NODs信号通路在RAW264.7细胞抗烟曲霉菌刺激中的作用

The role of NODs signaling pathway in RAW264.7 cells against the stimulate of Aspergillus fumigatus

摘要:

【目的】通过培养RAW264.7细胞, 并运用siRNA沉默NOD2基因来研究NODs信号通路在体外抗烟曲霉中的作用。【方法】体外培养RAW264.7细胞, 接种2×105个/孔细胞于六孔板中, 分为正常对照组(N)和正常沉默组[NOD2(RNAi), 正常+烟曲霉孢子刺激组(N+Af)和正常沉默+烟曲霉孢子刺激组[NOD2(RNAi)+Af], 每组三复孔。通过RT-PCR法检测细胞中NOD1、NOD2、RIP2 mRNA表达; Western blot法检测细胞中分泌蛋白TNF-α表达。【结果】与N组比较, N+Af 组NOD1、NOD2 mRNA和TNF-α蛋白表达显著上升。与阴性对照组(Nctrol)相比, NOD2(RNAi)组NOD2 mRNA表达明显受到抑制, 沉默效果达到80% 以上, 说明RAW264.7细胞中NOD2基因被成功沉默。与NOD2(RNAi)组比较, NOD2(RNAi)+Af组NOD1、RIP2 mRNA和TNF-α蛋白表达小幅上升, 但无显著性差异(P>0.05)。与NOD2基因沉默前比较发现: 与N组比较, NOD2(RNAi)组, TNF-α蛋白表达显著性升高(P<0.05)。与N+Af组比较, NOD2(RNAi)+Af组, TNF-α蛋白显著性降低(P<0.05); NOD1、RIP2 mRNA在各组中表达均未见显著性差异。【结论】NODs信号通路在 RAW264.7细胞抗烟曲霉中发挥作用, 尤以NOD2的作用较突出。

Abstract:

[Objective] To study the role of NODs signaling pathway against the stimulate of Aspergillus fumigatus in RAW264.7 cells by using siRNA to silence NOD2 gene. [Methods] RAW 264.7 cells were inoculated in six-well plate, and then divided into four groups: normal control group (N), normal control group with NOD2 gene silence group, normal with Aspergillus fumigatus spores stimulation group (N+Af) and normal with Aspergillus fumigatus spores stimulation with NOD2 gene silence group. Each groups has three duplicate wells. The expressions of NOD1、NOD2 and RIP2 mRNA in each group were studied by RT-PCR and the expression of TNF-α in each group was detected by western blot assay. [Results] Compared with the N group, expressions of NOD1、NOD2 mRNA and TNF-α protein increased significantly in N+Af group; Compared with negative control group, the expression of NOD2 mRNA was sig-nificantly suppressed in NOD2(RNAi) group, the silencing effect was reached to 80%, indicating that NOD2 gene in RAW264.7 cells was succsessfully silenced. Compared with NOD2(RNAi) group, the expressions of NOD1、NOD2 mRNA and TNF-α protein were slightly increased, but no siginifcant difference (P>0.05). Compared with N group, the ex-pression of TNF-α protein was increased significantly in NOD2(RNAi) group; Compared with N+Af group, the expression of TNF-α protein was lowered significantly in NOD2(RNAi)+ Af group. The expressions of NOD1、RIP2 mRNA in each group were changed slightly. [Conclusion] NODs and its signaling pathway play a role in resistance to Aspergillus fumiga-tus in RAW264.7 cells, particularly the role of NOD2 more prominent.