T4噬菌体诱导肿瘤相关巨噬细胞向M1型再极化

Re-polarization of tumor-associated macrophages to M1 macrophages induced by bacteriophage T4

目的:观察T4噬菌体对M2型巨噬细胞向M1型再极化的诱导作用,并检测再极化的M1型巨噬细胞诱导小鼠Lewis肺癌细胞凋亡和抑制其侵袭的作用效果。方法:小鼠巨噬细胞RAW264.7经白细胞介素-4(IL-4)诱导为替代性活化巨噬细胞(M2型),以T4噬菌体对M2型巨噬细胞再极化诱导;Real-time PCR检测IL-4诱导极化及T4噬菌体再极化后巨噬细胞中IL-12、TNF-α、Arg-1、TGF-β、IL-10和iNOS基因mRNA的变化,Western blotting检测巨噬细胞内iNOS和Arg-1蛋白表达变化,ELISA法检测巨噬细胞培养上清中IL-10和IL-12的含量;流式细胞术和Transwell法分别检测T4噬菌体再极化巨噬细胞诱导小鼠Lewis肺癌细胞凋亡和抑制其侵袭的作用效果。结果:RAW264.7细胞经IL-4处理后被诱导成为M2型巨噬细胞,其iNOS和IL-12 mRNA表达分别下降为对照组的1/2.5和1/6.2,而Arg-1和IL-10 mRNA表达分别增加了161.2和120.3倍。M2型巨噬细胞经野生型和突变型T4噬菌体处理后,IL-12、TNF-α、iNOS在mRNA和蛋白水平均明显逆转上调,IL-10、TGF-β、Arg-1则明显逆转下调,呈现M1型特征;其中突变型T4噬菌体的诱导作用显著强于野生型。野生型和突变型T4噬菌体诱导M1再极化的巨噬细胞致小鼠Lewis肺癌细胞的凋亡率较M2型巨噬细胞显著增高[(35.3±2.44)%、(39.1±2.08)%vs(4.68±0.56)%;均P<0.01)],同时,显著抑制了肺癌细胞的侵袭能力[侵袭细胞数:(43.8±7.51)、(23.2±4.33)个vs(177.5±12.33)个;均P<0.01]。结论:T4噬菌体能够诱导M2型巨噬细胞向M1型再极化,并增强巨噬细胞诱导肿瘤细胞凋亡和抑制肿瘤细胞侵袭的能力。

Objective： To analyse T4 phage-induced M1 re-polarization of tumor-associated M2 type macrophages,and to evaluate the ability of re-polarized M1 macrophages on apoptosis and metastasis of mouse Lewis lung cancer cells in vitro. Methods： Mouse macrophage RAW264. 7 cells were induced into M2 type macrophages by interleukin-4（ IL-4）, which were subsequently induced to re-polarize with T4-bacteriophage. In both IL-4-induced M2 macrophages and T4 repolariezed-macrophages,mRNA levels of IL-12,TNF-α,Arg-1,TGF-β,IL-10 and iNOS were analyzed by Real-time PCR,and protein levels of iNOS and Arg-1 were determined by Western blotting. The effect of T4-phage-induced re-polarization on the apoptosis and metastasis of Lewis lung cancer cells were evaluated by the flow cytometry and transwell assays,respectively. Results： RAW264. 7 cells were successfully induced into an M2 phenotype with a significant increase in mRNA levels of Arg-1 and IL-10（ 161. 2 and 120. 3 folds,respectively）,together with a significant decrease in iNOS and IL-12 mRNA levels（ 3. 3 and 7. 8 folds,respectively）. Both wild-type and the soc-hoc-T4 bacteriophages effectively induce M1 re-polarization of M2 type macrophages,but the induction activity of the soc-hoc-T4 phages was significantly stronger than that of the wild-type（ P 〈 0. 05）. Both wild-type and polarized M1 type macrophages more effectively induced apoptotic cell death（ [35. 3 ± 2. 44]%,[39. 1 ± 2. 08]% vs [4. 68 ± 0. 56]%,P 〈 0. 01） and suppressed the invasive capability in Lewis lung cancer cells（ [43. 8 ± 7. 51]%,[23. 2 ± 4. 33]% vs [177. 5 ± 12. 33]%,P 〈 0. 01）, as compared with M2 macrophages. Conclusion： T4 bacteriophages can induce M1 re-polarization of M2 type macrophages,which results in an increase in apoptotic cell death and a decrease in invasive activity in Lewis lung cancer cells in vitro.