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目的优化乙肝蛋白抗原表位特异性CTL体外扩增条件。方法选择急性自限性HBV感染者外周血,分离PBMC,体外用乙肝蛋白抗原表位多肽特异性刺激,特定天数后收集细胞,用CD8抗体与MHC-I类分子HBV抗原肽四聚体进行染色流式检测,检测扩增后乙肝蛋白抗原表位特异性CTL在CD8阳性细胞群中所占的频率。结果1)在不同多肽浓度刺激下,当浓度在20 ug/mL时,所测得特异性CTL占PBMC中CD8+T细胞的频率达到最高峰;2)固定刺激多肽浓度,在不同的刺激天数获得的特异性CTL频率第10天时最高;3)在不同的细胞因子培养条件中,当加入的细胞因子IL-2,IL-7,IL-15组合时所获得的特异性CTL频率最高。结论在刺激肽浓度为20μg/mL,并加入适量的细胞因子(IL-2,IL-7,IL-15),刺激10天的条件下,所获得的乙肝蛋白抗原特异性CTL频率最高。
Objective To optimize the conditions for the amplification of hepatitis B virus epitopes specific CTL in vitro. Methods PBMCs were isolated from peripheral blood of patients with acute self-limited HBV infection and stimulated with the specific antigen of hepatitis B virus epitopes in vitro. Cells were collected after a specific number of days and stained with CD8 antibody and MHC class I antigen HBV peptide tetramers Flow cytometry was used to detect the frequency of HBV CTL epitopes in CD8 positive cells after amplification. Results 1) Under the stimulation of different peptide concentrations, the specific CTLs accounted for the highest frequency of CD8 + T cells in PBMCs at a concentration of 20 ug / mL; 2) the concentration of immobilized stimulating peptides at different stimulation days The specific CTL frequency obtained was the highest on the 10th day; 3) The highest CTL frequency was obtained when the cytokines IL-2, IL-7 and IL-15 were added in different cytokine culture conditions. Conclusion The highest frequency of CTLs was obtained under stimulation of peptide concentration of 20 μg / mL with appropriate amount of cytokines (IL-2, IL-7, IL-15) for 10 days.