结肠癌DLD1细胞肿瘤干细胞富集模型的比较

冷政伟; 钟晓蓉; 熊永福; 周永红; 仲伟; 占志鹏; 林虹宇; 李敬东

doi:10.3760/cma.j.issn.1673-9752.2016.02.011

结肠癌DLD1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

摘要

摘要: 目的:比较3种富集结肠癌DLD1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DLD1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；Realtime 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±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(t=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)。Realtime 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DLD1细胞的肿瘤干细胞。

Abstract: Objective:To compare the effects of 3 enrichment models of cancer stem cells (CSCs) in colon cancer DLD1 cells.
Methods:DLD1 cells were cultured in 3 kinds of serumfree 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 realtime polymerase chain reaction (RTPCR). The capacity of selfrenewal was detected by sphereforming assay. The tumorigenesis in vivo was measured by colony formation assay. Measurement data with normal distribution were presented as ±s, comparisons among groups were analyzed using the oneway ANOVA, and pairwise comparisons were done by the pairedsamples t test. 〖HQK〗
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 RTPCR: mRNA expressions of G1, G2 and G3 groups were respectively 1.272±0.152, 11.636±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.

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