天然产物后，检测小鼠血清中免疫相关细胞因子分泌情况，判断天然产物的具体调控作用；该体内动物模型周期长、成本高、操作繁琐，且由于细胞因子往往并非单一细胞分泌，多种免疫细胞均可能产生同一种细胞因子，因此该体内动物实验的检测无法精准评估天然产物在体内的靶向免疫细胞及调控作用。体外巨噬细胞模型多是检测RAW264.7细胞吞噬或细胞因子分泌情况；该细胞模型仅仅表征了抗原进入机体内，部分天然免疫细胞对抗原的吞噬和消化过程，无法体现抗原进入机体后的吞噬、消化、递呈、T细胞分化等免疫过程，该体外巨噬细胞模型具有一定的片面性，无法准确评估活性物质的免疫调节活性。
发明内容
为了解决上述问题，本发明提出一种用于评价物质具有免疫调节功能的方法，该方法能够准确的评估活性物质是否具有免疫调节功能，该方法成本低、周期短。
为了实现上述目的，本发明的实施例提出了一种评价物质具有免疫调节功能的方法，其包括以下步骤：
(1)取后天免疫细胞缺陷小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液；
(2)将原代天然免疫细胞与OVA培养18～32h，获得培养体系；
(3)收集T细胞受体转基因小鼠的脾脏和淋巴结单细胞悬液，利用磁珠分选获得CD4+CD62L+Naive T细胞；
(4)将CD4+CD62L+Naive T细胞加入培养体系中，培养2～4天，构建T细胞体外分化模型；培养过程中，待评价物质以100～500μg/mL的浓度加入所述培养体系中；
(5)从所述T细胞体外分化模型中收集全部细胞，细胞用佛波醇和离子霉素刺激培养4～6h，破膜配合固定后添加荧光抗体，再利用流式细胞仪检测Th1、Th2或Treg的比例，以评估物质的免疫调节功能。
根据本发明实施例的一种评价物质具有免疫调节功能的方法，该方法建立原代的天然免疫细胞和CD4+Naive T细胞共培体系，利用T细胞受体转基因小鼠的CD4 T细胞对OVA蛋白的特异性(其可识别OVA蛋白323-339的氨基酸位点)，建成稳定的OVA吞噬递呈及T细胞分化模型，更接近真实在体情况，可以用于评价物质的免疫调节功能；在检测物质免疫调节功能时，通过检测模型中OVA诱导的Th1、Th2或Treg细胞分化的变化，可评价天物质对抗原递呈和T细胞分化的免疫调节作用，检测方法简单、精准度更高；整个方法的成本低、周期短、批间差异小，质量容易控制。
可选地，步骤(2)中，在96孔U型板中加入100～200μL/孔的1×106～3×106cells/mL的原代天然免疫细胞和1～10μg/mL的OVA。
可选地，步骤(4)中，在培养体系中加入100～200μL/孔的1×106～4×106cells/mL的CD4+CD62L+Naive T细胞。
可选地，步骤(4)中，待评价物质于培养的0～36h加入培养体系中。
可选地，步骤(5)中，荧光抗体包括CD4、CD44、CD25、IFN-γ、IL-4和Foxp3。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1为根据本发明实施例的OT-II小鼠淋巴细胞磁珠分选前后的流式检测图；
图2为根据本发明实施例的T细胞分化体外模型的示意图；
图3为根据本发明实施例的T细胞分化体外模型评价GLSO对Th1细胞的免疫调节作用；
图4为根据本发明实施例的T细胞分化体外模型评价GLSO对Th2细胞的免疫调节作用；
图5为根据本发明实施例的T细胞分化体外模型评价GLSO对Treg细胞的免疫调节作用。
图6为根据本发明实施例的T细胞分化体外模型评价坛紫菜多糖的免疫调节作用。
具体实施方式
以下通过特定的具体实例说明本发明的技术方案。应理解，本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤；还应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。而且，除非另有说明，各方法步骤的编号仅为鉴别各方法步骤的便利工具，而非为限制各方法步骤的排列次序或限定本发明可实施的范围，其相对关系的改变或调整，在无实质变更技术内容的情况下，当亦视为本发明可实施的范畴。
为了更好的理解上述技术方案，下面更详细地描述本发明的示例性实施例。虽然显示了本发明的示例性实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
本发明采用的试材皆为普通市售品，皆可于市场购得。
