PD-L1阻断对脓毒症小鼠T淋巴细胞凋亡及单核细胞功能的影响

Effect of PD-L1 blockade on T lymphocyte apoptosis and monocyte dysfunction of mice with sepsis

目的:探讨程序性死亡受体-1配体(PD-L1)阻断对脓毒症小鼠T淋巴细胞凋亡及单核细胞功能的影响。方法:30只8~10周龄清洁级C57BL/6雄性小鼠随机分为3组:sham组、模型组、PD-L1拮抗组,每组10只,模型组、PD-L1拮抗组小鼠采用盲肠结扎穿孔手术(CLP)构建脓毒症小鼠模型,sham组小鼠暴露盲肠后即进行伤口缝合,不结扎。PD-L1拮抗组小鼠术后腹腔注射抗PD-L1抗体(50μg/只),每6 h注射1次,连续4次,sham组和模型组小鼠腹腔注射等剂量生理盐水。流式标记法检测各组小鼠CD3+T细胞表面PD-1和单核细胞CD11b+表面PD-L1表达,HE染色观察组织病理学改变,电镜下观察脾脏和胸腺超微病理结构改变,TUNEL染色检测脾脏和胸腺组织细胞凋亡、流式细胞术检测胸腺组织T淋巴细胞凋亡,ELISA检测Caspase-3和TNF-α、IL-6及IL-10表达。结果:与sham组相比,模型组小鼠CD3^(+)、CD11b^(+)表面PD-1和PD-L1表达、脾脏和胸腺组织细胞凋亡率、T淋巴细胞凋亡率、Caspase-3、TNF-α、IL-6、IL-10蛋白表达显著升高(P<0.05);与模型组相比,PD-L1拮抗组小鼠CD3^(+)、CD11b^(+)表面PD-1和PD-L1表达、脾脏和胸腺组织细胞凋亡率、T淋巴细胞凋亡率、Caspase-3、TNF-α、IL-6蛋白表达明显降低,IL-10表达明显升高(P<0.05)。结论:PD-L1阻断可有效降低脓毒症小鼠T细胞凋亡率,改善其单核细胞功能。

Objective:To investigate effect of programmed death-1 ligand(PD-L1)blockade on T lymphocyte apoptosis and monocyte dysfunction of mice with sepsis.Methods:Thirty 8~10 weeks of clean grade C57BL/6 male mice were randomly divided into3 groups:sham group,model group and anti-PD-L1 antagonist group,with 10 mice in each group.Sepsis mouse model was replicated by cecal ligation and puncture(CLP)in model group and PD-L1 antagonist group.Wound was sutured only after exposure to cecum,and no ligation was performed in sham group.Mice in PD-L1 antagonist group were intraperitoneally injected with anti-PD-L1 antibody(50μg/mice),once every 6 h for 4 times.Mice in sham group and model group were intraperitoneally injected with same amount of normal saline.Flow cytometry to detect PD-1 expression on surface of CD3+T cells and PD-L1 expression on surface of monocytes CD11b+.HE staining to observe histopathological changes,and electron microscopy to observe ultrastructural pathological changes of spleen and thymus.TUNEL staining to detect apoptosis of spleen and thymus tissue cells,and flow cytometry to detect apoptosis of thymus T lymphocytes.Expressions of Caspase-3,TNF-α,IL-6 and IL-10 were detected by ELISA.Results:Compared with sham group,expressions of PD-1 and PD-L1 on surface of CD3^(+),CD11b^(+),apoptosis rates of spleen and thymus tissues,apoptosis rate of T lymphocytes,protein expressions of Caspase-3,TNF-α,IL-6 and IL-10 in model group were significantly increased(P<0.05).Compared with model group,expressions of PD-1 and PD-L1 on surface of CD3^(+)and CD11b^(+)in PD-L1 antagonistic group were significantly decreased,and apoptosis rates of spleen and thymus tissues,apoptosis rate of T lymphocytes,expressions of Caspase-3,TNF-αand IL-6 were significantly decreased,and expression of IL-10 was significantly increased(P<0.05).Conclusion:PD-L1 blockade can effectively decrease apoptotic rate of T cells and improve function of monocytes of septic mice.