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目的:体外分离扩增HBV多肽特异性IFN-γ分泌细胞,并进行自体扩增,检测其扩增后的多肽特异和IFN-分泌功能。方法:酶联免疫斑点法筛选HBV自然感染献血个体,分离外周血单核细胞,体外刺激并分选IFN-γ分泌细胞,并进行自体细胞体外扩增,流式细胞术检测扩增后细胞的CD4、CD8表型,多肽负载自体淋巴瘤细胞系(lymphoblastoid cell lines,LCLs)细胞检测其多肽特异性和IFN-γ分泌能力。结果:经过4周体外自体扩增,HBV特异性IFN-γ分泌细胞扩增数量达1000多倍,CD4和CD8比例变化不显著,4周扩增后的T淋巴细胞能有效识别HBV特异性多肽并分泌IFN-γ。结论:HBV多肽特异性IFN-γ分泌细胞能在体外有效扩增并保持功能表型不变。
OBJECTIVE: To isolate and amplify HBV polypeptide-specific IFN-γ secreting cells in vitro and to perform self-amplification to detect the specific and IFN-secreting function of the amplified polypeptide. Methods: Human peripheral blood mononuclear cells were screened by enzyme-linked immunosorbent assay (EMSA) for spontaneous infection of HBV. IFN-γ secreting cells were stimulated and sorted in vitro, and the autologous cells were expanded in vitro. Flow cytometry CD4, CD8 phenotype and polypeptide-loaded lymphoblastoid cell lines (LCLs) cells were tested for their polypeptide specificity and IFN-γ secretion ability. Results: After 4 weeks of autologous expansion, the number of HBV-specific IFN-γ-secreting cells expanded more than 1000 times and the ratio of CD4 and CD8 did not change significantly. After 4 weeks of expansion, T lymphocytes could effectively recognize and secrete HBV specific peptides IFN-γ. CONCLUSION: HBV polypeptide-specific IFN-γ secreting cells can effectively expand in vitro and maintain the functional phenotype.