CN114686431A - 一种免疫毒性的评价方法及模型 - Google Patents
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Abstract
本发明公开了一种免疫毒性的评价方法及模型。一种免疫毒性的评价模型的构建方法,其包括以下步骤:(1)将PBMC细胞(外周血单核细胞)与KLH共同孵育,获得孵育溶液;(2)选择评价指标,对(1)中的所述孵育溶液进行检测,获取各评价指标的检测结果;(3)根据(2)得到的各种评价指标的检测结果,分析并评价免疫毒性;其中,(2)中所述评价指标包括:细胞活力和/或髓样分化因子(MyD88)的表达水平。本发明将
Description
技术领域
本发明属于药物免疫毒性检测领域,具体涉及一种免疫毒性的评价方法及模型。
背景技术
免疫毒性是由于外源物质作用于机体免疫系统或细胞,出现免疫功能抑制、免疫增强、自身免疫、超敏反应及免疫原性等毒性反应。免疫毒性可表现为形态学变化,也可表现为功能性改变。按照ICH S8的标准,采用组织病理学、免疫组化、血液学和血清生化检查等可以进行初步的免疫毒性评价。而功能性变化需通过一定的技术才能检测到,如免疫细胞表型、细胞因子、局部淋巴结试验、淋巴细胞增殖试验、以及T细胞依赖性抗原抗体反应(TDAR)等进行评价。其中基于玥孔戚血蓝蛋白(KLH)的TDAR经过多个实验室的联合验证,是目前公认接受度最高的免疫毒性评价方法。
TDAR是B细胞接触T细胞依赖性抗原后产生的抗体反应,在Th2细胞的参与下并产生特异性抗体的一种免疫功能反应。近年来TDAR试验被认为是检测药物潜在免疫毒性预测性较好的功能性试验,通过人为引入外来抗原KLH并检测KLH特异性IgG抗体生成水平,反映待测物对免疫系统整体的影响,预测免疫功能的改变,并能对药物的免疫调节和免疫毒性特点进行早期预测,因此该方法在药物免疫毒理学研究中逐渐得到广泛应用。
目前的TDAR检测方法是在猴体内注射一定量(10mg/只)的KLH,KLH是T细胞依赖性抗原,首先通过抗原提呈细胞提呈并激活T细胞,进而激活B细胞,产生针对KLH的特异性抗体,通过检测特异性anti-KLH IgM和anti-KLH IgG的水平,从而评估T细胞和B细胞的功能。但是这种方法需要使用较多的实验动物,且用药量较大,评价时间较长(IgG型抗体的产生和消除均需要数周的时间)。
发明内容
本发明要解决的技术问题是为了克服现有技术中常规的TDAR试验是基于动物试验,耗时较长,还未见体外基于KLH和猴PBMC共孵育的TDAR方法的缺陷,提供一种免疫毒性的评价方法及模型。本发明的免疫毒性评价方法通过将食蟹猴外周血中的淋巴细胞与KLH共培养,最大程度的模拟了体内TDAR的情况,达到替代和优化动物使用,并且可以实现高通量筛选。
本研究首先提取猴体内的PBMC,由于PBMC中包含了不同种类的淋巴细胞,能较好的模拟体内过程,将KLH与PBMC共孵育,通过PBMC的活化效应,进而评价PBMC的功能状态。
本发明是通过下述技术方案来解决上述技术问题:
本发明第一方面提供一种免疫毒性的评价模型的构建方法,其包括以下步骤:
(1)将PBMC细胞与KLH共同孵育,获得孵育溶液;
(2)选择评价指标,对(1)中的所述孵育溶液进行检测,获取各评价指标的检测结果;
(3)根据(2)得到的各种评价指标的检测结果,分析并评价免疫毒性;
其中,(2)中所述评价指标包括:细胞活力和/或髓样分化因子的表达水平。
本发明所述的PBMC细胞较佳地来自猴血,例如食蟹猴的猴血。
在某一较佳实施方案中,所述细胞活力通过CCK-8试剂盒或MTT试剂盒进行检测。
本发明所述的CCK-8试剂盒或MTT试剂盒可为市面常见的CCK-8或MTT试剂盒。
上述构建方法中,进行共同孵育后,所述细胞活力较佳地增加30%~110%,例如36%、90%或106%。
在某一较佳实施方案中,所述髓样分化因子的表达水平通过qPCR技术进行检测。
上述构建方法中,进行共同孵育后,所述髓样分化因子的表达水平较佳地提高2~240倍,例如238倍、200倍、150倍、100倍。
所述孵育溶液中KLH的浓度较佳地为1×10-3~1×10-1mg/mL。
所述共同孵育的时间较佳地为48~72h。
本发明第二方面提供一种免疫毒性的评价模型,其通过如本发明第一方面所述的构建方法获得。
本发明第三方面提供一种免疫毒性的评价方法,其通过本发明第二方面所述的评价模型进行评价。
本发明的积极进步效果在于:利用体外猴PBMC细胞,开发体外TDAR检测,模拟体内多种类型淋巴细胞的相互作用,检测其活化作用,包括增殖作用和髓样分化因子(MyD88)表达作用,通过免疫细胞的功能检测,从而评价机体的免疫毒性。
附图说明
图1为实施例1中猴血中分离PBMC的分离流程示意图。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。
实施例1
1.猴血中分离PBMC细胞
(2)取两支15mL离心管,先加入5mL Ficoll溶液(商品名:GE Ficoll-Paque PLUS淋巴细胞分离液)。然后将稀释的血液轻轻加到Ficoll上层,一定要轻柔,避免两种溶液混合在一起,每只离心管各加10mL稀释的血液;
(3)2600rpm,离心30min,离心完毕后得到如图1所示分层;
(4)PBMC所在细胞层为白色。用吸管将该层细胞吸入另一干净的离心管中;
(5)加入PBS至10mL,1500rpm,10min离心后去掉上清,再加入培养基(RPMI1640培养基+10%FBS+1%双抗(商品名:Penicillin-Streptomycin Solution))清洗一遍;
2.加适量培养基重悬细胞,进行细胞计数,然后铺板:96孔板,2×105个/100μl 10孔。
3.加KLH孵育,每孔加10μL(10%)。
KLH共设10个浓度梯度(工作浓度):1、0.9、0.8、0.7、0.6、0.5、0.4、0.3、0.2、0.1mg/mL,供试品每个浓度设5个复孔,另设溶媒组5个复孔,空白组5个复孔。
