结肠癌干细胞样细胞的体外培养、鉴定及n-3多不饱和脂肪酸对结肠癌干细胞样细胞的抗增殖作用

 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。

Cultivation and identification of human colon cancer stem cell-like cells and antiproliferative effects of n-3 polyunsaturated fatty acids

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 eicosapentaenoic 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-4genes and were more resistant to 5-FU and mitomycin C treatments. These cells exhibited a greater ability in clone formation and tumorigenicity, indicating that these cells carried stem cell-like features, hence were considered SW620-derived CSCLC. DHA and/or EPA suppressed 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 resistance to chemotherapeutic drugs, can be obtained from SW620 spheres cultured in serum-free condition. DHA and EPA induce apoptosis in SW620-derived CSCLC and sensitize them to chemotherapeutic drugs.