【摘要】 目的建立真核表达的人CD1d（hCD1d）二聚体的纯化方法,并制备人工抗原提呈细胞,研究其对iNKT细胞的刺激效应。方法提取pFastBac^TMDual+[β2m+CD1d/IgG1-Fc]昆虫杆状病毒质粒并转染至sf9细胞中,收集病毒感染上清,通过ELISA法鉴定融合蛋白中人IgG-Fc、人CD1d和人β2m等组分并测定hCD1d二聚体浓度。探索保持hCD1d二聚体完整构象的亲和层析条件,对hCD1d二聚体进行纯化和浓缩。应用加载α-GalCer的hCD1d二聚体制备人工抗原提呈细胞刺激健康个体外周血单个核细胞（PBMC）,检测Th1、Th2、Th17细胞因子分泌水平与iNKT细胞的扩增效率。结果收集的第4代病毒感染上清中目的蛋白表达量较高。抗Ig-Fc抗体ELISA、抗CD1d抗体与抗β2m抗体双抗夹心ELISA检测显示sf9细胞表达的hCD1d二聚体构象正确,两种方法检测的蛋白浓度分别为（1.05±0.20）μg/mL和（0.85±0.01）μg/mL。亲和层析纯化结果显示用pH2.5的0.1 mol/L柠檬酸可较好地实现hCD1d二聚体有效纯化。健康个体iNKT细胞经人工抗原提呈细胞刺激3 d后主要产生Th1型细胞因子,上清中IFN-γ、TNF-α含量分别为（1876.26±96.73）pg/mL和（1186.25±98.63）pg/mL,明显高于对照组（79.98±18.52）pg/mL和（29.96±4.43）pg/mL,差异具有统计学意义（均P<0.01）,其余Th2型（IL-4）、Treg型（IL-10）和Th17型（IL-17）细胞因子未见明显升高,差异均无统计学意义（均P>0.05）,与iNKT细胞接收刺激后分泌细胞因子谱基本一致。健康个体PBMC接受刺激后,iNKT细胞迅速扩增,刺激第7天iNKT细胞占PBMC（4.17±0.76）%,而未包被hCD1d二聚体的微球对照组iNKT细胞占PBMC的比例仅为（0.41±0.09）%。这些结果说明包被有hCD1d二聚体的人工抗原提呈细胞可特异性地刺激iNKT细胞分泌Th1型细胞因子并有效扩增。结论成功制备hCD1d二聚体,并建立基于人工抗原提呈细胞的iNKT细胞有效的刺激与扩增体系,为后续对iNKT细胞深入研究打下了基础。更多还原

【Abstract】 Objective To establish a method for purification of human CD1 d（hCD1 d）dimer expressed in eukaryotic cells and prepare artificial antigen-presenting cells to study their stimulation and expansion effects on iNKT cells.Methods pFastBac^TMDual+[β2 m+CD1 d/IgG1-Fc]insect baculovirus plasmid was extracted and transfected into sf9 cells,and the virus infection supernatant was collected.The components of human IgG-Fc,human CD1 d and human β2 m in the fusion protein were identified by ELISA.Different eluting conditions were tried to set up optimized affinity chromatography method with high purity and complete conformation of hCD1 d dimer.Artificial antigen-presenting cells（aAPCs）were prepared by coating α-GalCer-loaded hCD1 d dimer and anti-CD28 mAb on the microbeads to stimulate peripheral blood mononuclear cells（PBMC）in healthy individuals,followed by detection of Th1/Th2/Th17 cytokine levels and iNKT cell expansion rate.Results The supernatant of the fourth-generation virus infection had highest expression of the hCD1 d dimer and were selected for further test.Sandwich ELISA by anti-Ig-Fc antibodies or by antibodies against CD1 d and β2 m was performed and showed（1.05±0.20）μg/mL and（0.85±0.01）μg/mL of hCD1 d dimer,respectively.Affinity chromatography purification showed that 0.1 mol/L citric acid with pH 2.5 maintained conformation of hCD1 d dimer and achieved effective purification.Human iNKT cells mainly produced Th1-type cytokines after stimulation by α-GalCer-loaded hCD1 d aAPCs for three days.The contents of IFN-γ and TNF-α supernatants were（1876.26±96.73）pg/mL and（1186.25±98.63）pg/mL,which were significantly higher than the control group（79.98±18.52）pg/mL and（29.96±4.43）pg/mL,with significant difference（P<0.01）.The remaining Th2（IL-4）,Treg（IL-10）and Th17（IL-17）cytokines were not significantly increased.The difference was not statistically significant（P>0.05）.This is consistent with the spectrum of cytokines secreted form iNKT cells after receiving stimulation.In the experimental group,after healthy individuals received PBMC stimulation,iNKT cells expanded rapidly,iNKT cells accounted for（4.17±0.76）% of PBMC on the seventh day of stimulation.However,the percentage of iNKT cells in the microsphere control group not coated with hCD1 d dimer in PBMC was only（0.41±0.09）%.In addition,human iNKT cells expanded rapidly upon α-GalCer-loaded hCD1 d aAPCs stimulation.These results indicate that purified hCD1 d dimer on aAPCs efficiently stimulated iNKT cell responses.Conclusion This study set up a method for preparation of purified hCD1 d dimer and established an α-GalCer-loaded hCD1 d aAPCs stimulation system to promote iNKT cell cytokine production and expansion.更多还原