慢性乙型肝炎患者外周血中记忆T细胞亚群分布研究

目的了解慢性乙型病毒性肝炎患者外周血中CD4+、CD8+T细胞的分布,以及相应记忆细胞亚群的表达水平,为研究记忆T淋巴细胞在抗乙肝病毒感染中的作用提供基础。方法采用多色流式细胞仪检测技术,检测慢性乙肝病例和健康对照外周血单个核细胞(PBMC)中T淋巴细胞表面标志CD3、CD4、CD8、CD45RO、CD62L、CCR7的表达情况。结果乙肝组CD4+、CD8+T淋巴细胞分别占外周血PBMC的(11.56±5.78)%、(13.11±6.64)%,均低于对照组,差异均有统计学意义(P值均<0.05)。乙肝组和对照组外周血PBMC中,CD4+TEM的表达水平高于CD4+TCM,CD8+TEM的表达水平高于CD8+TCM,差异均有统计学意义(P值均<0.05);CD4+TEM、CD4+TCM所占CD4+Tm的比例,差异均无统计学意义(P值均>0.05);CD8+TEM、CD8+TCM所占CD8+Tm的比例差异无统计学意义(P值均>0.05)。结论慢性乙肝病例外周血PBMC中,CD4+、CD8+T淋巴细胞均减少。对照组和乙肝组外周血PBMC中,CD4+、CD8+T记忆淋巴细胞均以TEM为主。

IL-7/IL-15/IL-21/IL-23体外联合刺激有效促进人CD8+中央记忆性T细胞生成

目的比较不同细胞因子组合联合抗CD3/CD28磁珠体外诱导CD8^(+)中央记忆性T细胞(Tcm)体生成的作用,为CD8^(+)Tcm过继性免疫疗法的临床应用提供实验依据。方法分离健康供者外周血单个核细胞(PBMC),应用免疫磁珠法分离纯化初始CD8^(+)T细胞,CD3/CD28抗体偶联磁珠刺激48 h,同时根据细胞因子种类和组合进行分组:白细胞介素2(IL-2)、IL-7/IL-15、IL-7/IL-15/IL-21、IL-7/IL-15/IL-21/IL-23。采用全自动血球计数仪进行细胞计数;5,6-羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)染色检测不同细胞因子组合诱导CD8^(+)T细胞的增殖水平,流式细胞术检测不同细胞因子组合诱导的CD8^(+)T细胞免疫记忆表型,细胞凋亡及细胞γ干扰素(IFN-γ)、穿孔素以及颗粒酶B水平,Western blot法检测不同细胞因子组合诱导的CD8^(+)T细胞的B细胞淋巴瘤因子2(Bcl2)蛋白表达。结果与其他组合相比,IL-7/IL-15/IL-21/IL-23处理CD8^(+)T细胞后,促进T细胞增殖,CD62L^(+)/CD45RA^(-)的CD8^(+)中央记忆性T细胞亚群的表达显著增加。同时,IL-7/IL-15/IL-21/IL-23处理组显著降低细胞的IFN-γ、穿孔素以及颗粒酶B的分泌水平,细胞的凋亡水平也显著降低。结论IL-7/IL-15/IL-21/IL-23联合刺激有效诱导初始CD8^(+)T细胞向CD8^(+)Tcm分化,有助于优化过继免疫治疗中CD8^(+)Tcm的体外扩增策略。

Photoprotective Effects of Hydroxychloroqine and TCMs on Human Keratinocytes Damaged from Ultraviolet Irradiation

Objective: To investigate damage effects of ultraviolet irradiation on eternal keratinocyte-HaCaT cells and to evaluate photo-protective efficiency of hydroxychloroqine and Traditional Chinese Medicines(epigallocatechingallate[EGCG], baikal skullcap root and szechwan lovge rhizome) on HaCaT cells damaged by middle wave ultraviolet(UVB) irradiation. Methods: Subconfluent HaCaT cells were sham or UVB irradiated and treated with above TCM agents. The damage degree of HaCaT cells was observed by a light microscop. Cell growth was recorded by cell count and cellular activity was detected by MTT method. The secretion amount of IL-6 and TNF-α was measured by ELISA. Results: The irradiation damage of HaCaT cells was depended on the irradiated dosages and cellular activity was reduced by 36%-80%, with a maximum decrease over 90% after 72 h. The intervention of the above drugs may increase the cellular activity by 10%-72%. The photo-protective efficiency was more apparent in EGCG (from 1.19±0.07 to 1.28±0.06, P<0.01) than that in hydroxychloroqine (from 0.43±0.04 to 0.96±0.04, P<0.05). The other two tested drugs also showed photo-protective effect(from 0.44±0.07 to 1.21±0.02, P<0.05). As to cytokine secretion, EGCG could decline the secretion amount of IL-6 and TNF-α apparently. Hydroxychloroqine and baikal skullcap root baikal skullcap root could only reduce the secretion of IL-6. The secretion of IL-6 and TNF-α could not be inhibited by szechwan lovge rhizome. Conclusion: The injury effect of UVB irradiation on cultured keratinocytes is dose-dependent and the tested drugs have photo-protective potency. Inhibition of cytokine secretion may be one of the mechanisms related to reducing the damage effect of UVB irradiation.

丁基羟基茴香醚对CD4^+中央记忆性T细胞体外扩增的影响

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。