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目的探讨丁基羟基茴香醚(BHA)在体外诱导扩增CD4~+中央记忆性T细胞(TCM)中的作用,提高过继免疫疗法的有效性。方法将健康人外周血单个核细胞使用密度梯度离心的方法分离,磁珠分选得到CD4~+T淋巴细胞,将细胞分为实验组和对照组,实验组加入不同浓度的BHA(10、20、40μmol/L)),对照组加入等体积的PBS,经过细胞培养使用流式细胞仪对其TCM细胞亚群的分化扩增进行检测。结果流式结果显示,实验组的CD4~+TCM中有扩增活性的细胞比例明显增加,其中对照组的比例为73.2%,低剂量组的比例为78.8%,中剂量组的比例为87.7%,高剂量组为89.9%,并有剂量依赖效应。对照组的CD45RA-CD62L+CCR7+TCM细胞占总CD4~+T细胞的比例为10.60%;BHA浓度为10、20、40μmol/L时比例分别为23.52%、32.39%、28.41%。对体外培养15 d后的细胞进行了凋亡检测,结果显示,与对照组比较,实验组中PI+Annexin V+发生凋亡的细胞从27.1%下降到了12.5%。结论在CD4~+T细胞的体外扩增实验中,BHA可以显著提高TCM细胞亚群的比例,在CD4~+T的体外培养过程中,BHA可以显著抑制细胞的凋亡。
Objective To investigate the role of butylated hydroxyanisole (BHA) in inducing the expansion of CD4 ~ + central memory T cells (TCM) in vitro and to improve the effectiveness of adoptive immunotherapy. Methods Peripheral blood mononuclear cells were isolated by density gradient centrifugation. CD4 ~ + T lymphocytes were sorted by magnetic beads. The cells were divided into experimental group and control group. The experimental group was given different concentrations of BHA (10, 20, , 40μmol / L). The control group was added with equal volume of PBS, and the differentiation and expansion of its TCM cell subpopulation were detected by flow cytometry. Results The results of flow cytometry showed that the proportion of cells with activity of amplification in CD4 ~ + TCM group increased significantly, which was 73.2% in control group, 78.8% in low dose group and 87.7% in middle dose group. , High-dose group was 89.9%, and a dose-dependent effect. The proportion of CD45RA-CD62L + CCR7 + TCM cells in the control group was 10.60% of the total CD4 + T cells. The ratios of BHA at 10, 20 and 40μmol / L were 23.52%, 32.39% and 28.41% respectively. Apoptosis assay was performed on the cells cultured in vitro for 15 days. The results showed that compared with the control group, the apoptosis rate of PI + Annexin V + in experimental group decreased from 27.1% to 12.5%. Conclusion BHA can significantly increase the proportion of TCM cell subsets during the in vitro expansion of CD4 ~ + T cells. BHA can significantly inhibit the apoptosis of CD4 ~ + T cells in vitro.