CAJ | 학술논문

目的：采取无血清培养法培养出SW620细胞球，并对细胞球细胞进行干细胞鉴定；在细胞水平研究n-3多不饱和脂肪酸（ n-3 PUFAs）对结肠癌干细胞样细胞的作用。方法：正常培养人结肠癌细胞株SW620并使其逐步适应无血清培养条件，经无血清培养1周后收集SW620细胞球。用免疫荧光法检测胚胎干细胞标志物SSEA-1和TRA-1-81；采用real-time PCR的方法检测干细胞相关基因Sox-2和Oct-4的表达情况；对比SW620贴壁细胞和干细胞样细胞（ CSCLC）在软琼脂上的克隆形成能力；采用裸鼠移植瘤模型比较2种细胞的成瘤能力；用MTS法对比2种细胞在递增浓度的5-氟尿嘧啶（5-FU）或mitomycin C处理下的生长抑制情况；用MTS法、Annexin V／PI染色和台盼蓝染色分别观察递增浓度二十二碳六烯酸（ DHA）和二十碳五烯酸（ EPA）作用于SW620 CSCLC后细胞生长抑制情况、凋亡情况和死亡情况；MTS法检测5-FU或mitomycin C联合n-3 PUFAs对结肠癌CSCLC增殖的影响。结果：无血清培养法成功从SW620中培养出细胞球。细胞球细胞高表达SSEA-1和TRA-1-81并一过性表达Sox-2和Oct-4基因；对5-FU及mitomycin C相对抵抗；在软琼脂上克隆形成率及在裸鼠皮下的成瘤率均显著高于贴壁细胞，表明这些细胞具有干细胞特性，即为来自于SW620的CSCLC。 DHA和（或） EPA作用于SW620 CSCLC能抑制细胞生长、诱导细胞凋亡，并能增强5-FU及mitomycin C对其抑制作用。结论：无血清培养法能够从SW620细胞中培养出具有干细胞特性的细胞，它们具有高克隆形成能力及高致瘤性，对化疗药物相对抗拒；DHA和EPA能够诱导SW620来源CSCLC发生凋亡并增强化疗药物的抗肿瘤活性。
목적：채취무혈청배양법배양출SW620세포구，병대세포구세포진행간세포감정；재세포수평연구n-3다불포화지방산（ n-3 PUFAs）대결장암간세포양세포적작용。방법：정상배양인결장암세포주SW620병사기축보괄응무혈청배양조건，경무혈청배양1주후수집SW620세포구。용면역형광법검측배태간세포표지물SSEA-1화TRA-1-81；채용real-time PCR적방법검측간세포상관기인Sox-2화Oct-4적표체정황；대비SW620첩벽세포화간세포양세포（ CSCLC）재연경지상적극륭형성능력；채용라서이식류모형비교2충세포적성류능력；용MTS법대비2충세포재체증농도적5-불뇨밀정（5-FU）혹mitomycin C처리하적생장억제정황；용MTS법、Annexin V／PI염색화태반람염색분별관찰체증농도이십이탄륙희산（ DHA）화이십탄오희산（ EPA）작용우SW620 CSCLC후세포생장억제정황、조망정황화사망정황；MTS법검측5-FU혹mitomycin C연합n-3 PUFAs대결장암CSCLC증식적영향。결과：무혈청배양법성공종SW620중배양출세포구。세포구세포고표체SSEA-1화TRA-1-81병일과성표체Sox-2화Oct-4기인；대5-FU급mitomycin C상대저항；재연경지상극륭형성솔급재라서피하적성류솔균현저고우첩벽세포，표명저사세포구유간세포특성，즉위래자우SW620적CSCLC。 DHA화（혹） EPA작용우SW620 CSCLC능억제세포생장、유도세포조망，병능증강5-FU급mitomycin C대기억제작용。결론：무혈청배양법능구종SW620세포중배양출구유간세포특성적세포，타문구유고극륭형성능력급고치류성，대화료약물상대항거；DHA화EPA능구유도SW620래원CSCLC발생조망병증강화료약물적항종류활성。
[ ABSTRACT] AIM:To cultivate stem-like spheres from SW620 cell line in the specific serum-free medium and evaluate the features of the cancer stem cells, and to investigate the effects of docosahexaenoic acid ( DHA) and eicosapen-taenoic acid ( EPA) on the growth of SW620 stem cell-like cells.METHODS: Human colon cancer stem cell-like cells ( CSCLC) were obtained from SW620 spheres cultured in serum-free medium.These cells were tested for the expression of SSEA-1 and TRA-1-81 by immunofluorescence staining.The mRNA expression of Sox-2 and Oct-4 was detected by real-time PCR.The efficiency of colony formation on a soft agar gel and tumor formation in the nude mice was compared between SW620 adherent cells and CSCLC.The inhibitory effects of 5-fluorouracil (5-FU) and mitomycin C on both types of cells were measured by MTS assay.MTS assay, Annexin V/PI staining and trypan blue staining were used to determine the effects of DHA and EPA on both types of cells.MTS assay was also used to analyze the combined effect of DHA or EPA with chemotherapeutic drugs on SW620 CSCLC.RESULTS:SW620 cells formed spheres in serum-free culture.The cells from spheres highly expressed SSEA-1 and TRA-1-81, transiently expressed Sox-2 and Oct-4 genes and were more resistant to 5-FU and mitomycin C treatments.These cells exhibited a greater ability in clone formation and tumorigenicity, indica-ting that these cells carried stem cell-like features, hence were considered SW620-derived CSCLC.DHA and/or EPA sup-pressed SW620 CSCLC by inhibiting cell growth, inducing cell apoptosis and sensitizing them to chemotherapeutic drugs. CONCLUSION:The cells with stem cell-like features, such as high efficiency in clonogenicity, tumorigenicity and resist-ance to chemotherapeutic drugs, can be obtained from SW620 spheres cultured in serum-free condition.DHA and EPA in-duce apoptosis in SW620-derived CSCLC and sensitize them to chemotherapeutic drugs.