供试品溶液的配制(现用现配):母液:10mg KLH+1mL培养基(RPMI1640培养基+10%FBS+1%双抗),得到浓度为10mg/mL的KLH母液,再按下表1稀释。
表1
4.KLH孵育72h后,加CCK-8孵育2h,酶标仪测定各孔的吸光度值,计算细胞活力,结果见表2。
5.采用下列PCR引物,开展实时荧光PCR检测,检测MyD88的表达情况。
待测基因MyD88-F(SEQ ID NO:1):tct ctc cag gtg ccc atc aga ag;MyD88-R(SEQ ID NO:2):gca agg cga gtc cag aac caa g。
参比基因β-actin-F(SEQ ID NO:3):cca ggt cat cac cat cgg;β-actin-R(SEQID NO:4):tgt cca cgt cgc act tca。
扩增获得的待测基因MyD88的序列如SEQ ID NO:5所示,参比基因β-actin的序列如SEQ ID NO:6所示。
体外KLH可刺激猴PBMC的活性,包括增殖作用和Toll样受体信号通路分子MyD88的表达。
PBMC铺板后24h,加KLH孵育48h。表2显示了CCK-8检测结果(2h)。
表2
根据表2结果,增殖曲线斜率最大出现在10-5到10-2范围内,因此采用上述浓度开展qPCR检测,反应体系为25μL;反应程序:50℃2min;95℃10min;95℃15sec,60℃60sec,40个循环。结果如下表3所示。
表3
综上所述,在一定浓度范围内,KLH的浓度越高,刺激作用越大。在1×10-2mg/mL的浓度下,细胞的增殖作用和MyD88均最为明显,可以用于免疫毒性评价。
虽然以上描述了本发明的具体实施方式,但是本领域的技术人员应当理解,这仅是举例说明,本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下,可以对这些实施方式做出多种变更或修改,但这些变更和修改均落入本发明的保护范围。
SEQUENCE LISTING
<110> 益诺思生物技术南通有限公司
<120> 一种免疫毒性的评价方法及模型
<130> P210110134C
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> MyD88-F
<400> 1
tctctccagg tgcccatcag aag 23
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> MyD88-R
<400> 2
gcaaggcgag tccagaacca ag 22
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> β-actin -F
<400> 3
ccaggtcatc accatcgg 18
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> β-actin-R
<400> 4
tgtccacgtc gcacttca 18
<210> 5
<211> 142
<212> DNA
<213> Artificial Sequence
<220>
<223> 基因MyD88
<400> 5
ggcttgcagg tgcccatcag aagcgactga tccccatcaa gtacaaggca atgaagaaag 60
agttccccag catcctgagg ttcatcactg tctgcgatta caccaacccc tgcaccaaat 120
cttggttctg gactcgcctt gc 142
<210> 6
<211> 131
<212> DNA
<213> Artificial Sequence
<220>
<223> 参比基因β-actin
<400> 6
ccaggtcatc accattggca atgagcggtt ccgctgccct gaggcactct tccagccttc 60
cttcctgggc atggagtcct gtggcatcca cgaaactacc ttcaactcca tcatgaagtg 120
tgacgtggac a 131
Claims (10)
1.一种免疫毒性的评价模型的构建方法,其包括以下步骤:
(1)将PBMC细胞与KLH共同孵育,获得孵育溶液;
(2)选择评价指标,对(1)中的所述孵育溶液进行检测,获取各评价指标的检测结果;
(3)根据(2)得到的各种评价指标的检测结果,分析并评价免疫毒性;
其中,(2)中所述评价指标包括:细胞活力和/或髓样分化因子的表达水平。
2.如权利要求1所述的构建方法,其特征在于,所述PBMC细胞来自猴血,例如食蟹猴的猴血。
3.如权利要求1所述的构建方法,其特征在于,所述细胞活力通过CCK-8试剂盒或MTT试剂盒进行检测。
4.如权利要求3所述的构建方法,其特征在于,进行共同孵育后,所述细胞活力增加30%~110%,例如36%、90%或106%。
5.如权利要求1所述的构建方法,其特征在于,所述髓样分化因子的表达水平通过qPCR技术进行检测。
6.如权利要求5所述的构建方法,其特征在于,进行共同孵育后,所述髓样分化因子的表达水平提高2~240倍,例如238倍。
7.如权利要求1所述的构建方法,其特征在于,所述孵育溶液中KLH的浓度为1×10-3~1×10-1mg/mL。
8.如权利要求1~7任一项所述的构建方法,其特征在于,所述共同孵育的时间为48~72h。
9.一种免疫毒性的评价模型,其特征在于,通过如权利要求1~8任一项所述的构建方法获得。
10.一种免疫毒性的评价方法,其特征在于,通过如权利要求9所述的评价模型进行评价。
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