CAJ | 학술논문

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TLR4-siRNA转染RAW264.7细胞24h后给予烟曲霉孢子刺激12h,将细胞随即分为正常组(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Western blot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88 mRNA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、MyD88 mRNA及TNF-α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、MyD88 mRNA的表达量均显著降低(P＜0.05);与N+Af组比较,TLR4( RNAi)+Af组的TLR2、MyD88 mRNA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)+ Af组MyD88 mRNA表达量显著升高(P＜0.05),而TLR2 mRNA及TNF-α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.
목적 연구단핵거서세포RAW264.7수연곡매포자자격시,TLR2화TLR4신호통로발휘적작용,이급침묵TLR4기인후,대TLR2신호통로적영향.방법 이용RNAi기술장TLR4-siRNA전염RAW264.7세포24h후급여연곡매포자자격12h,장세포수즉분위정상조(N조)、정상+연곡매포자자격조(N+Af조)、정상+TLR4-siRNA조[TLR4(RNAi)조]、정상+TLR4-siRNA+연곡매포자자격조[ TLR4(RNAi) +Af조],RT-PCR화Western blot법검측세포수연곡매포자자격후TLR2、TLR4、MyD88 mRNA급TNF-α단백적표체변화.결과 (1)TLR4기인침묵전:여N조비교,N+Af조TLR2、TLR4、MyD88 mRNA급TNF-α단백표체량균현저승고(P＜0.05).(2)TLR4-siRNA( 100nmoL/L)전염RAW264.7세포,침묵효솔체83％.(3)TLR4기인침묵후:여N조비교,TLR4( RNAi)조TLR2、MyD88 mRNA적표체량균현저강저(P＜0.05);여N+Af조비교,TLR4( RNAi)+Af조적TLR2、MyD88 mRNA화TNF-α단백적표체량균현저강저(P＜0.05);여TLR4( RNAi)조비교,TLR4(RNAi)+ Af조MyD88 mRNA표체량현저승고(P＜0.05),이TLR2 mRNA급TNF-α단백표체량각무현저변화(P＞0.05).결론 RAW264.7세포수연곡매포자자격시,TLR2화TLR4신호통로피격활,통과석방촉염세포인자TNF-α발휘항연곡매포자자격작용;당침묵TLR4기인후,TLR2신호통로불능피흔호지격활래저항연곡매포자대세포적자격작용,침묵TLR4기인하조료TLR2신호통로재RAW264.7세포중적항연곡매포자자격작용,가능TLR4교TLR2재저항연곡매포자자격시발휘경중요적작용.
Objective To study the role of TLR2 and TLR4 signal transduction in RAW264.7 monocyte-macrophages stimulated by Aspergillus fumigatus 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 Aspergillus fumigatus conidia ( N +Af group ),normal + transfected with TLR4-siRNA [ TLR4 (RNAi) group ],normal+transfected with TLR4-siRNA +stimulated with Aspergillus fumigatus 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-α 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-αprotein in N+Af group significantly increased before silencing gene of TLR4.(2) Silencing efficiency of macrophates 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 +Af group.Compared with TLR4 (RNAi) group,the expression of MyD88 mRNA in TLR4 (RNAi) +Af group significantly increased.However,the expression of TLR2 mRNA and TNF-α protein have no significant change after silencing gene of TLR4.Conclusion Signaling pathway of TLR2 and TLR4 in macrophages was activated by given stimulus of Aspergillusfumigatus conidia and exerted the effect of anti-Aspergillus fumigatus 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-Aspergillus fumigatus conidia stimulation,and it found that TLR4 played an more important role by contrast with TLR2.