肿瘤相关巨噬细胞自噬状态对人大肠癌细胞凋亡的影响

Effects of autophagy regulation of tumor-associated macrophages on apoptosis of colorectal cancer cells

目的 观察肿瘤相关巨噬细胞自噬调控后对大肠癌loVo细胞凋亡的影响。方法使用佛波酯PMA、人重组白细胞介素（IL）-4诱导人单核白血病细胞THP-1分化为肿瘤相关巨噬细胞（TAM）后,使用流式细胞仪检测其表面CD68、CD204、CD206分子表达,分别使用一定浓度的雷帕霉素（RAD001）,巴弗洛霉素（Bafilomycin a1）对TAM的自噬状态进行调控,应用磺酰尸胺（MDC）荧光染色法观察TAM自噬囊泡形成,免疫荧光检测自噬特异性微管相关蛋白1轻链3（LC3）表达,利用Transwell小室将不同自噬状态的TAM与大肠癌细胞行非接触式共培养,实验分组为TAM自噬上调组、下调组、未调组及单独大肠癌LoVo细胞的空白对照组4组,各组经4 Gy射线照射后分别对大肠癌细胞凋亡细胞比例、存活素（Survivn）蛋白、B细胞淋巴瘤/白血病-2（bcl-2）蛋白,半胱天冬蛋白酶激活剂（Smac）蛋白进行检测。结果 TAM表面CD68、CD204、CD206表达水平显著高于未经处理的THP-1细胞和只经PMA作用得到的未活化巨噬细胞。TAM自噬上调组中MDC染色的自噬囊泡数目多于自噬未调组,LC3荧光也强于自噬未调组,自噬下调组中MDC染色的自噬囊泡数目少于自噬未调组,LC3荧光强度则弱于自噬未调组。共培养体系中细胞同时接受照射处理后,膜联蛋白V/碘化丙锭（Annexin V/PI）双染检测大肠癌细胞凋亡显示：共培养组中大肠癌细胞凋亡率分别为（36.84±0.37）%、（43.23±1.34）%、（29.37±0.82）%,均高于对照组（27.23±0.63%）（P〈0.05）,其中,TAM自噬上调组中大肠癌细胞凋亡率[（43.23±1.34）%]高于自噬下调组[（29.37±0.82）%]和自噬未调组[（36.84±0.37）%,P〈0.05]。各组大肠癌细胞凋亡相关蛋白的检测结果显示：TAM自噬未调组中bcl-2表达量为0.24±0.02、上调组为0.08±0.01、下调组为0.42±0.02,均低于对照组（0.61±0.05）,TAM可下调大肠癌细胞的bcl-2表达（P〈0.05）,共培养组中Smac表达水平（分别为1.26±0.03、1.49±0.24、0.85±0.03）均高于对照组（0.68±0.03）（P〈0.05）,共培养组中Survivin表达水平（分别为0.48±0.01、0.23±0.02、0.55±0.05）均低于对照组（0.80±0.05）（P〈0.05）。结论 上调TAM自噬水平可显著促进大肠癌细胞凋亡,改变大肠癌细胞凋亡相关蛋白的表达,提高射线对大肠癌细胞的杀伤作用。

Objective To investigate the effects of autophagy regulation of tumor-associated mac rophages on apoptosis of colorectal cancer cells. Methods Human mononuclear THP-1 cells was induced and differentiated into tumor associated macrophages （TAM）with the treatment of phorbol ester phorbol ester （PMA） and human recombinant interleukin （IL）-4, the expression of cell surface iconic molecule CD68, CD204, CD206 were detected by flow cytometry. We used RAD001 and bafilomycin A1 to regulate the autophagy degree of TAM respectively ; the expression of autophagy was monitored through monodansylcadaverin （MDC） staining, MAP 1-microtubule-associated protein 1 light chain 3 （LC3） which is the specific protein of autouphagy was detected through immune fluorescence. Experiment groups were divided into up-regulation autophagy TAM group; down-regulation autophagy TAM group; no regulation autophagy TAM grouo and colorectal LoVo cells cultured alone was the control aroud. We used transwell chamber to con-struct the TAM/colorectal cancer cells co-cultured training system, and after 4 Gy-ray irradiation of each group respectively; we detected the apoptosis radio of colorectal cancer cells and the expression levels of apoptosis related proteins such as B cell lymphoma/leukemia-2 （bcl-2） , Survivin, and Same proteins by Western blotting. Results The expression levels of CD68, CD204, CD206 of TAM were significantly higher than that of untreated THP-1 cells, also higher than the M0 cells. The number of Avs （autophagic vacuoles） and fluorescence inteusityof LC3 in up-regulation autophagy group increased than the control group ; in down- regulation autophagy group, the number of Avs and fluorescence intensity of LC3 decreased than the control group. After radiotherapy, by Annexin V/proliferation index （Annexin V/PI） flow cytometry, we found that significantly apoptosis occurred in groups which colorectal cancer cells co-cuhured with the different autoph- agy degree TAM [ Percentage of apoptotic cells followed by （ 36. 84 ± 0. 37 ） %, （43.23 ± 1.34 ） % , （29. 37 ± 0. 82）% ± compared with the control group （Percentage of apoptotic was [ （27.23 ± 0. 63 ）%, P 〈 0. 05 ] ; the most obviously apoptosis was occurred in the group which colorectal cancer cells co-cul- tured with autophagy up-regulated TAM （43.23 ± 1.34） % （P 〈 0. 05 ）. Western blotting showed that the expression level of apoptosis related proteins bcl-2 in co-cultured groups （ followed by 0. 24 ± 0. 02, 0. 08 ± 0. 01, 0.42 ±0. 02） were lower than that of the control group （0. 61 ±0. 05） （P 〈0. 05） ; the expression of Smac in co-cultured groups （ followed by 1.26 ± 0. 03, 1.49 ± 0. 24, 0. 85 ± 0. 03 ） were higher than that of the control group （0. 68 ±0. 03） （P 〈0. 05） ; the expression of Survivin in co-cultured groups （followed by 0. 48 ± 0. 01, 0. 23 ± 0. 02, 0. 55 ± 0. 05 ） were lower than that of the control group （ 0. 80 ± 0. 05） （ P 〈 0. 05 ）. Conclusion Up-regulation of TAM autophagy degree induced apoptosis of colon cancer cells significantly, changed the expression of apoptosis related proteins, improved the radiosensitivity of coloreetal cancer cells.