CN114317667A - 一种鉴定过敏原的方法 - Google Patents
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Abstract
本发明属于过敏原鉴定技术领域,本发明提供了一种鉴定过敏原的方法。本发明用待鉴定样品刺激未成熟的树突细胞,采用流式细胞术检测细胞表面标记物(MHCⅡ、CD86)的表达,采用ELISA法检测细胞因子(IL‑6、IFN‑γ、TNF‑α)的产生情况。同时,根据待鉴定过敏原体外刺激树突细胞后,对T细胞分化的作用来判断待鉴定样品是否为过敏原。本发明方法简单有效,为快速鉴定过敏原提供了可参考的依据。
Description
技术领域
本发明涉及过敏原鉴定技术领域,尤其涉及一种鉴定过敏原的方法。
背景技术
食物过敏是人体免疫系统对部分食品成分产生的不良反应,已经成为全球关注的食品安全问题。超过全世界人口40%的人受到食物过敏的困扰。食物过敏症状包括皮肤瘙痒、呕吐、腹泻等胃肠道症状,严重的会导致休克甚至死亡。随着人民生活水平的提高和国际食品贸易的普及,迫切需要对食物过敏原进行风险评估和机制探索。
在食物引发的致敏过程中,位于免疫系统前端的树突细胞(Dendritic cells,DC)和T细胞的作用是尤为重要的一环。树突细胞因其表面呈星型多形体或树突而得名。它能够摄取抗原,并对抗原进行加工处理后递呈给不同的免疫细胞。因此,DC在细胞免疫和体液免疫的启动和调节中发挥着重要作用。DC是最有效的抗原递呈细胞,对T细胞介导的免疫应答的启动和调控发挥着关键作用:食物过敏原诱导DC细胞活化和成熟的程度,决定了T细胞的激活与分化。目前,常用的过敏原鉴定方法仍主要针对过敏反应的效应细胞(肥大细胞和嗜碱性粒细胞),完全忽略了DC细胞和T细胞分化对食物过敏反应的影响,目前此阶段的发明研究仍为空白。因此,针对食物过敏启动阶段的研究,如何开发一种有效、快速的鉴定过敏原的方法是至关重要的。
发明内容
为克服现有技术中存在的上述缺陷,本发明提供了一种有效、快速的鉴定过敏原的方法。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种鉴定过敏原的方法,包括如下步骤:
(1)用GM-CSF和IL-4诱导骨髓细胞分化,当树突细胞比例超过90%时,得未成熟的树突细胞;
(2)用待鉴定样品刺激上述所得未成熟的树突细胞45~50h,若树突细胞的MHCⅡ和CD86表达上调6%以上,且树突细胞分泌IL-6的水平上升15%以上,分泌IFN-γ的水平上升4%以上,同时树突细胞分泌TNF-α的水平下降1%以上,则对待鉴定样品进行下一步鉴定,否则说明待鉴定样品不是过敏原;
(3)将通过上述鉴定的刺激完成的树突细胞与T细胞进行共培养,若共培养体系中IL-4的分泌水平与IFN-γ的分泌水平的比例≥0.8,则表示T细胞发生了Th2向偏移,认为待鉴定样品是过敏原,否则说明待鉴定样品不是过敏原。
优选的,所述过敏原包括食物过敏原或非食物过敏原。
优选的,步骤(1)中所述GM-CSF的浓度为18~22ng/mL,所述IL-4的浓度为8~12ng/mL。
优选的,步骤(2)中所述待鉴定样品的浓度为0.05~0.5mg/mL。
本发明的有益效果如下:
本发明用待鉴定样品刺激未成熟的树突细胞,采用流式细胞术检测细胞表面标记物(MHCⅡ、CD86)的表达,采用ELISA法检测细胞因子(IL-6、IFN-γ、TNF-α)的产生情况。同时,根据待鉴定过敏原体外刺激树突细胞后,对T细胞分化的作用来判断待鉴定样品是否为过敏原。本发明方法简单有效,为快速鉴定过敏原提供了可参考的依据。
附图说明
