结肠癌DLD-1细胞肿瘤干细胞富集模型的比较

Comparison of enrichment model of cancer stem cells in colon cancer DLD-1 cells

目的 比较3种富集结肠癌DLD-1细胞肿瘤干细胞的培养模型的效果.方法 用含不同浓度生长因子的无血清培养基分为3组悬浮培养DLD-1细胞获得细胞球,其中G1组方案：10 ng/mL上皮生长因子（EGF）＋10 ng/mL成纤维细胞生长因子（bFGF）;G2组方案：20 ng/mL EGF＋ 10 ng/mL bFGF;G3组方案：20 ng/mL EGF ＋20 ng/mL bFGF.流式细胞术检测细胞球中CD133＋ CD44＋、CD133＋、CD44＋、ALDH＋细胞比例变化;Real-time PCR检测细胞球中干细胞基因（Oct4、Sox2、Nanog）及CSCs基因（CD133、CD44、CD166、ALDH、CD66c） mRNA表达变化;成球实验检测细胞球自我更新能力变化;平板克隆形成实验检测细胞体外成瘤能力变化.正态分布的计量资料以x-±s表示,多组间比较采用单因素方差分析,两两比较采用配对样本t检验.结果 流式细胞术检测结果：G1组、G2组和G3组细胞中CD133＋ CD44＋细胞比例分别为0.030±0.010、0.113±0.012、0.043±0.006,3组比较,差异有统计学意义（F=67.625,P＜0.05）;G2组中CD133＋ CD44＋细胞比例分别与G1组和G3组比较,差异均有统计学意义（=9.449,12.124,P＜0.05）;G1组与G3组比较,差异无统计学意义（t=-4.000,P＞0.05）.G1组、G2组和G3组细胞中CD133＋细胞比例分别为0.037±0.015、0.027 ±0.006、0.023 ±0.006,3组比较,差异无统计学意义（F=1.444,P＞0.05）.G1组、G2组和G3组细胞中CD44＋细胞比例分别为20.103±4.761、44.600±2.138、44.387±2.117,3组比较,差异有统计学意义（F=56.269,P＜0.05）;G1组中CD44＋细胞比例分别与G2组和G3组比较,差异均有统计学意义（t=-10.024,-14.696,P＜0.05）;G2组与G3组比较,差异无统计学意义（t=0.131,P＞0.05）.G1组、G2组和G3组细胞中ALDH＋细胞比例分别为0.060 ±±0.010、0.627±0.068、0.043 ±±0.021,3组比较,差异有统计学意义（F =192.097,P＜0.05）;G2组中ALDH＋细胞比例分别与G1组和G3组比较,差异均有统计学意义（t=12.962,21.380,P＜0.05）;G1组与G3组比较,差异无统计学意义（t=2.500,P＞0.05）.Real-time PCR检测结果：G1组、G2组和G3组干细胞基因Oct4、Sox2、Nanog及CSCs基因CD133、CD44、CD166、ALDH、CD66c mRNA表达分别为1.272±0.152、11.636±3.164、1.420 ±0.201,4.717±0.409、8.262±1.079、4.827±0.446,1.836±0.417、12.883±4.030、1.636±0.589,1.555±0.397、4.367±1.109、1.208 ±0.165,25.853 ±4.544、59.483±8.950、26.924±4.134,21.223±1.088、57.847±5.880、21.710±4.010,19.378±2.479、78.211±9.879、18.475±3.571,4.737±2.105、18.003±3.826、5.211±1.474;各组上述基因mRNA表达比较,差异有统计学意义（F=31.529,23.865,21.557,19.102,27.837,76.608,90.530,23.997,P＜0.05）.上述基因mRNA表达中,G2组分别与G1组和G3组比较,差异均有统计学意义（t=5.950,8.997,5.257,5.507,5.766,9.158,11.507,6.730;5.428,7.782,4.340,5.583,4.979,6.829,7.937,8.445,P＜0.05）;G1组与G3组比较,差异无统计学意义（t=-0.797,-0.555,0.392,2.539,-1.004,-0.232,0.265,-0.675,P＞0.05）.成球实验检测结果：G1组、G2组和G3组细胞球数分别为（182±34）个、（396±22）个、（217 ±22）个,3组比较,差异有统计学意义（F =56.128,P＜0.05）.G2组中细胞球数分别与G1组和G3组比较,差异均有统计学意义（t=6.698,9.483,P＜0.05）;G1组与G3组比较,差异无统计学意义（t=-1.904,P＞0.05）.平板克隆形成实验检测结果：G1组、G2组和G3组细胞体外形成克隆数分别为（230±33）个、（403±32）个、（266±36）个,3组比较,差异有统计学意义（F =22.111,P＜0.05）.G2组中细胞体外形成克隆数分别与G1组和G3组比较,差异均有统计学意义（t=5.053,4.913,P＜0.05）;G1组与G3组比较,差异无统计学意义（t=-2.073,P＞0.05）.结论 20 ng/mL EGF＋ 10 ng/mL bFGF方案更能有效富集结肠癌DLD-1细胞的肿瘤干细胞.

