结肠癌细胞调节M2型巨噬细胞极化促进化疗耐药的研究

Colon cancer cells regulate polarization of M2 macrophages to promote chemoresistance

目的探讨结肠癌细胞调节M2型巨噬细胞极化促进化疗耐药的作用。方法用收集的结肠癌细胞系HCT116细胞和HCT8细胞上清液,培养由人源单核细胞系THP-1诱导的M0巨噬细胞96 h,对其细胞形态进行记录;用流式细胞术检测其M2型巨噬细胞极化标志因子组CD11b^(+)CD206^(+)的表达;用实时荧光定量聚合酶链反应(Real-time PCR)和酶联免疫吸附(ELISA)法检测M2型巨噬细胞极化标志因子的表达。用细胞计数法-8(CCK-8)检测M2型巨噬细胞条件培养基处理后结肠癌细胞对奥沙利铂的耐药性变化。结果通过形态学观察发现悬浮细胞THP-1细胞经PMA诱导后变为贴壁的M0巨噬细胞,呈圆饼状,用结肠癌细胞条件培养基刺激M0巨噬细胞后,细胞呈双极形态,变为长条梭形细胞。流式细胞术结果表明,HCT116、HCT8细胞上清液中CD11b^(+)CD206^(+)表达水平分别为42.63±0.38和40.27±2.60,明显高于M0组12.33±0.64和11.90±0.95,差异均有统计意义(均P<0.01)。HCT116、HCT8细胞上清组M2型巨噬细胞标志物IL-10 mRNA表达水平分别为11.29±0.86,13.64±1.20,TGF-βmRNA表达水平分别为3.38±0.23,2.88±0.13,与M0组(1.00)相比,差异均有统计意义(均P<0.01)。ELISA实验结果显示,HCT116、HCT8细胞上清刺激后的巨噬细胞上清中M2型巨噬细胞标志物IL-10表达水平分别为(10.69±0.06),(11.59±0.03)pg·mL^(-1),明显高于M0组(5.03±0.03),(5.78±0.07)pg·mL^(-1),而TGF-β表达水平分别为(26.63±0.52),(31.60±1.38)pg·mL^(-1),明显高于M0组(17.23±0.29),(21.63±0.11)pg·mL^(-1),差异均有统计意义(P<0.01或P<0.05)。CCK-8实验结果显示,M2型巨噬细胞条件培养基处理的HCT116、HCT8细胞对于化疗药物奥沙利铂的IC_(50)分别为(82.72±4.80)和(309.69±7.62)μmol·L^(-1),明显高于普通培养的HCT116(13.60±2.37)μmol·L^(-1)、HCT8(124.70±5.48)μmol·L^(-1),差异均有统计意义(均P<0.01)。结论结肠癌细胞可以调节M2型巨噬细胞极化促进化疗耐药。

Objective To explore the role of colon cancer cells in regulating the polarization of M2 macrophages to promote chemoresistance.Methods The collected colon cancer cell line HCT116 and HCT8 cell supernatants were used to culture M0 macrophages induced by human monocyte line THP-1 for 96 hours,and cells morphology was recorded.Flow cytometry was used to detect the expression of CD11b^(+)CD206^(+)in the M2 macrophage;real-time polymerase chain reaction(PCR)and enzyme-linked immunosorbent assay(ELISA)were used to detect theexpression of M2 macrophage polarization marker factor.The cell counting kit-8(CCK-8)method was used todetect the changes in the resistance of colon cancer cells to oxaliplatin after treatment with the conditioned medium of M2 macrophages.Results Through morphological observation,it is found that suspension cell THP-1 becomeadherent M0 macrophages after PMA induction,which is in the shape of a disc.After the M0 macrophages arestimulated with colon cancer cell conditioned medium,the cells show a bipolar morphology.It becomes a long spindlecell.The results showed that the expression levels of CD11b^(+)CD206^(+)in the HCT116 and HCT8 cell supernatantgroups were 42.63±0.38 and 40.27±2.60,significantly higher than those in the M0 group,which were12.33±0.64 and 11.90±0.95,the difference was statistical significance(P<0.01).Real-time PCR resultsshowed that the expression levels of M2 macrophage marker IL-10 mRNA(11.29±0.86,13.64±1.20)and TGF-βexpression levels(3.38±0.23,2.88±0.13)in the supernatant group of HCT116 and HCT8 cells were significantlyincreased,compared with M0 group(1.00),the difference was statistically significant(allP<0.01).ELISA resultsshowed that the expression level of M2 macrophage marker IL-10[(10.69±0.06),(11.59±0.03)pg·mL^(-1)]inthe macrophage supernatant stimulated by HCT116 and HCT8 cell supernatants was significantly higher than that in theM0 group[(5.03±0.03),(5.78±0.07)pg·mL^(-1)],and the expression level of TGF-β[(26.63±0.52),(31.60±-1.38)pg·mL^(-1)]was also significantly higher than that of the M0 group[(17.23±0.29),(21.63±0.11)pg·mL^(-1)],the differences were statistically significant(P<0.01 orP<0.05).CCK-8 experiment results showedthat the IC_(50) of HCT116 and HCT8 cells treated with M2 macrophage conditioned medium to the chemotherapy drugoxaliplatin were(82.72±4.80)μmol·L^(-1) and(309.69±7.62)μmol·L^(-1),respectively,which were significantlyhigher than those of ordinary cultured HCT116 and HCT8 cells,which were(13.60±2.37)μmol·L^(-1),(124.70±5.48)μmol·L^(-1),the differences were statistically significant(P<0.01).Conclusion Colon cancercells can regulate the polarization of M2 macrophages to promote chemoresistance.