其中，当抗原进入机体后，经由天然免疫细胞(包括巨噬细胞、树突状细胞、自然杀伤细胞等)的吞噬、消化或递呈，传递至初始T细胞，促使初始T细胞分化为不同类型，与自身免疫性疾病、炎症、过敏反应、肿瘤反应等息息相关的是不同类型CD4+T细胞的分化情况，能够调控CD4+T细胞的分化过程可认定为具有免疫调节活性。辅助性T细胞(Helper T cells，Th)-1型细胞通常分泌IFN-γ细胞因子，对细菌、病毒具有很强的杀伤力，具有促炎功能；Th2型细胞一般产生IL-4/13等细胞因子，Th2细胞的极化会引起一系列过敏性疾病的产生；调节性T细胞(Regulatory T，Treg)细胞是区别于机体促炎反应的一类免疫细胞，在多种疾病中发挥抑制免疫反应和维持免疫平衡的作用，通常以核内表达Foxp3转录因子为检测指标。通过检测CD4+T细胞的胞内IFN-γ和IL-4水平判断Th1和Th2的分化情况，通过检测CD4+T细胞核内Foxp3表达情况评估Treg细胞的分化情况，可以利用Th1/Th2/Treg细胞分化的变化来进行免疫调节活性物质的筛选及药效学评价。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例1 T细胞体外模型评价龙须菜硫酸寡糖对Th1细胞分化的影响
1)取Rag1小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液，96孔U底板中每孔加入1×105个细胞，培养体系中加入2μg/mL的OVA和300μg/mL的龙须菜硫酸寡糖(sulfated oligosaccharide of Gracilaria lemaneiformis，GLSO)，37℃、5％CO2的培养箱中培养24h，其中以不加龙须菜硫酸寡糖作为对照组。
2)收集OT-II小鼠脾脏单细胞悬液，利用磁珠分选法获得CD4+CD62L+的Naive T细胞，经过分选后，96.3％的细胞是表达CD4+CD44+CD62L+的Naive T细胞(结合图1)，按照2×105个细胞/每孔加入上述培养体系中，继续培养3天，构建T细胞分化体外模型，如图2所示。
3)于培养体系的第4天收集全部细胞，添加佛波醇(50ng/mL)和离子霉素(1μg/mL)刺激培养4h，再对细胞进行破膜配合固定后添加荧光抗体(CD4-eF450，CD44-PE，IFN-γ-APC)，利用流式细胞仪(BD LSR Fortessa，488nm、561nm、633nm三激光)检测培养体系中Th1的比例，以评估GLSO对Th1细胞分化的调控影响。
结果如图3所示，OVA经由天然免疫细胞的吞噬递呈后，刺激Th1细胞的分化(26.8％)，300μg/mL的GLSO能够有效抑制Th1细胞分化，其比例为16.8％。
实施例2 T细胞体外模型评价龙须菜硫酸寡糖对Th2细胞分化的影响
1)取Rag1小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液，96孔U底板中每孔加入1×105个细胞，培养体系中加入2μg/mL的OVA和300μg/mL的龙须菜硫酸寡糖(sulfated oligosaccharide of Gracilaria lemaneiformis，GLSO)，37℃、5％CO2的培养箱中培养24h，其中以不加龙须菜硫酸寡糖作为对照组。
2)收集OT-II小鼠脾脏单细胞悬液，利用磁珠分选法获得CD4+CD62L+的Naive T细胞，经过分选后，96.3％的细胞是表达CD4+CD44+CD62L+的Naive T细胞(结合图1)，按照2×105个细胞/每孔加入培养体系中，继续培养3天，构建T细胞分化体外模型，如图2所示。
3)于培养体系的第4天收集全部细胞，添加佛波醇(50ng/mL)和离子霉素(1μg/mL)刺激培养4h，再对细胞进行破膜配合固定后添加荧光抗体(CD4-eF450，CD44-PE，IL-4-PE)，利用流式细胞仪检测培养体系中Th2的比例，以评估GLSO对Th2细胞分化的调控影响。
结果如图4所示，OVA经由天然免疫细胞的吞噬递呈后，刺激Th1细胞的分化，其分群比例达到8.23％，300μg/mL的GLSO能够有效抑制Th1细胞分化，其比例为8.04％。
实施例3 T细胞体外模型评价龙须菜硫酸寡糖对Treg细胞分化的影响
1)取Rag1小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液，96孔U底板中每孔加入1×105个细胞，培养体系中加入2μg/mL的OVA和300μg/mL的龙须菜硫酸寡糖(sulfated oligosaccharide of Gracilaria lemaneiformis，GLSO)，37℃、5％CO2的培养箱中培养24h，其中以不加龙须菜硫酸寡糖作为对照组。