Objective To compare the effects of 3 enrichment models of cancer stem cells （CSCs） in colon cancer DLD-1 cells.Methods DLD-1 cells were cultured in 3 kinds of serum-free medium containing various growth factors to generate spheroid cells,including G1 group using 10 ng/mL EGF ＋ 10 ng/mL bFGF,G2 group using 20 ng/mL EGF ＋ 10 ng/mL bFGF and G3 group using 20 ng/mL EGF ＋ 20 ng/mL bFGF.The cell proportion of CD133 ＋ CD44 ＋,CD133 ＋,CD44 ＋ and ALDH ＋ were confirmed by flow cytometery.The mRNA expression of stem cells related genes （Oct4,Sox2,Nanog） and colon CSCs genes （CD133,CD44,CD166,ALDH,CD66c） were detected by real-time polymerase chain reaction （RT-PCR）.The capacity of self-renewal was detected by sphere-forming assay.The tumorigenesis in vivo was measured by colony formation assay.Measurement data with normal distribution were presented as x-± s,comparisons among groups were analyzed using the one-way ANOVA,and pairwise comparisons were done by the paired-samples t test.Results The results of flow cytometery：the proportion of CD133 ＋ CD44 ＋ cells in the G1,G2 and G3 groups were 0.030 ± 0.010,0.113 ± 0.012 and 0.043 ± 0.006,respectively,with a significant difference among the 3 groups （F =67.625,P ＜ 0.05）.The proportion of CD133 ＋ CD44＋ cells in the G2 group was significantly different from those in the G1 and G3 groups （t =9.449,12.124,P ＜ 0.05）,with no significant difference between G1 and G3 groups （t =-4.000,P ＞0.05）.The proportion of CD133 ＋ cells in the G1,G2 and G3 groups were respectively 0.037 ± 0.015,0.027 ±0.006 and 0.023 ± 0.006,with no significant difference among the 3 groups （F =1.444,P ＞0.05）.The proportions of CD44＋ cells in the G1,G2 and G3 groups were 20.103 ±4.761,44.600 ±2.138 and 44.387 ± 2.117,respectively,with a significant difference among the 3 groups （F =56.269,P ＜ 0.05）.There were significant differences in the proportions of CD44 ＋ cells between G1 group and G2 or G3 group （t =-10.024,-14.696,P ＜ 0.05）,and no significant difference between G2 and G3 groups （t =0.131,P ＞ 0.05）.The proportion of ALDH ＋ cells in the G1,G2 and G3 groups were 0.060 ±0.010,0.627 ±0.068 and 0.043 ±0.021,respectively,with a significant difference among the 3 groups （F =192.097,P ＜ 0.05）.The proportion of ALDH ＋ cells in the G2 group was significantly different from those in the G1 and G3 groups （t =12.962,21.380,P ＜ 0.05）,with no significant difference between G1 and G3 groups （t =2.500,P ＞ 0.05）.The results of RT-PCR：mRNA expressions of G1,G2 and G3 groups were respectively 1.272 ±0.152,1 1.636 ±3.164 and 1.420 ± 0.201 in stem cell related genes Oct4,4.717 ±0.409,8.262 ± 1.079 and 4.827 ± 0.446 in stem cell related genes Sox2,1.836 ± 0.417,12.883 ± 4.030 and 1.636 ± 0.589 in stem cell related genes Nanog,1.555 ± 0.397,4.367 ± 1.109 and 1.208 ± 0.165 in colon CSCs genes CD133,25.853 ± 4.544,59.483 ±8.950 and 26.924 ±4.134 in colon CSCs genes CD44,21.223 ± 1.088,57.847 ±5.880 and 21.710 ±4.010 in colon CSCs genes CD166,19.378 ±2.479,78.211 ±9.879 and 18.475 ± 3.571 in colon CSCs genes ALDH,4.737 ± 2.105,18.003 ± 3.826 and 5.211 ± 1.474 in colon CSCs genes CD66c,showing significant differences among the 3 groups （F =31.529,23.865,21.557,19.102,27.837,76.608,90.530,23.997,P ＜0.05）.There were significant differences in the mRNA expression of genes between G2 group and G1 or G3 group （t =5.950,8.997,5.257,5.507,5.766,9.158,11.507,6.730;5.428,7.782,4.340,5.583,4.979,6.829,7.937,8.445,P ＜ 0.05）,and no significant difference between G1 and G3 groups （t =-0.797,-0.555,0.392,2.539,-1.004,-0.232,0.265,-0.675,P ＞ 0.05）.The results of sphereforming assay：the numbers of spheres in the G1,G2 and G3 groups were 182 ± 34,396 ± 22 and 217 ± 22,respectively,showing a significant difference among the 3 groups （F =56.128,P ＜ 0.05）.The number of spheres in the G2 group was significantly different from those in the G1 and G3 groups （t =6.698,9.483,P ＜0.05）,with no significant difference between G1 and G3 groups （t =-1.904,P ＞ 0.05）.The results of colony formation assay：the numbers of colonies in the G1,G2 and G3 groups were 230 ±33,403 ±32 and 266 ±36,showing a significant difference （F =22.111,P ＜ 0.05）.The number of colonies in the G2 group was significantly different from those in the G1 and G3 groups （t =5.053,4.913,P ＜ 0.05）,with no significant difference between G1 and G3 groups （t =-2.073,P ＞ 0.05）.Conclusion The culturing cells using 20 ng/mL EGF ＋ 10 ng/mL bFGF could effectively promote the enrichment of CSCs in colon cancer DLD-1 cells.