图1为大菱鲆小清蛋白分离纯化及三维结构模拟图;
图2为大菱鲆小清蛋白、卵清蛋白对BMDC形态及增殖的影响;
图3为大菱鲆小清蛋白、卵清蛋白、马铃薯酸性磷酸酶对BMDC表型分子影响的流式细胞图谱;
图4为大菱鲆小清蛋白、卵清蛋白、马铃薯酸性磷酸酶对BMDC表型分子的定量分析图;
图5为大菱鲆小清蛋白、卵清蛋白、马铃薯酸性磷酸酶对BMDC分泌细胞因子的影响;
图6为大菱鲆小清蛋白、卵清蛋白、马铃薯酸性磷酸酶对细胞共培养体系分泌细胞因子的影响。
具体实施方式
本发明提供了一种鉴定过敏原的方法,包括如下步骤:
(1)用GM-CSF和IL-4诱导骨髓细胞分化,当树突细胞比例超过90%时,得未成熟的树突细胞;
(2)用待鉴定样品刺激上述所得未成熟的树突细胞45~50h,若树突细胞的MHCⅡ和CD86表达上调6%以上,且树突细胞分泌IL-6的水平上升15%以上,分泌IFN-γ的水平上升4%以上,同时树突细胞分泌TNF-α的水平下降1%以上,则对待鉴定样品进行下一步鉴定,否则说明待鉴定样品不是过敏原;
(3)将通过上述鉴定的刺激完成的树突细胞与T细胞进行共培养,若共培养体系中IL-4的分泌水平与IFN-γ的分泌水平的比例≥0.8,则表示T细胞发生了Th2向偏移,认为待鉴定样品是过敏原,否则说明待鉴定样品不是过敏原。
在本发明中,所述过敏原优选包括食物过敏原或非食物过敏原。
在本发明中,步骤(1)中所述GM-CSF的浓度优选为18~22ng/mL,进一步优选为20ng/mL;所述IL-4的浓度优选为8~12ng/mL,进一步优选为10ng/mL。
在本发明中,步骤(2)中所述用待鉴定样品刺激步骤(1)所得未成熟的树突细胞45~50h,进一步优选用待鉴定样品刺激步骤(1)所得未成熟的树突细胞48h。
在本发明中,步骤(2)中所述待鉴定样品的浓度优选为0.05~0.5mg/mL,进一步优选为0.2mg/mL。
下面结合实验例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
以下实验例采用大菱鲆小清蛋白(PV)、卵清蛋白(OVA)、马铃薯酸性磷酸酶(ACP)作为待鉴定样品,其中,卵清蛋白和马铃薯酸性磷酸酶均购买自美国的sigma公司。
实验例1
(1)大菱鲆小清蛋白的分离、纯化和鉴定:
使用提取缓冲液(Tris 0.1mol·L-1,甘氨酸0.5mmol·L-1,DTT 0.1mmol·L-1)对大菱鲆背脊肉进行粗提取,收集粗提液并过夜透析。将透析液在水中煮沸5分钟,离心收集上清液。在上清液中加入硫酸铵,使体系中硫酸铵的终浓度为60%和100%,获得纯化的大菱鲆小清蛋白提取物。采用BCA法测定蛋白浓度。利用SDS-PAGE对不同提取物中提取的蛋白质组分进行分离和鉴定。电泳分离凝胶浓度为12%(w/v),负载凝胶浓度为5%(w/v)。
通过Westernblot分别检测纯化PV的IgG和IgE结合能力。利用iBlot 2凝胶转移装置将蛋白条带转移到PVDF膜上(室温)。使用Tanon-4200SF凝胶成像仪进行化学显影拍照,以观察不同曝光时间下的显色情况,并选择最佳图像进行保存。条带的灰度表明PV的结合能力。间接ELISA法检测纯化PV与IgG的结合能力。用0.05M(pH 9.6)CBS稀释PV为30μg/ml作为实验组,用0.05M(pH 9.6)CBS稀释BSA为30μg/ml作为阴性对照组,以0.05μM(pH 9.6)CBS作为空白对照组。第二天,取出平板,弃去包被液,用PBST清洗3次,每次5分钟。用1%BSA+5%脱脂奶粉作为封闭液进行封闭,37℃孵育2.5h,然后弃去封闭液并洗涤3次。兔抗PV抗体用PBS稀释10000倍,每孔加入100μL,孵育1.5h,然后弃去,用PBST洗涤3次。每孔添加100μLHRP标记的山羊抗兔IgG抗体(10000倍稀释),在37℃下孵育1.5h,然后进行洗涤步骤。向孔中加入100μL TMB基质溶液,在37℃下培养5分钟。最后,添加50μL终止溶液,在450nm条件下测定吸光度。
