干扰素-γ作用后的小鼠肺微血管内皮细胞对共培养T淋巴细胞增殖的影响及机制

Effect of pulmonary microvascular endothelial cells inducing by interferon-γon proliferation of co-cultured T lymphocytes and its mechanism

目的探讨干扰素-γ(IFN-γ)作用后的小鼠肺微血管内皮细胞(PMVECs)对共培养T淋巴细胞增殖的影响,并探讨其可能的机制。方法分离雌性Balb/C小鼠肺组织,采用组织块贴壁法培养PMVECs;经倒置显微镜、透射电镜观察及内皮细胞表面标志CD31荧光检测鉴定,证实为PMVECs且纯度>95%。在Transwell板下层小室中加入PMVECs,随机分为空白对照组、10 ng/m L组、20 ng/m L组、50 ng/m L组、80 ng/m L组,加入终浓度分别为0、10、20、50、80 ng/m L的IFN-γ,Transwell板上层小室中加入T淋巴细胞进行共培养,采用流式细胞术检测上层小室T淋巴细胞增殖指数。将第3代PMVECs随机分为观察组和对照组,分别加入终浓度为80 ng/m L的IFN-γ及等量PBS;采用反相高效液相色谱法检测细胞培养上清中色氨酸、犬尿氨酸水平及吲哚胺2,3-加氧酶(IDO)活性,RT-PCR法及Western blotting法检测细胞IDO mRNA及蛋白表达。结果空白对照组、10 ng/m L组、20 ng/m L组、50 ng/m L组、80ng/m L组T淋巴细胞增殖指数分别为3.06±0.07、2.93±0.09、2.46±0.28、2.21±0.41、2.20±0.05,20 ng/m L组、50ng/m L组、80 ng/m L组T淋巴细胞增殖指数均低于空白对照组(P均<0.01),20 ng/m L组、50 ng/m L组、80 ng/m L组T淋巴细胞增殖指数比较差异均无统计学差异(P均>0.05)。观察组细胞培养上清中色氨酸水平低于对照组,犬尿氨酸水平及IDO活性均高于对照组(P均<0.01)。观察组和对照组细胞IDO mRNA相对表达量分别为0.68±0.02、0,IDO蛋白相对表达量分别为0.49±0.02、0;两组比较P均<0.01。结论IFN-γ作用后的PMVECs可抑制共培养的T淋巴细胞增殖,其机制可能与促进色氨酸降解、犬尿氨酸累积及IDO表达有关。

Objective To investigate the effect of pulmonary microvascular endothelial cells(PMVECs)induced by interferon-γ(IFN-γ)on the proliferation of co-cultured T lymphocytes,and to explore its possible mechanism.Methods The lung tissues of female Balb/C mice were isolated,and mouse PMVECs were cultivated by tissue block method.After the identification of inverted microscope,transmission electron microscope observation and endothelial cell surface marker CD31 fluorescence detection identification,PMVECs with high purity were confirmed.PMVECs were added to the lower chamber of the Transwell plate and then were randomly divided into five groups:the control group,10 ng/m L group,20 ng/m L group,50 ng/m L group,and 80 ng/m L group,which were added with 0,10,20,50,and 80 ng/m L IFN-γ,respectively.T-lymphocytes were added to the upper chamber of the transwell plate for co-culture,and flow cytometry was used to detect the T-lymphocyte proliferation index(PI)of the upper chamber.PMVECs of the third generation were randomly divided into two groups:the observation group and control group,which were added with 80 ng/m L IFN-γand an equal amount of PBS,respectively;reverse-phase high-performance liquid chromatography was used to detect tryptophan,kynurenine and indoleamine 2,3-dioxygenase(IDO)activity in the cell culture supernatant.The expression levels of IDO mRNA and protein were detected by RT-PCR and Western blotting,respectively.Results The T-lymphocyte PI of the control group,10 ng/m L group,20 ng/m L group,50 ng/m L group,and 80 ng/m L group were 3.06±0.07,2.93±0.09,2.46±0.28,2.21±0.41,and 2.20±0.05,respectively;T-lymphocyte PI of the 20 ng/m L group,50 ng/m L group,and 80 ng/m L group was lower than that of control group(all P<0.01),but there was no significant difference in T lymphocyte PI among 20 ng/m L group,50 ng/m L group,and 80 ng/m L group(all P>0.05).The observation group had a significantly lower tryptophan level in the cell culture supernatant than the control group,and the kynurenine level and IDO activity were higher than those of the control group(all P<0.01).The relative expression levels of IDO mRNA in the observation group and the control group were 0.68±0.02 and 0,and the relative expression levels of IDO protein were 0.49±0.02 and 0,respectively,with significant difference between every two groups(all P<0.01).Conclusion PMVECs treated with IFN-γcan inhibit the proliferation of co-cultured T lymphocytes by promoting tryptophan degradation,kynurenine accumulation,and IDO expression.