调控肿瘤相关巨噬细胞自噬对大肠癌移植瘤放疗后凋亡的影响

Effects of autophagy regulation of tumor associated macrophages on the apoptosis of colorectal cancer xenografted tumor after radiotherapy

目的观察调控肿瘤相关巨噬细胞自噬后对大肠癌移植瘤放疗后凋亡的影响。方法药物诱导人单核白血病细胞THP-1(中国科学院上海细胞研究所)分化为肿瘤相关巨噬细胞(TAM)后,流式细胞术检测表面分子;使用雷帕霉素(RAD001),巴弗洛霉素(Bafilomycin a1)上调、下调TAM自噬,蛋白质印迹法(Western blot)检测自噬相关蛋白表达;TAM与大肠癌LoVo(美国组织细胞库)细胞混合接种于BALB/c-nu/nu裸鼠(苏州大学实验动物中心),分为TAM自噬上调组、下调组、未调组,LoVo细胞单独接种为对照组。对荷瘤鼠行8 Gy局部外照射,比较照光前后瘤体病理改变、瘤细胞凋亡变化。涉及数据应用SPSS 17.0统计软件分析,计量资料以均值±标准差(Mean±SD)表示,统计分析采用单因素方差分析(One-way ANOVA),以P<0.05为差异有统计学意义。结果TAM表面CD68、CD204、CD206[(62.32±1.58)%、(33.49±2.11)%、(52.42±2.65)%]表达显著高于THP-1细胞[(39.69±1.59)%、(8.56±1.25)%、(7.93±0.53)%]和仅由PMA作用得到的未活化巨噬细胞[(49.72±1.82)%、(7.90±1.19)%、(14.39±0.85)%,P<0.05]。自噬相关蛋白LC3BⅠ、Ⅱ、ATGs、Beclin-1表达结果证明,利用药物成功上调,下调了TAM自噬。照光后凋亡检测结果表明,TAM自噬未调节组[(25.18±0.37)%]高于LoVo细胞单独接种组[(13.74±0.61)%],上调组[(41.75±1.15)%]高于LoVo细胞单独接种组,下调组[(19.34±0.72)%]高于LoVo细胞单独接种组;自噬上调组凋亡率[(41.75±1.15)%]高于未调组[(25.18±0.37)%]与下调组[(19.34±0.72)%];免疫组织化学结果显示,存活素(Survivin)与Livin在上调组表达最少;照光后凋亡相关蛋白表达显示:与单独接种组比较,混合接种组p53、Smac表达均升高,而Livin表达下降,上调组下降最为显著;混合接种后MRP4表达显著上升,调控TAM自噬后,LoVo细胞中MRP4表达下降,其中上调组中表达显著降低。结论上调TAM自噬可改变大肠癌细胞凋亡相关蛋白表达,诱导放疗后大肠癌细胞凋亡发生,增强大肠癌细胞的放疗敏感性。

Objective To observe Effect of regulating autophagy of tumor-associated macrophages(TAM)on apoptosis of transplanted colorectal cancer in nude mice after radiotherapy.Methods THP-1 cells(Shanghai Institute of cell,Chinese Academy of Sciences)were induced into TAM with drugs,cell surface iconic molecule were detected by flow cytometry.RAD001 and bafilomycin A1 were used to regulate autophagy of TAM respectively;Autophagy related proteins were detected by Western blotting.TAM were mixed with LoVo cells(American tissue cell bank),inoculated on nude mice(Experimental animal center of Suzhou University).Mice were divided into TAM autophagy up-regulation group,down-regulation group,no regulation group,and LoVo cells inoculated alone as the control group.After 8Gy irradiation The apoptosis of LoVo cells were detected by Immunohistochemistry and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL),apoptosis related proteins were detected by Western blotting.SPSS 17.0 software was used to analyze the data.The data were expressed as mean±standard deviation(Mean±SD).One way ANOVA was used for statistical analysis.The difference was statistically significant when P<0.05.Results CD68,CD204,CD206 of TAM[(62.32±1.58)%,(33.49±2.11)%,(52.42±2.65)%]were significantly higher than that of THP-1[(39.69±1.59)%,(8.56±1.25)%,(7.93±0.53)%]and M0 cells[(49.72±1.82)%,(7.90±1.19)%,(14.39±0.85)%].LC3BⅠ,Ⅱ,ATGs and Beclin-1 were the highest In the up-regulation group,and was the lowest in the down-regulation group(P<0.05).The apoptosis rate of LoVo cells in TAM autophagy unadjusted group[(25.18±0.37%]was higher than that of LoVo cells inoculated alone,TAM autophagy up-regulated group[(41.75±1.15)%]was higher than that of LoVo cells inoculated alone,TAM autophagy down-regulation group[(19.34±0.72)%]was higher than that of LoVo cells inoculated alone;The apoptotic rate of up-regulated group[(41.75±1.15)%]was higher than that of non-regulation group[(25.18±0.37)%]and down-regulation group[(19.34±0.72)%].Immunohistochemistry showed that Survivin and Livin were least expressed in the TAM autophagy up-regulation group.After 8 Gy irradiation,compared with the control group,the expression of p53 increased in the mixed inoculation groups,while the expression of Livin decreased,and the up-regulation group decreased most significantly.After Mixed Inoculation with TAM,the expression of MRP4 increased significantly.After regulating TAM autophagy,the expression of MRP4 decreased in LoVo cells,especially in the up-regulation group.Conclusion Upregulation of TAM autophagy can significantly change the expression of apoptosis-related proteins to promote the apoptosis of colorectal cancer cells after,enhance the killing effect of radiotherapy.