肠神经胶质细胞源性神经营养因子对肠T淋巴细胞的影响及其在葡聚糖硫酸钠诱导的小鼠结肠炎中的作用

Effect of enteric glial cell line derived neurotrophic factor on intestinal T lymphocytes and its role in dextran sodium induced colitis in mice

目的探讨肠神经胶质细胞(EGCs)源性神经营养因子(GDNF)对肠T淋巴细胞(简称T细胞)的影响及其在葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎中的作用。方法取4只C57BL/6小鼠提取脾脏原代T细胞,将其与EGCs随机分为T细胞1组、T+EGCs组及T+EGCs+anti-GDNF组,行Transwell共培养。采用蛋白质印迹法(Western Blot)及免疫荧光检测GDNF受体(GFR-α1/RET),酶联免疫吸附试验(ELISA)检测IL-10的表达,流式细胞术检测Foxp3+调节性T细胞(Foxp3+Treg)比例。再取脾脏T细胞及EGCs并将其随机分为T细胞2组与T+GDNF组,检测IL-10的表达及Foxp3+Treg比例。另取18只C57BL/6雄性小鼠随机分为对照组、DSS组及DSS+GDNF组,每组6只。对照组连续7日饮用无菌水,其余两组连续7日饮用2.5%DSS,DSS+GDNF组再连续7日腹腔注射GDNF 5μg/kg。对所有小鼠进行疾病活动指数(DAI)评分及HE染色后组织学评分,采用流式细胞术检测小鼠肠系膜淋巴结Foxp3+Treg比例,ELISA检测结肠组织中IL-10和肿瘤坏死因子(TNF)-α的表达。结果C57BL/6小鼠脾脏原代T细胞中存在GFR-α1/RET。T+EGCs组Foxp3+Treg比例及IL-10水平均高于其余两组(P<0.05)。T+GDNF组Foxp3+Treg比例及IL-10水平均高于T细胞2组(P<0.05)。DSS组小鼠DAI、组织学评分及TNF-α水平均高于对照组,IL-10水平低于对照组(P<0.05)。DSS+GDNF组小鼠DAI、组织学评分及TNF-α水平均低于DSS组,IL-10水平及Foxp3+Treg比例均高于DSS组(P<0.05)。DSS+GDNF组的IL-10、TNF-α水平及Foxp3+Treg比例均高于对照组(P<0.05)。结论GDNF可通过诱导T细胞向Foxp3+Treg分化来间接调节肠道免疫反应、减轻肠道炎症。

Objective To investigate the effect of enteric glial cell(EGCs)derived neurotrophic factor(GDNF)on intestinal T lymphocytes and its role in dextran sulfate sodium(DSS)induced colitis in mice.Methods Four C57BL/6 mice were randomly divided into T cell group 1,T+EGCs group,T+EGCs+anti-GDNF group.Western Blotting and immunofluorescence were used to detect GDNF receptor(GFR-α1/RET),ELISA was used to detect IL-10 levels,and flow cytometry was used to detect Foxp3+Treg ratio.Spleen T cells and EGCs were randomly divided into T cell group 2 and T+GDNF group.IL-10 levels and Foxp3+Treg ratio were detected.Eighteen C57BL/6 mice were randomly divided into Control group,DSS group and DSS+GDNF group,with 6 mice in each group.Control group drank sterile water for 7 consecutive days,other two groups drank 2.5%DSS for 7 consecutive days,and DSS+GDNF group received intraperitoneal injection of GDNF 55μg/kg for 7 consecutive days.DAI score and histological score after HE staining were performed on all mice.The Foxp3+Treg ratio of mesenteric lymph nodes in mice was detected by flow cytometry,and the expression of IL-10 and TNF-αin colon tissue was detected by ELISA.Results There were GFR-α1/RET in primary splenic T cells of C57BL/6 mice.Foxp3+Treg ratio and IL-10 level in T+EGCs group were higher than those in the other two groups(P<0.05).Foxp3+Treg ratio and IL-10 level in T+GDNF group were higher than those in T cell 2 group(P<0.05).The DAI and histological scores and TNF-αlevel in DSS group were higher than those in control group,and IL-10 level in DSS group was lower than that in control group(P<0.05).The DAI and histological scores and TNF-αlevel in the DSS+GDNF group were lower than those in the DSS group,while IL-10 level and the proportion of Foxp3+Treg in DSS+GDNF group were higher than those in DSS group(P<0.05).IL-10 and TNF-αlevel and the percentage of Foxp3+Treg cells in DSS+GDNF group were higher than those in control group(P<0.05).Conclusion GDNF can indirectly regulate the intestinal immune response by inducing T cells to differentiate into Foxp3+Treg to alleviate intestinal inflammation.