Lewis肺癌细胞TLR4对Foxp3表达的调控

目的:探讨小鼠Lewis肺癌(Lewis lung cancer,LLC)细胞中TLR4对Foxp3表达的调控作用。方法:选择LPS为配体活化TLR4,采用RT-PCR方法检测LPS在不同浓度(0,1,10μg/ml)和不同时间点(12,24,36,48小时)Foxp3 mRNA的表达量的变化,流式细胞术检测有效浓度LPS 10μg/ml和最佳作用时间点24小时Foxp3蛋白和TLR4蛋白表达量的变化,RT-PCR和流式细胞术检测anti-TLR4/MD2抗体阻断TLR4后再用LPS刺激LLC细胞Foxp3表达量的变化。结果:LPS在10μg/ml浓度作用LLC细胞后可显著上调Foxp3 mRNA的表达量,与未刺激组相比差异显著(P<0.05);LPS最佳作用时间为24小时;LPS在10μg/ml浓度作用LLC细胞24小时后可显著上调Foxp3蛋白和TLR4蛋白的表达量,与对照组相比差异显著(P<0.05);阻断TLR4后再用LPS刺激LLC细胞,Foxp3的表达量较未阻断组明显降低(P<0.05)。结论:LLC细胞TLR4蛋白参与对Foxp3的表达调控,TLR4可能是Foxp3的上游信号分子。

Regulation of Foxp3 expression by TLR4 in Lewis lung cancer cells

Objective:To investigate whether the expression of Foxp3 in Lewis lung cancer(LLC)cells could be regulated by TLR4 activation.Methods:Lipopolysaccharide(LPS) was employed as exogenous ligands to activate TLR4.The mRNA level of Foxp3 was detected by RT-PCR at different time points(12,24,36,48 h)upon LPS stimulation(0,1,10 μg/ml).The protein level of Foxp3 and TLR4 was detected by FCM at 24 h time point upon 10 μg/ml LPS stimulation.Foxp3 mRNA and protein were detected by RT-PCR and FCM respectively after LPS activation following blockade of TLR4 by anti-TLR4/MD2 antibody.Results:The mRNA level of Foxp3 was significantly increased by 10 μg/ml LPS stimulation at 24 hours(P < 0.05).The protein level of Foxp3 and TLR4 were significantly up-regulated at 24 h time point upon 10 μg/ml LPS stimulation(P < 0.05).The expression of Foxp3 protein was significantly decreased in LLC cells with TLR4 blockade followed by LPS stimulation than that of without TLR4 blocking ones(P < 0.05).Conclusion:These results indicate that TLR4 could be involved in the regulation of Foxp3 expression in LLC cells and TLR4 might be the upstream signaling molecules of Foxp3.