肺腺癌细胞对RAW264.7巨噬细胞分化的影响

Effect of Lung Adenocarcinoma Cells on the Polarization of Mouse Macrophage RAW264.7 Cells

[目的]体外探究Lewis肺腺癌(Lewis lung carcinoma,LLC)细胞培养上清液对小鼠RAW264.7巨噬细胞极化的影响及其可能机制。[方法]RAW264.7细胞按如下分组:空白对照组、IL-4处理组、LLC细胞培养上清液条件培养基(LLC-CM)培养组和TC-1细胞培养上清液条件培养基(TC-1-CM)培养组。各组培养48h后收集上清液和细胞,酶联免疫吸附测定(ELISA)检测空白对照组、IL-4组、LLC-CM组和TC-1-CM组巨噬细胞及LLC细胞和TC-1细胞培养上清液中补体C1q的含量,实时荧光定量PCR(qRT-PCR)检测巨噬细胞CD68、CD80、CD206、Arg-1、IL-10和IL-27 mRNA,Western blot检测CD68、CD80、CD206、Arg-1、JAK2、p-JAK2、STAT5和p-STAT5的表达。[结果]巨噬细胞经IL-4处理后,上清液中补体C1q含量较空白对照组升高(P<0.001);与空白对照组及IL-4处理组相比,LLC-CM组巨噬细胞培养上清液中补体C1q含量明显升高(P均<0.001)。与空白对照组相比,LLC-CM组巨噬细胞CD80表达明显降低(P<0.001),CD206、Arg-1、IL-10和IL-27表达明显升高(P均<0.001),p-JAK2、p-STAT5(P均<0.001)含量降低。此外,LLC-CM组巨噬细胞p-JAK2、p-STAT5表达量低于IL-4处理组(P均<0.001)。[结论]LLC细胞培养上清液可以通过抑制RAW264.7细胞JAK2/STAT5通路诱导其发生M2型极化并促进补体C1q高表达,高水平补体C1q可能参与促进RAW264.7细胞的M2型极化。

[Objective]To explore the effect of Lewis lung adenocarcinoma(LLC)cells on the polarization of the mouse macrophages in vitro and its mechanism.[Methods]Mouse macrophage RAW264.7 cells were cultured with conditioned medium(control group)and the cultured cells were treated with IL-4(IL-4 group),LLC cells culture supernatant(LLC-CM group)or TC-1 cells culture supernatant group(TC-1-CM group),respectively.The content of complement C1q in the culture supernatant of 4 groups were detected with ELISA;the expression of CD68,CD80,CD206,Arg-1,IL-10,IL-27 mRNA was detected with real-time fluorescence quantitative RT-PCR(qRT-PCR);and the expression of CD68,CD80,CD206,Arg-1,JAK2,p-JAK2,STAT5 and p-STAT5 proteins were detected by Western blot.[Results]After RAW264.7 cells were treated with IL-4,the content of complement C1q in the supernatant was higher than that in the blank control group(P<0.001).Compared with the blank control group and the IL-4 group,the content of complement C1q in LLC-CM group was significantly increased(P<0.001).Compared with the blank control group,the expressions of CD80 in LLC-CM group were significantly decreased(P<0.001),while the expressions of CD206,Arg-1,IL-10 and IL-27 were significantly increased(all P<0.001),and the levels of p-JAK2 and p-STAT5 were reduced(both P<0.001).Compared with the IL4 group,the levels of p-JAK2 and pSTAT5 in the LLC-CM group were decreased(both P<0.001).[Conclusion]LLC cell culture supernatant can induce M2 polarization in RAW264.7 cells by inhibiting the JAK2/STAT5 pathway and promoting the expression of complement C1q.