敲低Krüppel样因子4(KLF4)促进RAW264.7巨噬细胞向M1型极化

Knockdown of Krüppel like factor 4(KLF4) promotes RAW264.7 macrophages into M1 polarization

目的研究敲低Krüppel样因子4(KLF4)基因对RAW264.7巨噬细胞极化的影响。方法采用RNA干涉技术(RNAi)构建敲低KLF4的慢病毒载体,转染RAW264.7巨噬细胞获得KLF4基因沉默的稳定细胞株。采用白细胞介素4(IL-4)刺激野生型、KLF4敲低组和阴性对照组巨噬细胞,反转录PCR检测细胞诱导型一氧化氮合酶(iNOS)、IL-1β、肿瘤坏死因子α(TNF-α)及精氨酸酶1(Arg1)、IL-10、转化生长因子β(TGF-β)的mRNA水平,免疫细胞化学染色检测和定位巨噬细胞iNOS及Arg1蛋白。结果敲低KLF4基因后,RAW264.7细胞的iNOS、IL-1β的mRNA含量明显增高,而TNF-α、Arg1、IL-10及TGF-β的mRNA含量则明显降低;IL-4处理野生型RAW264.7细胞后KLF4、Arg1、IL-10及TGF-β的mRNA的表达增加,iNOS、IL-1β及TNF-αmRNA的表达减少;IL-4处理敲低KLF4的细胞,其iNOS、IL-1β及TNF-αmRNA含量低于野生型组,但高于阴性对照组,而Arg1、IL-10及TGF-βmRNA含量较高于野生型组,而Arg1及IL-10低于阴性对照组;IL-4处理敲低KLF4细胞12h后,与野生型细胞相比,iNOS蛋白表达显著减少,而Arg1的蛋白的表达则明显增加。结论敲低KLF4促进RAW264.7巨噬细胞向M1型极化、抑制巨噬细胞向M2型极化。

Objective To investigate the effects of Krüppel like factor 4(KLF4)gene knockdown on the polarization of RAW264.7 macrophages.Methods KLF4 knockdown lentiviral vector was constructed by RNA interfering.The lentiviral vector was transfected into RAW264.7 cells to realize stable KLF4 gene silencing in RAW264.7 cells.Interleukin-4(IL-4)was used to stimulate macrophages in wild type group,KLF4 knockdown group and negative control group.The mRNA expression of inducible nitric oxide synthase(iNOS),IL-1β,tumor necrosis factorα(TNF-α)and Arg1,IL-10,transforming growth factorβ(TGF-β)of the cells was detected by reverse transcription-PCR.Immunocytochemical staining was used to detect and localize iNOS and Arg1 protein in RAW264.7 cells.Results Levels of iNOS and IL-1βmRNA in RAW264.7 cells were significantly raised,while levels of Arg1,IL-10 and TGF-βmRNA were significantly reduced after KLF4 gene knockdown.Levels of KLF4,Arg1,IL-10 and TGF-βmRNA went up,while the relative levels of iNOS,IL-1βand TNF-αmRNA went down in wild-type RAW264.7 cells after IL-4 intervention.After shKLF4 group was intervened by IL-4,levels of iNOS,IL-1βand TNF-αmRNA in shKLF4 group(lentivirus group)were lower than those in wild-type group and higher than those in negative control group.Levels of Arg1,IL-10 and TGF-βmRNA in shKLF4 group after IL-4 treatment were higher than those in wild-type group,while Arg1 and IL-10 were lower than those in negative control group.Compared with wide-type group,the expression of iNOS protein significantly decreased,while Arg1 protein significantly increased in shKLF4 group 12 hours after IL-4 treatment.Conclusion Knockdown of KLF4 promotes the polarization of RAW264.7 macrophages into M1 as well as inhibits their polarization into M2.