LFA-1基因缺失对小鼠Naïve T细胞体外向Th17细胞分化的影响

摘要

背景：淋巴细胞功能相关抗原-1（ LFA-1）参与T细胞的活化和功能调节，与炎症性肠病的发病密切相关。目的：观察LFA-1基因缺失（ LFA-1-/-）对小鼠Naïve T细胞体外向Th17细胞分化的影响。方法：繁殖LFA-1-/-子代小鼠，提取鼠尾DNA，PCR法鉴定基因型。LFA-1-/-子代小鼠为实验组，野生型（ WT）C57BL/6J小鼠为对照组，磁珠分选脾脏单个核细胞中的CD4+CD62L+ Naïve T细胞并检测其纯度。体外建立不同Th17细胞诱导分化体系[转化生长因子-β（ TGF-β）、TGF-β+白细胞介素-6（ IL-6）和 TGF-β+IL-6+IL-23]，以流式细胞术检测两组分选得到的Naïve T细胞在不同体系中诱导出的Th17细胞比率，荧光定量PCR法和ELISA法检测Th17细胞特异性转录因子ROR-γt和特异性标记物 IL-17A表达。结果：15只子代小鼠均为 LFA-1-/-小鼠，磁珠分选得到的 CD4+CD62L+Naïve T细胞纯度大于95%。低剂量TGF-β+IL-6即能诱导出Th17细胞，在此基础上加入IL-23能促进更多Th17细胞产生。与WT对照组相比，LFA-1-/-组Naïve T细胞在TGF-β+IL-6+IL-23体系中诱导产生Th17细胞的效应更为明显（17.2%±1.4%对5.7%±0.2%，P＜0.001），ROR-γt、IL-17A mRNA表达上调（P＜0.001），细胞培养上清液中IL-17A浓度升高（ P＜0.01）。结论：LFA-1基因缺失能促进小鼠Naïve T细胞体外向Th17细胞分化。%BacKground:Lymphocyte function-associated antigen-1( LFA-1 ) pIays a cruciaI roIe in the pathogenesis of infIammatory boweI disease by reguIating the activation and function of T ceIIs. Aims:To investigate the effect of LFA-1 deficiency( LFA-1-/-)on differentiation of mice naïve T ceIIs into Th17 ceIIs in vitro. Methods:LFA-1-/- mice were breeded and the progeny genome DNA was extracted from the taiIs for genotyping by PCR. CD4+CD62L+ naïve T ceIIs were separated from spIenic mononucIear ceIIs of LFA-1-/- progeny mice and wiId type ( WT ) C57BL/6J mice, respectiveIy,by magnetic-activated ceII sorting( MACS),and then the purity of separated ceIIs was determined. Naïve T ceIIs obtained were cuItured in different inducing systems[ transforming growth factor-β( TGF-β),TGF-β + interIeukin-6 (IL-6),and TGF-β + IL-6 + IL-23]in vitro for Th17 ceII differentiation;ceIIs in each inducing system were coIIected for anaIyzing the ratio of Th17 ceIIs by fIow cytometry,and the expressions of Th17-specific transcription factor ROR-γt and Th17-specific marker IL-17A were measured by qRT-PCR and ELISA methods. Results:AII fifteen progeny mice were identified as LFA-1-/- genotype. Purity of CD4+CD62L+ naïve T ceIIs separated by MACS was above 95%. Th17 ceIIs couId be induced by Iow-dose TGF-βcombined with IL-6,and the differentiation ratio was increased obviousIy when IL-23 was added. In inducing system containing TGF-β,IL-6 and IL-23,naïve T ceIIs from LFA-1-/- mice produced more Th17 ceIIs than those from WT mice(17. 2% ± 1. 4% vs. 5. 7% ± 0. 2%,P<0. 001),expressions of ROR-γt mRNA and IL-17A mRNA were up-reguIated(P<0. 001),and IL-17A concentration in ceII cuIture supernatant was increased(P<0. 01). Conclusions:Deficiency of LFA-1 promotes the differentiation of mice naïve T ceIIs into Th17 ceIIs in vitro.