TRAIL基因敲除对葡聚糖硫酸钠诱导的实验性结肠炎小鼠调节性T细胞的影响

Impact of TRAIL gene knockout on regulatory T cells in mice with dextran sodium sulphate-induced experimental colitis

目的在葡聚糖硫酸钠(DSS)诱导的实验性结肠炎小鼠中探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因敲除(TRAIL-/-)对结肠炎症反应及调节性T细胞(Treg)的影响。方法选取C57BL/6品系的TRAIL-/-小鼠和野生型(WT)小鼠,随机分为WT对照组、WT结肠炎组,TRAIL-/-对照组和TRAIL-/-结肠炎组,每组各10只。口服3.5%的DSS诱导实验性结肠炎模型并评估结肠炎症程度。收集外周血单个核细胞(PBMCs)和肠系膜淋巴结(MLNs),经流式细胞术检测Treg细胞占CD4+T细胞的比例,采用实时荧光定量PCR法检测Treg细胞相关转录因子(Foxp3)和细胞因子(IL-10)的mRNA表达水平,分别采用蛋白质印迹法和酶联免疫吸附试验检测Foxp3和IL-10蛋白表达水平。结果与WT对照组相比,WT结肠炎小鼠PBMCs中Treg细胞比例显著降低[(1.85±0.38)%比(3.12±0.69)%,P<0.05],而MLNs中Treg细胞比例显著增高[(11.79±1.18)%比(6.24±1.04)%,P<0.05]。与WT结肠炎小鼠相比,TRAIL-/-结肠炎小鼠结肠炎症更为严重,PBMCs中Treg细胞比例显著增高[(3.15±0.64)%比(1.85±0.38)%,P<0.05],而MLNs中Treg细胞比例显著降低[(9.80±0.50)%比(11.79±1.18)%,P<0.05]。WT结肠炎小鼠PBMCs中Foxp3、IL-10的mRNA和蛋白表达水平均显著低于WT对照组小鼠[Foxp3 mRNA:0.48±0.21比1.06±0.31,IL-10 mRNA:0.23±0.07比1.22±0.38;Foxp3蛋白:0.68±0.12比1,IL-10蛋白:(4.91 ±0.72)pg/ml比(21.86±2.40)pg/ml,P均<0.05],而MLNs中Foxp3、IL-10的mRNA和蛋白表达水平均显著高于WT对照组小鼠[Foxp3 mRNA:3.71±0.49比1.03±0.15,IL-10 mRNA:11.98±6.10比1.01±0.31;Foxp3蛋白:1.60±0.03比1,IL-10蛋白:(1 260.00±18.02)pg/ml比(1 184.00 ± 38.62)pg/ml,P均<0.05]。与WT结肠炎小鼠相比,TRAIL-/-结肠炎小鼠PBMCs中Foxp3、IL-10的mRNA及蛋白表达水平显著增高[Foxp3 mRNA:1.80±0.49比0.48±0.21,IL-10 mRNA:1.67±0.99比0.23±0.07;Foxp3蛋白:1.10±0.01比0.68±0.12,IL-10蛋白:(31.33±25.02)pg/ml比(4.58±3.73)pg/ml,P均<0.05],而MLNs中Foxp3、IL-10的mRNA及蛋白表达水平显著降低[Foxp3 mRNA:0.49±0.21比3.71±0.49,IL-10 mRNA:2.80±1.82比11.98±6.10;Foxp3蛋白:1.21±0.12比1.60±0.03,IL-10蛋白:(1 158.00±26.48)pg/ml比(1 190.00±37.19)pg/ml,P均<0.05]。结论TRAIL基因敲除可能引起外周血及肠系膜淋巴结中Treg细胞数量改变,从而导致小鼠结肠炎加重。

Objective To investigate the impact of knocking out tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)gene(TRAIL-/-)on colonic inflammation and regulatory T cells(Treg)in mice with dextran sulfate sodium(DSS)-induced experimental colitis. Methods C57BL/6 mice were randomly assigned into four groups with 10 in each group: wild-type(WT)control, WT colitis, TRAIL-/- control and TRAIL-/- colitis. The mouse model of colitis was induced by oral administration of 3.5% DSS and the severity of colonic inflammation was assessed. Peripheral blood mononuclear cells(PBMCs)and mesenteric lymph nodes(MLNs)were collected. The ratios of Treg cells to CD4+ T cells in PBMCs were detected by flow cytometry. Expression of Treg cell-associated transcription factor(Foxp3)and cytokine(IL-10)at mRNA level was measured by real-time fluorescent quantitative polymerase chain reaction. Western blot and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of Foxp3 and IL-10 at protein level, respectively. Results Compared with the WT control group, the WT colitis group showed significantly decreased proportions of Treg cells in PBMCs [(1.85±0.38)% vs(3.12±0.69)%, P<0.05], but increased proportions in MLNs [(11.79±1.18)% vs(6.24±1.04)%, P<0.05]. Compared with the WT mice with colitis, the TRAIL-/- mice with colitis had more severe colonic inflammation and significantly increased proportions of Treg cells in PBMCs [(3.15±0.64)% vs(1.85±0.38)%, P<0.05], but decreased Treg cells in MLNs [(9.80±0.50)% vs(11.79±1.18)%, P<0.05]. Expression of Foxp3 and IL-10 at mRNA and protein levels in PBMCs of the WT mice with colitis was significantly lower than that in the WT control mice [Foxp3 mRNA: 0.48±0.21 vs 1.06±0.31, IL-10 mRNA: 0.23±0.07 vs 1.22±0.38;Foxp3 protein: 0.68±0.12 vs 1, IL-10 protein:(4.91± 0.72)pg/ml vs(21.86±2.40)pg/ml;all P<0.05], while in MLNs, the expression of Foxp3 and IL-10 at mRNA and protein levels was significantly higher than that of the WT control group [Foxp3 mRNA: 3.71±0.49 vs 1.03±0.15, IL-10 mRNA: 11.98±6.10 vs 1.01±0.31;Foxp3 protein: 1.60±0.03 vs 1, IL-10 protein:(1 260.00±18.02)pg/ml vs(1 184.00±38.62)pg/ml;all P<0.05]. Compared with the WT mice with colitis, the TRAIL-/- mice with colitis showed significantly increased expression of Foxp3 and IL-10 at mRNA and protein levels [Foxp3 mRNA: 1.80±0.49 vs 0.48±0.21, IL-10 mRNA: 1.67±0.99 vs 0.23±0.07;Foxp3 protein: 1.10±0.01 vs 0.68±0.12, IL-10 protein:(31.33± 25.02)pg/ml vs(4.58±3.73)pg/ml;all P<0.05], while decreased expression in MLNs [Foxp3 mRNA: 0.49±0.21 vs 3.71±0.49, IL-10 mRNA: 2.80±1.82 vs 11.98±6.10;Foxp3 protein: 1.21±0.12 vs 1.60±0.03, IL-10 protein:(1 158.00±26.48)pg/ml vs(1 190.00±37.19)pg/ml;all P<0.05]. Conclusions Knocking out the expression of TRAIL might affect the ratios of Treg cells in peripheral blood and MLNs, thereby aggravating the colitis in mice.