纳米佐剂对树突状细胞活化效果的评估  

Evaluation of the activation efficacy of nano-adjuvant on dendritic cells

作　　者：赵莹 时洪艳[2] 李明蔚 吴洋 闫浩鑫 郭龙军[2] 冯力 ZHAO Ying;SHI Hong-yan;LI Ming-wei;WU Yang;YAN Hao-xin;GUO Long-jun;FENG Li(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163000,China;Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163000 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第8期853-858,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2021YFD1801105)。

摘　　要：为评价新型纳米佐剂乳化抗原的免疫效果,本研究以FITC标记的卵清蛋白(FITC-OVA)为抗原,以新型纳米佐剂乳化制备OVA抗原(Nano-OVA),并将ISA 201佐剂乳化制备OVA抗原(201-OVA),抗原浓度均为100μg/mL。将10μL Nano-OVA和201-OVA分别加入小鼠骨髓源树突状细胞(DC2.4)中,2 h后收集细胞样品,利用流式细胞术检测各组DC2.4对OVA抗原的吞噬水平(FITC-OVA+%)及交叉递呈OVA抗原肽(SIINFEKL)水平(PE-25-D1.16+%)。将DC2.4分别与5μL Nano-OVA和201-OVA共孵育24 h后收集细胞样品,利用荧光定量PCR(qPCR)检测各组DC2.4共刺激分子CD86、细胞因子IL-12p40和IL-6 mRNA的转录水平。流式细胞术检测结果显示,与201-OVA组相比,Nano-OVA组DC2.4 FITC-OVA+%和PE-25-D1.16+%均极显著升高(P<0.001),且均极显著高于对照组(P<0.001)。qPCR结果显示,Nano-OVA组DC2.4中IL-12p40 mRNA的转录水平极显著高于对照组(P<0.01),显著高于201-OVA组(P<0.05);Nano-OVA组DC2.4中CD86及IL-6 mRNA的转录水平极显著高于201-OVA和对照组(P<0.01),但201-OVA组和对照组DC2.4中CD86、IL-12p40及IL-6 mRNA的转录水平均无显著差异(P>0.05)。将50μL Nano-OVA和201-OVA分别通过足垫皮下免疫BALB/c小鼠,12 h后剖杀各组小鼠分离其腘窝淋巴结,制备冷冻切片,利用免疫组化试验检测各组FITC-OVA在小鼠腘窝淋巴结中的分布,并利用Image J软件量化各组小鼠腘窝淋巴结FITC-OVA平均荧光强度。免疫组化试验结果显示,与201-OVA组相比,Nano-OVA组小鼠腘窝淋巴结FITC-OVA荧光分布更广;荧光强度观察结果显示,Nano-OVA和201-OVA组小鼠腘窝淋巴结中FITC-OVA的平均荧光强度差异不显著(P>0.05),但均显著强于对照组(P<0.05)。上述结果表明,Nano-OVA可更高效地被DC2.4吞噬、交叉递呈以及促进DC2.4的活化,诱导机体产生更强的免疫应答及靶向淋巴结的能力。本研究初步评估了新型纳米佐剂对DC的活化效果及其向小鼠淋巴结靶向引流FITC-OVA抗原的效果,为其�To evaluate the immune efficacy of a new nano-adjuvant emulsified antigen,FITC-labeled ovalbumin(FITC-OVA)was selected as the tested antigen to prepare OVA antigen(Nano-OVA)with new nano-adjuvant emulsification,and the ISA 201 adjuvant was emulsified to prepare OVA antigen(201-OVA),both with antigen concentration of 100μg/mL.Nano-OVA and 201-OVA with volume of 10μL were separately added to mouse bone marrow-derived dendritic cells(DC2.4).After incubation for 2 hours,cell samples were collected and flow cytometry was used to detect the phagocytic levels of OVA internalization(FITC-OVA+%)and cross-presentation of OVA antigen peptide(SIINFEKL)levels(PE-25-D1.16+%)by DC2.4 in each group.After co-incubation of DC2.4 with 5μL Nano-OVA and 201-OVA for 24 hours respectively,the cell samples were collected,and the transcriptional levels of costimulatory molecules CD86,cytokines IL-12p40 and IL-6 mRNA in each group were detected by fluorescent quantitative PCR(qPCR).The flow cytometry results showed that compared with 201-OVA group,FITC-OVA+%and PE-25-D1.16+%of DC2.4 in Nano-OVA group were significantly increased(P<0.001),and were significantly higher than those in the control group(P<0.001).The qPCR results revealed a significant upregulation level of IL-12p40 mRNA transcription in DC2.4 treated with Nano-OVA compared to untreated controls(P<0.01)or those treated with 201-OVA(P<0.05).Furthermore,the transcriptional level of CD86 and IL-6 mRNA in Nano-OVA group were significantly higher than those in 201-OVA and control group(P<0.01),while but there was no significant difference in the transcription levels of CD86,IL-12p40 and IL-6 mRNA in DC2.4 between 201-OVA and control group(P>0.05).BALB/c mice were immunized subcutaneously with 50μL of Nano-OVA and 201-OVA through foot pad,respectively.After 12 hours,the popliteal lymph nodes of each group were isolated,and frozen sections were prepared to examine the distribution of FITC-OVA by immunohistochemical method and to quantify the average fluorescence intensity of FITC-

关 键 词：纳米佐剂 交叉递呈 CD86 IL-12P40 IL-6 

分 类 号：S852.6[农业科学—基础兽医学]