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

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,为其临床应用提供参考依据。

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-OVA by Image J software.The results of immunohistochemistry showed a wider distribution in the popliteal lymph nodes of mice in Nano-OVA group compared with the 201-OVA group,while there was no significant difference in the average fluorescence intensity of FITC-OVA between Nano-OVA and 201-OVA groups(P>0.05),but both were significantly stronger than the control group(P<0.05).These findings suggest that Nano-OVA can be more efficiently engulfed and cross-presented by DC2.4,resulting in enhanced activation capacity of DC2.4 and potentially leading to a robust immune response in the body,particularly with advantages in targeting lymph nodes.This study preliminarily evaluated the activation efficacy of the novel nano adjuvant on DC and its targeted drainage of FITC-OVA antigen to mouse lymph nodes,providing a reference for its clinical application.