TLR4基因沉默下调TLR2信号通路在RAW264．7细胞中的抗烟曲霉孢子刺激作用  

Silencing gene of TLR4 down-regulates the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus couldia stimulation

作　　者：饶振华[1] 朱根华[1] 谢卫华[2] 苏明声[1] 龙凯[2] 罗闳丹[1] 谢小梅[1] 

机构地区：[1]江西中医学院现代中药制剂教育部重点实验室,南昌330004 [2]江西中医学院药学院

出　　处：《中华微生物学和免疫学杂志》2012年第2期108-113,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：国家自然科学基金（30560147,30760236）

摘　　要：目的研究单核巨噬细胞RAW264．7受烟曲霉孢子刺激时，TLR2和TLR4信号通路发挥的作用，以及沉默TLR4基因后，对TLR2信号通路的影响。方法利用RNAi技术将11LR4-siRNA转染RAW264．7细胞24h后给予烟曲霉孢子刺激12h，将细胞随机分为正常组（N组）、正常＋烟曲霉孢子刺激组（N＋Af组）、正常＋TLR4-siRNA组[TLR4（RNAi）组]、正常＋TLR4-siRNA＋烟曲霉孢子刺激组[TLR4（RNAi）＋Af组]，RT-PCR和Westernblot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88mRNA及TNF-α蛋白的表达变化。结果（1）TLR4基因沉默前：与N组比较，N＋Af组TLR2、TLR4、MyD88mRNA及TNF．Ot蛋白表达量均显著升高（P〈0．05）。（2）TLR4-siRNA（100nmol／L）转染RAW264．7细胞，沉默效率达83％。（3）TLR4基因沉默后：与N组比较，TLR4（RNAi）组TLR2、MyD88mRNA的表达量均显著降低（P〈0．05）；与N＋缈组比较，rrLR4（RNAi）＋4厂组的TLR2、MyD88mRNA和TNF-α蛋白的表达量均显著降低（P〈0．05）；与TLR4（RNAi）组比较，TLR4（RNAi）＋A厂组MyD88mRNA表达量显著升高（P〈0．05），而TLR2mRNA及TNF．仪蛋白表达量却无显著变化（P〉0．05）。结论RAW264．7细胞受烟曲霉孢子刺激时，TLR2和TLR4信号通路被激活，通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用；当沉默TLR4基因后，TLR2信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用，沉默TLR4基因下调了TLR2信号通路在RAW264．7细胞中的抗烟曲霉孢子刺激作用，可能TLR4较TLR2在抵抗烟曲霉孢子刺激时发挥更重要的作用。Objective To study the role of TLR2 and TLR4 signal transduction in RAW264.7 monocyte-macrophages stimulated by Aspergillusfumigatus conidia, and to investigate the expression of TLR2 signal transduction after silencing gene of TLR4. Methods Macrophages were randomly divided into normal group （ N group）, normal＋stimulated with AspergiUus fumigatus conidia （ N ＋Af group） , normal ＋ transfected with TLR4-siRNA[ TLR4（RNAi） group], normal＋transfected with TLR4-siRNA＋stimulated with Aspergillus fumigatns conidia [ TLR4 （RNAi） ＋Af group ]. RT-PCR and Western blot were used to assay expression levels of TLR2, TLR4, MyD88 mRNA and pro-inflammatory cytokines TNF-ct protein when macrophages were stimulated 12 h by Aspergillus fumigatus conidia after tranfected 24 h with TLR4-siRNA by technology of RNAi. Results （ 1 ） Compared with N group, the expression of TLR2, TLR4, MyD88 mRNA and TNF-α protein in N＋Af group significantly increased before silencing gene of TLR4. （2）Silencing efficiency of mac- rophates was up to 83% after transfected with TLR4-siRNA. （3）The expression of TLR2, MyD88 mRNA in TLR4 （RNAi） group significantly decreased contrast with normal group. Meanwhile the expression of TLR2, MyD88 mRNA and TNF-α protein also obviously reduced in TLR4 （RNAi）＋Af group when compared with N ＋Afgroup. Compared with TLR4 （RNAi） group, the expression of MyD88 mRNA in TLR4 （RNAi） ＋Afgroup significantly increased. However, the expression of TLR2 mRNA and TNF-α protein have no signifi- cant change after silencing gene of TLR4. Conclusion Signaling pathway of TLR2 and TLR4 in macropha- ges was activated by given stimulus of Aspergillus fumigatus conidia and exerted the effect of anti-Aspergillus fumigat spores stimulation through the release of pro-inflammatory cytokines TNF-α. Meanwhile, silencing gene of TLR4 down-regulate the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillusfu- migatus conidia stimulation, and it found that TLR4 played a

关 键 词：TLR2 TLR4 TLR4-siRNA 烟曲霉孢子 调控机制 

分 类 号：R363[医药卫生—病理学]