上述结果如图1所示。由图1中的A可知,在10kDa附近有清晰、高纯度的电泳条带。大菱鲆小清蛋白的蛋白条带有α和β亚基组成,其中α亚基为10.4kDa,β亚基为8.1kDa。图1中的B为通过在线软件SAVES(http://servicesn.mbi.ucla.edu/SAVES/)模拟小清蛋白的三维结构。图1中的C显示纯化PV的IgG/IgE的结合能力。PV的α亚基和β亚基具有相同的IgG结合能力,表明纯化的PV具有免疫活性,可用于后续小鼠实验。间接ELISA(图1中的D)显示纯化PV对兔IgG抗体的识别和结合能力强于阴性对照的BSA。
(2)选取6周龄BALB/c雌性小鼠(购自北京维通利华实验动物技术有限公司,许可证号SCXK2016-0006),室内温度控制在25±2℃,相对湿度50±5%,12小时光照/黑暗循环。喂食含有64%碳水化合物,19%蛋白质和17%脂肪的标准商品化小鼠饲料(不含鱼肉和鱼粉),不限制食物摄入量及供水。本发明中小鼠的养殖与繁育符合欧盟实验动物指南(Directive2010/63/EU)。BMDCs的提取方法如下:无菌取出小鼠股骨和胫骨,去除骨表面肌肉和肌腱,尽可能少的剪去腿骨两端,用1mL RPMI 1640冲出骨髓,并反复吹打制成单细胞悬液。将得到的单细胞悬液300×g离心5min,PBS洗涤一次。2mL红细胞裂解液重悬细胞,室温下静置4min后加入10mLPBS中和红细胞裂解液,300×g离心5min,再用PBS洗涤一次。细胞在添加20ng/mL GM-CSF、10ng/mL IL-4、10%FBS、100IU/ml penicillin/streptomycin(Gibco,Thermo Scientific),100μg/ml Primocin(InvivoGen)的RPMI 1640培养基中重悬,以1×106细胞/mL的密度铺6孔板,将6孔板置于37℃、5%CO2的细胞培养箱中培养。每2天进行一次换液。
用20ng/mL GM-CSF、10ng/mL IL-4诱导骨髓细胞分化,细胞在第2天贴壁生长。第三天悬浮细胞增多并开始聚集。第6天悬浮细胞明显增多,细胞形态不规则,部分细胞呈簇状生长,细胞表面出现短突起,呈现出典型的未成熟DC形态。流式细胞术检测CD11c+细胞比例超过90%,说明DC细胞纯度可以满足实验需要。用LPS(1μg/mL)刺激2天,细胞表面突起明显增多变长,细胞成熟进程增快,符合成熟DC形态特征。第6天收集BMDC,调整细胞密度为2×105个/mL。
(3)根据实验设计,分别添加不同浓度的PV、OVA、LPS(PV的浓度为5和500μg/mL,OVA的浓度为5和500μg/mL,LPS的浓度为0.1和1μg/mL)刺激48h,并设置Control为空白对照组,LPS为阳性对照组,在倒置显微镜下观察各组BMDC分化和成熟情况。结果如图2所示。图2中的A是诱导第6天的未成熟BMDC形态;图2中的B是对未成熟BMDC的鉴定,证明细胞纯度大于90%,符合要求;图2中的C是用LPS诱导48小时的BMDC形态,主要用于观察形态;由图2中的D可以看出,与对照组相比,在LPS、PV、OVA处理48h后,BMDC呈现出不同大小的悬浮细胞样,并分化出大量的细长突起。
