碱性成纤维细胞生长因子在富集结直肠癌肿瘤干细胞中的作用

目的 研究不同浓度碱性成纤维细胞生长因子（bFGF）作用于结直肠癌细胞不同时间，富集肿 瘤干细胞的效果。方法? 结直肠癌DLD-1细胞分别在含5 ng/mL、10 ng/mL、20 ng/mL bFGF的无血清培 养基中悬浮培养，分为G1、G2、G3组，设置培养时间梯度为10 d、20 d、30 d，获得球细胞。采用流式 细胞术（FCM）检测细胞球中的CD44+、CD133+及CD44+CD133+双阳性的细胞表达比例，Real-time PCR 检测球细胞中的干细胞基因（KLF4、Nanog）；上皮间质转化基因（E-cadherin、Snail）；Wnt/β-catenine通路 基因（Wnt-3a）的 mRNA表达情况，成球实验检测细胞球的自我更新能力。结果 FCM检测结果：CD44+ 阳性表达的细胞表达以G2组20 d最高，CD133+及CD44+CD133+双阳性的细胞表达均以G2组20 d及G3 组10 d最高，差异有统计学意义（F=98.895、147.641、13.321，P<0.05）。 Real-time PCR检测结果：各组中 KLF4、Nanog、Snail以及Wnt-3a mRNA的表达均以G2组20 d表达最高（F=2.424、7.694、2.951、3.771， 均P<0.05）， E-cadherin基因G2组20 d mRNA表达最低（G2组30 d、G1组10 d除外）（F=10.620，P<0.05）。 成球实验结果：各组比较G1组20 d和G2组20 d成球数量明显多于其他组，差异具有统计学意义（F=14.279， P<0.01）；但 G1组20 d和G2组20 d比较，无统计学差异（t=0.605，P=0.578）。 结论 碱性成纤维细胞生长 因子无血清悬浮培养能够有效富集肿瘤干细胞，其中G2组培养20 d较其余组能更有效地富集结直肠癌肿 瘤干细胞。

Role of basic fibroblast growth factor in enrichment of colorectal cancer stem cells

objective To investigate the effect of different concentrations of basic fibroblast growth factor (bFGF) on enriching colorectal cancer stem cells with different lengths of time. Methods DLD-1 cells were cultured in three kinds of serum-free medium containing different concentrations of bFGF, to generate spheroid cells, G1 group (5 ng/mL bFGF), G2 group (10 ng/mL bFGF) and G3 group (20 ng/mL bFGF), and the culture time was 10 d, 20 d and 30 d, respectively. The percentage of CD44+, CD133+ and CD44+CD133+ cells in each group was detected by flow cytometry (FCM). The mRNA expressions of stem cells related genes (KLF4, Nanog), epithelial-mesenchymal transformation (EMT) genes (E-cadherin, Snail), and Wnt/β-catenine pathway genes (Wnt-3a) were detected by real-time polymerase chain reaction (qRT-PCR). The self-renewal capacity was detected by sphere-forming assay. Results The results of FCM: the G2 group on the 20th day had the highest expression of CD44+ cells. Both the G3 group on the 10th day and the G2 group on the 20th day had the highest expression of CD133+ and CD44+CD133+ cells (F=98.895, 147.641, 13.321, all P<0.05). The results of RT-PCR: the mRNA expressions of KLF4, Nanog, Snail and Wnt-3a in G2 group on the 20th day were higher than those in other groups (F=2.424, 7.694, 2.951, 3.771, P<0.05). While E-cadherin had the lowest mRNA expression in G2 group on the 20th day (except for G2 group on the 30th day and G1 group on the 10th day) (F=10.620, P<0.05). The results of sphere-forming assay: the number of spheroid cells in G2 group on the 20th day and in G1 group on the 20th day were higher than those in other groups (F=14.279, P<0.01). The difference of spheroid cells number in G2 group on the 20th day and G1 group on the 20th day was not significant (t=0.605, P=0.578). Conclusion The serum-free suspension culture with bFGF could effectively enrich cancer stem cells, and G2 group on the 20th day is more effective than other groups in enriching colorectal cancer stem cells.