摘要
目的改良胶原蛋白诱导性关节炎(CIA)大鼠脾脏来源耐受性树突状细胞(DC)的制备技术,以获取高质量的实验细胞。方法在提取脾脏单个核细胞过程中,对制备单细胞悬液流程以及单个核细胞铺板后孵育时间进行改进,培养初期加入核因子κB(NF-κB)寡核苷酸诱骗剂诱导为耐受性DC。显微镜观察细胞形态,台盼蓝染色观察细胞活力,流式细胞术检测细胞CD103(OX62)、CD80和CD86表达,噻唑蓝(MTT)法检测该DC刺激淋巴细胞的增殖能力。结果DC的细胞活力>90%,OX62表达率>87.4%;共刺激分子低表达,DC刺激淋巴细胞增殖能力低。结论将CIA大鼠脾脏单个核细胞的制备方法进行改良,经NF-κB寡核苷酸诱骗剂诱导可获得高活力、高稳定性的耐受性DC,且细胞纯度明显提高。
Objective To yield high-quality tolerogenic dendritic cells(TolDCs)for therapeutic intervention by a refined technique of TolDC preparation from the spleen of modified collagen-induced arthritis(CIA)rats.Methods The refinements took place amid monocellular cell isolation process,including both the preparation of single cell suspension and the adjustment of incubation time after plate seeding of mononuclear cells.TolDCs were induced by administration of NF-κB oligonucleotide(ODN)decoy at the initiation of cell culture.Cell morphology was examined under a microscope and cell viability was revealed by trypan blue staining.Expression of classical cell identity and activation makers,CD103(OX62),CD80 and CD86 was determined by flow cytometry;meanwhile,DC-stimulated lymphocyte proliferation was measured by MTT assay following mixed lymphocyte reaction(MLR).Results With the established approach,the viability ratio of resulting cells reached over 90%and the proportion of OX62 positive ones was above 87.4%,which altogether confirmed their ideal DC phenotype.Functionally,the tolerogenic nature of the NF-κB ODN-treated DCs was further unveiled by the low expression of costimulatory molecules(CD80 and CD86)and the abrogated capacity of lymphocyte stimulation effect.Conclusion Compared with TolDCs generated by conventional protocol,CIA rat spleen-derived TolDCs isolated by the optimized splenic mononuclear cell preparation procedure and induced by NF-κB ODN decoy show the higher regulatory activity,higher phenotypic stability,higher purity and tolerogenic properties.
作者
谭艳
田娅玲
宁丽常
蒋红梅
TAN Yan;TIAN Yaling;NING Lichang;JIANG Hongmei(Department of Microbial Immunology,Medical Laboratory,Guizhou Medical University,Guiyang 550025;Department of Clinical Laboratory,Minda Hospital,Hubei University for Nationalities,Enshi 445000;Department of Laboratory Medicine,First Affiliated Hospital,Nanhua University,Hengyang 421001,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第12期1076-1081,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金地区科学基金(81660361,81960393)。