(4)采用流式细胞术检测不同浓度ACP、PV、OVA刺激下BMDC细胞表型分子的变化(Control为空白对照组,LPS为阳性对照组),研究ACP、PV、OVA对BMDC细胞表型分子的影响。结果如图3所示。图3为不同抗原刺激后细胞表型鉴定的流式图。进一步定量分析,得到图4。图4显示了经过48h处理后,不同浓度ACP、PV、OVA处理对BMDC细胞表型分子(MHCⅡ、CD80、CD83、CD86)的影响。通过统计发现,ACP可诱导MHCⅡ上调6%,PV、OVA可诱导MHCⅡ上调10%(图4中的A)。并且随着样品浓度的增加,其上调水平也会随之提高。由图4中的B和C可知,两种浓度的ACP、PV、OVA刺激后,BMDC的CD80和CD83未产生显著变化。由图4中的D可知,与空白对照组相比,当ACP、PV、OVA的浓度均为5μg/mL时,分别可以使BMDC的CD86上调6.6%、10%、8.6%;当ACP、PV、OVA的浓度均为500μg/mL时,分别可以使BMDC的CD86上调6.1%、18%、11.1%。
使用不同浓度ACP、PV、OVA刺激BMDC细胞上清液(Control为空白对照组,LPS为阳性对照组),采用ELISA方法测定各因子水平(IL-6、IFN-γ、IL-10、IL-12p70、TNF-α),结果如图5所示。由图5可知,与空白对照组相比,在浓度为0.05mg/mL时,ACP、PV、OVA可分别诱导BMDC细胞分泌IL-6的水平上升15.5%、26.5%、41.4%,分别诱导BMDC细胞分泌IFN-γ的水平上升4.8%、11.4%、5.4%,同时还可分别诱导BMDC细胞分泌TNF-α的水平下降3.1%、10%、1.3%。对ACP、PV、OVA进行下一步鉴定。
(5)收集ACP、PV、OVA刺激完成的BMDC,并与CD4+T细胞以1:10的个数比进行共培养,48h后收集细胞培养上清液,检测其中IL-4和IFN-γ的浓度。结果如图6所示。由图6可知,在ACP、PV、OVA刺激后,共培养体系中IL-4的分泌水平与IFN-γ的分泌水平的比例分别为0.8、1.1、1.1。这表示T细胞发生了Th2向偏移,说明ACP、PV、OVA是过敏原。这与现有技术中对ACP、PV、OVA的鉴定结果一致。说明本发明鉴定方法有效可行。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种鉴定过敏原的方法,其特征在于,包括如下步骤:
(1)用GM-CSF和IL-4诱导骨髓细胞分化,当树突细胞比例超过90%时,得未成熟的树突细胞;
(2)用待鉴定样品刺激上述所得未成熟的树突细胞45~50h,若树突细胞的MHCⅡ和CD86表达上调6%以上,且树突细胞分泌IL-6的水平上升15%以上,分泌IFN-γ的水平上升4%以上,同时树突细胞分泌TNF-α的水平下降1%以上,则对待鉴定样品进行下一步鉴定,否则说明待鉴定样品不是过敏原;
(3)将通过上述鉴定的刺激完成的树突细胞与T细胞进行共培养,若共培养体系中IL-4的分泌水平与IFN-γ的分泌水平的比例≥0.8,则表示T细胞发生了Th2向偏移,认为待鉴定样品是过敏原,否则说明待鉴定样品不是过敏原。
2.根据权利要求1所述的一种鉴定过敏原的方法,其特征在于,所述过敏原包括食物过敏原或非食物过敏原。
3.根据权利要求1或2所述的一种鉴定过敏原的方法,其特征在于,步骤(1)中所述GM-CSF的浓度为18~22ng/mL,所述IL-4的浓度为8~12ng/mL。
4.根据权利要求3所述的一种鉴定过敏原的方法,其特征在于,步骤(2)中所述待鉴定样品的浓度为0.05~0.5mg/mL。
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