内毒素耐受树突状细胞免疫功能变化及其对脓毒症模型小鼠的作用

Changes of endotoxin tolerant dendritic cell immune function and its effect on sepsis in mouse model

目的探讨内毒素耐受树突状细胞(endotoxin tolerant dendritic cell,ETDC)免疫功能的改变,观察其对脓毒症模型小鼠的作用。方法以脂多糖刺激小鼠来源骨髓树突状细胞制备ETDC,流式细胞仪检测ETDC表面标志物主要组织相容性复合体(major histocompatibility complex,MHC)Ⅱ、CD86和CD11c表达水平,细胞计数试剂盒检测T淋巴细胞增殖率,ELISA检测细胞上清液中细胞因子水平。将36只BALB/c小鼠分为4组,空白对照组不做任何处理,假手术组行单纯切开缝合术,脓毒症组和ETDC回输治疗组行盲肠结扎穿孔术建立脓毒症模型前,分别尾静脉回输0.9%氯化钠溶液和ETDC与0.9%氯化钠混悬液。检测各组小鼠血丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspatate,AST)、肿瘤坏死因子(tumor necrosis factro,TNF)-α、白细胞介素(interleukin,IL)-6、IL-10水平和脾脏辅助性T-淋巴细胞(help T cells,Th)17/调节性T淋巴细胞(regulatory T cells,Treg)比例,观察小鼠肝脏、肾脏和回肠病理学表现。组间比较采用t检验或χ^2检验。结果ETDC表面MHCⅡ、CD86和CD11c表达分别为70.4%、43.1%、73.1%,较成熟树突状细胞(mature dendritic cell,mDC)的96.1%、89.5%和84.6%显著下降(χ^2值分别为56.47、83.78和23.29,均P<0.01)。ETDC细胞上清液中IL-10、TNF-α和IL-6分别为(978.04±56.70)、(980.34±111.96)和(12 743.03±865.81)ng/L,mDC分别为(741.35±99.23)、(1 703.11±117.00)和(19 052.28±1 145.84)ng/L,差异均有统计学意义(t值分别为5.073、10.93和10.76,均P<0.01)。ETDC和T淋巴细胞1∶5、1∶10共培养的增殖率分别为(676.95±85.99)%和(514.00±106.39)%,mDC为(956.25±127.12)%和(772.07±214.08)%。ETDC回输治疗组小鼠肝脏、肾脏和回肠病理学改变明显轻于脓毒症组。ETDC回输治疗组小鼠血清ALT和AST为(299.71±36.91)和(690.39±154.92)U/L,TNF-α、IL-6和IL-10分别为(0.067±0.005)、(0.428±0.051)和(0.058±0.005)ng/L,脓毒症组ALT和AST为(620.67±69.27)和(1 430.17±134.05)U/L,TNF-α、IL-6和IL-10分别为(0.085±0.007)、(0.774±0.088)和(0.036±0.005)ng/L,差异均有统计学意义(t值分别为11.60、10.96、5.991、8.657和8.042,均P<0.01)。ETDC回输治疗组小鼠Treg/Th17比例为23.4%,脓毒症组为60.8%(χ^2=28.69,P<0.01)。结论ETDC回输对脓毒症模型小鼠具有保护作用,可能是通过调节T淋巴细胞分化发挥了负向免疫调控作用。

Objective To study the changes of immune function of endotoxin tolerant dendritic cell(ETDC)and to observe its effect on sepsis in mouse model.Methods ETDC were prepared by pretreated bone marrow dendritic cells derived from BALB/c mice with lipopolysaccharide stimulation.The cells were collected and the expressions of surface markers including major histocompatibility complex(MHC)Ⅱ,CD86 and CD11c were detected by flow cytometry.The proliferation rate of T lymphocytes was evaluated by cell counting kit-8 and the concentrations of cytokines in the supernatant were detected by enzyme linked immuno sorbent assay.Afterwards,36 mice were randomly assigned into 4 groups.The blank control group did not receive any treatment,the sham-operated group underwent simple incision suture,the sepsis group and ETDC reinfusion group underwent cecal ligation and puncture to establish sepsis.Before sepsis model establishment,0.9%sodium chloride solution or suspension of ETDC and 0.9%sodium chloride were reinfused by tail vein.The serum levels of alanine aminotransferase(ALT)and aspartate transaminase(AST),tumor necrosis factor(TNF)-α,interleukin(IL)-6 and IL-10,and the proportion of help T cell(Th)17/regulatory T cell(Treg)in spleen of each group were detected.The pathological manifestations of liver,kidney and ileum in each group were observed.T test andχ^2 test were used for comparisons between groups.Results The results of flow cytometry showed that MHCⅡ,CD86 and CD11c of ETDC were 70.4%,43.1%,and 73.1%,respectively,which were significantly lower than those of mature dendritic cell(mDC)(96.1%,89.5%,and 84.6%,respectively)(χ^2=56.47,83.78,and 23.29,respectively,all P<0.01).The concentrations of IL-10,TNF-αand IL-6 in the supernatant of ETDC were(978.04±56.70),(980.34±111.96)and(12 743.03±865.81)ng/L,respectively,and those of mDC were(741.35±99.23),(1 703.11±117.00)and(19 052.28±1 145.84)ng/L,respectively.The differences were all statistically significant(t=5.073,10.93,and 10.76,respectively,all P<0.01).The proliferation rates of T lymphocytes co-cultured with ETDC in 1∶5 and 1∶10 ratio group were(676.95±85.99)%and(514.00±106.39)%,respectively,which were lower than those of the mDC group(956.25±127.12)%and(772.07±214.08)%,respectively.The pathological injuries of liver,kidney and ileum in ETDC treatment group were significantly lighter than those in sepsis group.Serum ALT and AST levels in the ETDC reinfusion group were(299.71±36.91)and(690.39±154.92)U/L,respectively,and TNF-α,IL-6 and IL-10 were(0.067±0.005),(0.428±0.051)and(0.058±0.005)ng/L,respectively.Serum ALT and AST levels in the sepsis group were(620.67±69.27)and(1 430.17±134.05)U/L,respectively,and TNF-α,IL-6 and IL-10 were(0.085±0.007),(0.774±0.088)and(0.036±0.005)ng/L,respectively.The differences were all statistically significant(t=11.60,10.96,5.991,8.657,and 8.04,respectively,all P<0.01).The proportion of Treg/Th17 in the ETDC reinfusion group was 23.4%,and that in the sepsis group was 60.8%(χ^2=28.69,P<0.01).Conclusion The reinfusion of ETDC has a protective effect on sepsis in mouse model,which may play a negative immune regulatory role by regulating the differentiation of T cells.