肿瘤相关成纤维细胞上调自噬促进结直肠癌耐药  

Cancer associated fibroblasts promote chemoresistence through up - regulation of autophage in colorectal cancer

作　　者：张容圣 李庚 张小超 胡俊波[1] 冯永东[1] Zhang Rongsheng;Li Geng;Zhang Xiaochao;Hu Junbo;Feng Yongdong(Department of Gastrointestinal Surgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院胃肠外科,武汉430030

出　　处：《中华实验外科杂志》2018年第10期1878-1880,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81372323、81572725）

摘　　要：目的探讨结直肠癌中肿瘤相关成纤维细胞（CAFs）促进结直肠癌肿瘤细胞（CRC）对5-氟尿嘧啶（5-Fu）耐药及其机制。方法体外培养原代结直肠癌组织CAFs及普通纤维细胞（NFs），收集肿瘤相关成纤维细胞来源上清（CAFCM）或普通纤维细胞来源上清（NFCM）与5-Fu共同处理人类结肠癌细胞株SW48，利用Hoeehst33342染色观察凋亡细胞形态变化、膜联蛋白V-异硫氰酸荧光素（AnnexinV-FITC）／碘化丙锭（PI）双染检测肿瘤细胞早期凋亡比例的变化，细胞计数试剂盒（CCKl8）绘制毒性曲线，Westernblot法检测甘油醛-3-磷酸脱氢酶（GAPDH）、LC3、Beelin-1的表达水平，利用自噬抑制剂3-甲基腺嘌呤（3-MA）检测自噬在此过程中的作用。结果Hoechst33342染色显示，肿瘤细胞5-Fu较空白对照组及5-Fu＋CAFCM组凋亡细胞比例明显上升，AnnexinV-FITC／PI双染示，3-MA组、5．Fu组、5-Fu＋CAFCM组、5-Fu＋CAFCM＋3-MA组凋亡百分比分别为（7．21±1．3）％、（35．4％±2．6）％、（19．1±0．5）％、（30．4±2．1）％，Western法检测发现5-Fu＋CAFCM组较5-Fu＋NFCM组的LC3-B上升（1．52±0．4）倍、beclin-1表达水平上升（1．76±0．3）倍，与5-Fu比较，5-Fu＋CAFCM组在各时问点细胞活性均明显上升，而5-Fu＋CAFCM＋3-MA组与5-Fu组比，细胞活力差异无统计学意义。以上结论显示CAFCM＋5-Fu组凋亡率较5-Fu组明显减少，差异有统计学意义（P=0．000），3-MA＋CAFCM＋5．Fu组凋亡率较CAFCM＋5-Fu组明显上升，差异有统计学意义（P=0．008），CAFCM＋5-Fu组较NFCM＋5-Fu组LC3-B、beclin-l表达增强，差异统计学意义（P=0．040、0．001）。结论肿瘤相关成纤维细胞可以减少结直肠癌对5-Fu的药物敏感性，而这-效应可能依赖保护性自噬的增强。Objective To investigate phenomenon and mechanism of cancer associated fibroblasts （CAFs） promote chemoresisitence to 5 - fluorouracil （ 5 - Fu） in colon - rectal cancer （CRC） cells. Methods Isolation and cultivation of primary cancer associated fibroblasts and normal fibroblasts, supervision was collected to treat human colorectal cancer cell SW48 with or without 5 - Fu, Hoechst33342 stain was used to observe morphology changes of apoptosis cells, flow cytometry based Annexin V -fluoresceine isothiocyanate （FITC）/propidium iodide （PI） stain was used to evaluate the rate of early apoptpsis cells, cell counting kit - 8 （ CCK - 8 ） was used to depict survival curve, western - blot was used to detect expression of glycerahtehyde - 3 - phosphate dehydrogenase （GAPDH） , LC3, beclin - 1, autophage inhibitor 3 - methyladenine （3 - MA） was used to explore the underlying role of autophage in this phenomenon. Results Hoechst33342 shows the rate of apoptosis cells was decreased compared with 5 - Fu ± CAF CM and 5 - Fu alone, Annexin V - FITC/PI stain shows, apoptosis rate of 3 - MA, 5 - Fu, 5 - Fu ＋ CAF CM, 5-Fu±CAF CM±3 -MA were （7.21 ±1.3）%, （35.4% ±2.6）%, （19.1 ±0.5）%, （30.4 ± 2. 1 ） % , respectively. Expression of beclin - 1 and LC - 3B were increased 1.76 ± 0. 3 and 1.52 ± 0.4 fold compared with 5 - Fu ± CAF CM and 5 - Fu ± NF CM. CCK - 8 shows that cells viability was up - regulated compared with 5 - Fu ± CAF CM and 5 - Fu alone. The results shows that condition medium derived from CAFs can significantly decrease apoptosis rate （ P =0.000 ） , whereas 3 - MA can bloke this effect （ P =0.008 ） , the expression of LC3B and beclinl was up - regulated significantly compared with 5 - Fu ± CAF CM and 5 - Fu ± NF CM （ P = 0. 040,0.001 ）. Conclusion CAFs may promote chemore- sisitence to 5 - Fu in CRC cells, and the function may dependent on upregulation of protective autophage.

关 键 词：结直肠癌 肿瘤相关成纤维细胞 5-氟尿嘧啶 自噬 

分 类 号：R730.231[医药卫生—肿瘤]