肿瘤相关巨噬细胞自噬状态对人大肠癌细胞凋亡的影响
종류상관거서세포자서상태대인대장암세포조망적영향
Effects of autophagy regulation of tumor-associated macrophages on apoptosis of colorectal cancer cells
  • 万方数据
    中华实验外科杂志 중화실험외과잡지
    CHINESE JOURNAL OF EXPERIMENTAL SURGERY
    2014年 7期 1504-1507 ,共4页

    邵乐宁%杨晓东%邢春根%吴永友%赵奎%龚巍%钟丰云%曹建平

    소악저%양효동%형춘근%오영우%조규%공외%종봉운%조건평

    肿瘤相关巨噬细胞%自噬%凋亡%大肠癌%放疗 腫瘤相關巨噬細胞%自噬%凋亡%大腸癌%放療
    종류상관거서세포%자서%조망%대장암%방료
    Tumor-associated macrophages%Autophagy%Apoptosis%Colorectal cancer%Radiotherapy
    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4Gy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.
    목적 관찰종류상관거서세포자서조공후대대장암loVo세포조망적영향.방법 사용불파지PMA、인중조백세포개소(IL)-4유도인단핵백혈병세포THP-1분화위종류상관거서세포(TAM)후,사용류식세포의검측기표면CD68、CD204、CD206분자표체;분별사용일정농도적뢰파매소(RAD001),파불락매소(Bafilomycin a1)대TAM적자서상태진행조공,응용광선시알(MDC)형광염색법관찰TAM자서낭포형성,면역형광검측자서특이성미관상관단백1경련3(LC3)표체;이용Transwell소실장불동자서상태적TAM여대장암세포행비접촉식공배양,실험분조위TAM자서상조조、하조조、미조조급단독대장암LoVo세포적공백대조조4조,각조경4Gy사선조사후분별대대장암세포조망세포비례、존활소(Survivn)단백、B세포림파류/백혈병-2(bcl-2)단백,반광천동단백매격활제(Smac)단백진행검측.결과 TAM표면CD68、CD204、CD206표체수평현저고우미경처리적THP-1세포화지경PMA작용득도적미활화거서세포.TAM자서상조조중MDC염색적자서낭포수목다우자서미조조,LC3형광야강우자서미조조;자서하조조중MDC염색적자서낭포수목소우자서미조조,LC3형광강도칙약우자서미조조.공배양체계중세포동시접수조사처리후,막련단백V/전화병정(Annexin V/PI)쌍염검측대장암세포조망현시:공배양조중대장암세포조망솔분별위(36.84 ±0.37)％、(43.23±1.34)％、(29.37±0.82)％,균고우대조조(27.23±0.63％)(P＜0.05);기중,TAM자서상조조중대장암세포조망솔[(43.23±1.34)％]고우자서하조조[(29.37±0.82)％]화자서미조조[(36.84±0.37)％,P＜0.05].각조대장암세포조망상관단백적검측결과현시:TAM자서미조조중bcl-2표체량위0.24±0.02、상조조위0.08±0.01、하조조위0.42±0.02,균저우대조조(0.61±0.05),TAM가하조대장암세포적bcl-2표체(P＜0.05);공배양조중Smac표체수평(분별위1.26±0.03、1.49±0.24、0.85±0.03)균고우대조조(0.68±0.03)(P＜0.05);공배양조중Survivin표체수평(분별위0.48±0.01、0.23 ±0.02、0.55±0.05)균저우대조조(0.80±0.05) (P ＜0.05).결론 상조TAM자서수평가현저촉진대장암세포조망,개변대장암세포조망상관단백적표체,제고사선대대장암세포적살상작용.
    Objective To investigate the effects of autophagy regulation of tumor-associated macrophages 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-micmtubule-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 group and colorectal LoVo cells cultured alone was the control group.We used transwell chamber to construct 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 Smac 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 intensityof LC3 in up-regulation autophagy group increased than the control group; in downregulation 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-cultured with the different autophagy 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-cultured 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 colorectal cancer cells.
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