肺岩宁颗粒调节肿瘤相关巨噬细胞对Lewis肺癌细胞迁移能力影响的研究

Experimental study of Feiyanning granule modulating tumor associated macrophages and effect on the migration capability of Lewis lung cancer cells

目的观察肺岩宁颗粒对肿瘤相关巨噬细胞调节作用及对Lewis肺癌细胞迁移能力影响。方法采用肺岩宁颗粒给SD大鼠灌胃制备药物血清;利用Lewis肺癌细胞制作条件培养基(Conditioned medium,CM)并诱导小鼠巨噬细胞RAW 264.7细胞转化为肿瘤相关巨噬细胞;MTT法检测对肿瘤相关巨噬细胞增殖的影响;迁徙实验检测肺岩宁颗粒对Lewis肺癌细胞迁移能力的影响;刘氏染色的方法观察对肿瘤相关巨噬细胞形态的影响;PCR法检测肺癌细胞中MMP-2、MMP-9、NF-κBp65m RNA的表达。结果 MTT:培养24 h、48 h和72 h后,DDP组、肺岩宁颗粒组OD值均显著低于模型组(P<0.05)。迁徙实验:DDP组、肺岩宁颗粒组穿过膜细胞个数明显低于模型组(P<0.05),且肺岩宁颗粒组细胞个数明显低于DDP组(P<0.05)。肿瘤相关巨噬细胞刘氏染色:模型组、DDP组细胞显得粗糙和较多分支(M2型),肺岩宁颗粒组细胞呈不规则形或类圆形(M1型)。PCR检测MMP-2、MMP-9、NF-Bp65 m RNA的表达发现:DDP组、肺岩宁颗粒组在细胞中的表达减低,与模型组比较差异有统计学意义(P<0.05),且与DDP组比较,肺岩宁颗粒组MMP-2、MMP-9、NF-Bp65的表达更低(P<0.05)。结论肺岩宁颗粒能在体外能抑制Lewis肺癌细胞迁移能力,其机制可能是通过NF-κB的信号通路调节肿瘤相关巨噬细胞及其分泌的细胞因子。

Objective To observe the effects of Feiyanning granule on the tumor associated macrophages and the migration capability of Lewis lung cancer cells.Methods Serum containing Feiyanning was prepared in SD rats by oral gavage of the Feiyanning granule.The conditioned medium was prepared by Lewis lung cancer cells.And it could induce RAW264.7 cells into TAMs.The cytostatic effects on TAMs were evaluated by MTT assay.The effect of Feiyanning granule on the migration capability of Lewis lung cancer cells was measured by Transwell chamber assays.The shapes of TAMs were observed by LIU＇s stain.The expression of m RNA of MMP-2,MMP-9 and NF-κBp65 were detected by Real-time PCR.Results MTT： After being cultured for 24 h,48 h and 72 h,the OD of DDP group and the Feiyanning granule group were obviously lower,compared with model group（P 0.05）.Migration experiment： Reslut of migration test shows that the number of cells of DDP group and Feiyanning granule group were significantly lowercompared to model group（P 0.05）.Compared with DDP group,the number of cells of Feiyanning granule group was also significantly lower（P0.05）.Staining of tumor associated macrophages： the Feiyanning granule group appeared more round and smooth（M1 type）.On the contrary,the macrophages of model group and DDP group were rough and branched（M2 type）.Reslut of Real-time PCR shows that the positive expression of m RNA of MMP-2,MMP-9 and NF-κBp65 of DDP group and Feiyanning granule group were obviously lower,compared with model group（P 0.05）.Compared with DDP group,the positive expression of m RNA of MMP-2,MMP-9 and NF-κBp65 of Feiyanning granule were obviously lower（P 0.05）.Conclusion Feiyanning granule was able to inhibit migration ability in Lewis lung cancer cells in vitro.And its mechanisms may be modulating tumor associated macrophages and its cytokines through NF-κB pathway.