2)收集OT-II小鼠脾脏单细胞悬液，利用磁珠分选法获得CD4+CD62L+的Naive T细胞，经过分选后，96.3％的细胞是表达CD4+CD44+CD62L+的Naive T细胞(结合图1)，按照2×105个细胞/每孔加入培养体系中，继续培养3天，构建T细胞分化体外模型，如图2所示。
3)于培养体系的第4天收集全部细胞，添加佛波醇(50ng/mL)和离子霉素(1μg/mL)刺激培养4h，再对细胞进行破膜配合固定后添加荧光抗体(CD4-eF450，CD44-PE，Foxp3-APC)，利用流式细胞仪检测培养体系中Treg的比例，以评估GLSO对Treg细胞分化的调控影响。
结果如图5所示，OVA经由天然免疫细胞的吞噬递呈后，Treg细胞分群比例为1.51％，300μg/mL的GLSO能够显著促进Treg细胞分化(5.74％)。
实施例4 T细胞体外模型评价坛紫菜多糖的免疫调节活性
1)取Rag1小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液，96孔U底板中每孔加入1×105个细胞，培养体系中加入2μg/mL的OVA，37℃、5％CO2的培养箱中培养24h，其中以不加龙须菜硫酸寡糖作为对照组。
2)收集OT-II小鼠脾脏单细胞悬液，利用磁珠分选法获得CD4+CD62L+的Naive T细胞，按照2×105个细胞/每孔加入上述培养体系中，并在培养体系中添加坛紫菜多糖(porphyra haitanensis polysaccharide，PHP)200μg/mL，继续培养3天。
3)于培养体系的第4天收集全部细胞，添加佛波醇(50ng/mL)和离子霉素(1μg/mL)刺激培养4h，再对细胞进行破膜配合固定后添加荧光抗体(CD4-eF450，CD44-PE，IFN-γ-APC，IL-4-PE，Foxp3-APC)，利用流式细胞仪检测培养体系中Th1/Th2/Treg的比例，以评估GLSO对T细胞分化的调控影响。
结果如图6所示，在OVA诱导的T细胞分化体外模型中，坛紫菜多糖能够稍微促进Treg细胞的分化，对Th1/Th2细胞分化基本无影响。
综上，根据本发明的实施例的方法，建立原代的天然免疫细胞和CD4+Naive T细胞共培体系，利用OT-II小鼠CD4 T细胞对OVA蛋白的特异性，建成稳定的OVA吞噬递呈及T细胞分化模型，更接近在体内情况，可以用于评价天然产物的免疫调节功能；在检测天然产物免疫调节功能时，通过检测模型中OVA诱导的Th1/Th2/Treg细胞分化的变化，可评价天然产物对抗原递呈和T细胞分化的免疫调节作用，检测方法简单、精准度更高；该T细胞体外模型构建成本低，周期短，批间差异小，质量容易控制，应用广泛。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外，本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (2)
1.一种评价物质具有免疫调节功能的方法，其特征在于，包括以下步骤：
(1)取Rag1小鼠的脾脏，去除红细胞后，制成原代天然免疫细胞悬液；
(2)在96孔U型板中加入100～200μL/孔的1×106～3×106cells/mL的原代天然免疫细胞和1～10μg/mL的OVA培养18～32h，获得培养体系；
(3)收集OT-II小鼠的脾脏和淋巴结单细胞悬液，利用磁珠分选获得CD4+CD62L+Naive T细胞；
(4)在培养体系中加入100～200μL/孔的1×106～4×106cells/mL的CD4+CD62L+Naive T细胞，培养2～4天，构建T细胞体外分化模型；培养过程中，待评价物质以100～500μg/mL的浓度、于培养的0～36h加入所述培养体系中；
(5)从所述T细胞体外分化模型中收集全部细胞，细胞用佛波醇和离子霉素刺激培养4～6h，破膜配合固定后添加荧光抗体，再利用流式细胞仪检测Th1、Th2或Treg的比例，以评估物质的免疫调节功能。
2.如权利要求1所述的方法，其特征在于，步骤(5)中，荧光抗体包括CD4、CD44、CD25、IFN-γ、IL-4和Foxp3。
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