内毒素打击后小鼠脾脏T淋巴细胞功能恢复动态研究

Dynamic recovery of splenic T lymphocytes function in mice with endotoxin-induced sepsis

目的探讨脂多糖(LPS)打击后,小鼠脾脏T淋巴细胞亚群及功能的动态恢复过程。方法采用高剂量LPS(10 mg/kg)腹腔单次注射建立小鼠脓毒症模型,在LPS打击后第0 d、7 d,14 d和28 d,使用流式细胞术动态检测脾脏CD4^(+)T和CD8^(+)T细胞中初始T细胞、效应记忆T细胞和中央记忆T细胞的比例以及细胞表面活化分子(CD38和CD69)和共抑制分子(PD-1和TIGIT)的表达水平,分泌细胞因子IL-2和IFN-γ及释放颗粒酶B的能力。正态分布数据采用one-way ANOVA检验进行方差分析,Holm-Sidak’s进行多重比较;偏态分布数据则采用Kruskal-Wallis检验进行分析,Bonferroni法进行多重比较。结果LPS打击7 d后,与基线相比,小鼠脾脏初始CD4^(+)T和CD8^(+)T细胞比例均显著降低(F=52.22、P<0.0001,F=10.87、P=0.0019);效应记忆CD4^(+)T和CD8^(+)T细胞比例均显著增高(F=20.54、P<0.0001,F=26.03、P<0.0001);CD4^(+)CD38^(+)(F=35.40、P<0.0001)、CD4^(+)CD69^(+)(F=45.65、P<0.0001)、CD4^(+)PD-1^(+)(F=20.55、P<0.0001)、CD4^(+)TIGIT^(+)(F=19.20、P<0.0001)、CD8^(+)CD38^(+)(F=56.76、P<0.0001)、CD8^(+)CD69^(+)(F=59.47、P<0.0001)、CD8^(+)PD-1^(+)(F=11.15、P=0.0013)和CD8^(+)TIGIT^(+)(F=21.21、P<0.0001)细胞比例均显著增高,差异均有统计学意义。与基线相比,LPS打击14 d后细胞活化水平、共抑制分子表达水平逐渐恢复,但至28 d时,初始CD4^(+)T细胞(F=52.22、P<0.0001)、效应记忆CD4^(+)T细胞(F=20.54、P=0.0093)、CD4^(+)CD69^(+)(F=45.65、P=0.0037)、CD4^(+)PD-1^(+)(F=20.55、P=0.0255)和CD4^(+)TIGIT^(+)(F=19.20、P=0.0087)未恢复至基线水平;CD8^(+)IFN-γ^(+)T细胞比例(F=14.33、P=0.0343)仍高于基线水平,差异均有统计学意义。结论LPS打击后28 d小鼠脾脏CD4^(+)T和CD8^(+)T细胞恢复过程存在差异,CD4^(+)T细胞功能总体恢复较CD8^(+)T细胞更为缓慢。

Objective To investigate the dynamic recovery process of splenic T lymphocyte function and their subsets in mice after lipopolysaccharide(LPS)administration.Methods High dose LPS(10 mg/kg,once)was administered intraperitoneally to establish septic mouse model.On day 0,day 7,day 14,and day 28 after LPS treatment,flow cytometry was used to check the proportions of naive T cells,effector memory T cells,and central memory T cells in splenic CD4^(+)and CD8^(+)T cells.The expression of cell surface activation molecules(CD38,CD69)and co-inhibitory molecules(PD-1,TIGIT),as well as the ability of splenic CD4^(+)T cells to secrete cytokines IL-2,IFN-γand CD8^(+)T cells to release granzyme B.Normal distribution data used one-way ANOVA test for variance analysis and used holm-Sidak’s test for multiple comparison.Skewness distribution data used Kruskal-wallis test for variance analysis and used Bonferroni method for multiple comparison.Results On day 7 after LPS challenge,compared with the baseline,the proportions of splenic naive CD4^(+)and CD8^(+)T cells in mice were significantly decreased(F=52.22,P<0.0001;F=10.87,P=0.0019);the proportions of effector memory CD4^(+)and CD8^(+)T cells were significantly increased(F=20.54,P<0.0001;F=26.03,P<0.0001);the proportions of CD4^(+)CD38^(+)(F=35.40,P<0.0001),CD4^(+)CD69^(+)(F=45.65,P<0.0001),CD4^(+)PD-1^(+)(F=20.55,P<0.0001),CD4^(+)TIGIT^(+)(F=19.20,P<0.0001),CD8^(+)CD38^(+)(F=56.76,P<0.0001),CD8^(+)CD69^(+)(F=59.47,P<0.0001),CD8^(+)PD-1^(+)(F=11.15,P=0.0013)and CD8^(+)TIGIT^(+)(F=21.21,P<0.0001)cells were significantly increased.These indicators gradually recovered compared with the baseline within 14 days.On day 28 after LPS challenge,the proportions of naive CD4^(+)T cells(F=52.22,P<0.0001),effector memory CD4^(+)T cells(F=20.54,P=0.0093),CD4^(+)CD69^(+)(F=45.65,P=0.0037),CD4^(+)PD-1^(+)(F=20.55,P=0.0255)and CD4^(+)TIGIT^(+)(F=19.20,P=0.0087)cells were not returned to the baseline level;the proportion of CD8^(+)IFN-γ^(+)T cells(F=14.33,P<0.0001)was still higher than the baseline.Conclusions After LPS challenge,differences exist during the recovery process of splenic CD4^(+)T and CD8^(+)T cells in mice within 28 days.The overall recovery process of splenic CD4^(+)T cells function in septic mice was slower than that of CD8^(+)T cells after LPS treatment.