Lewis肺癌细胞TLR4对Foxp3表达的调控

Regulation of Foxp3 expression by TLR4 in Lewis lung cancer cells

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。

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.