无血清培养胆囊癌GBC-SD细胞形成肿瘤细胞球的鉴定

Identification of tumor spheres from human gallbladder carcinoma GBC-SD cells cultured in serum-free medium

目的:分离扩增肿瘤干细胞,并鉴定其生物学性质.方法:用无血清培养基培养人胆囊癌GBC-SD细胞得到肿瘤细胞球.将肿瘤细胞球传代扩增,并用含血清培养基培养促使其分化;将肿瘤球和普通GBC-SD细胞分别种入96孔板,MTT检测增殖能力,并将肿瘤球和普通GBC-SD细胞分别植入裸鼠皮下,观察移植瘤的形成;流式细胞术检测CD15s和CD24在肿瘤球细胞和普通GBC-SD细胞中的表达,筛选细胞表面标志物.结果:在无血清培养基中,胆囊癌细胞可以形成少量的肿瘤细胞球,并显示很强的自我更新和增殖能力,含血清环境能够诱导其分化而贴壁生长;在动物实验中,肿瘤球细胞较普通GBC-SD细胞显示更强的致瘤能力(80.00%vs10.00%,P<0.05);标志物CD15s在肿瘤球的表达较普通GBC-SD细胞明显增高(2.56%±0.38%vs10.77%±0.93%,t=18.25,P<0.05).结论:人胆囊癌细胞GBC-SD的肿瘤干细胞可以通过无血清培养环境来分离和扩增,CD15s可能为其细胞表面标志物.

AIM： To isolate and expand cancer stem cells in human gallbladder carcinoma cell line GBC-SD and to identify their biological properties. METHODS： GBC-SD cells were cultured in serum-free conditions to derive tumor spheres. Tumor spheres were then expanded and cultured in serum-containing medium to permit their differentiation. The proliferative capacity of tumor sphere-forming cells was tested by methyl thiazoly tetrazolium （MTT） assay. The tumorigenicity of tumor sphere-forming cells was evaluated using animal experiments. The expression of CD15s and CD24 on the surface of tumor sphere-forming cells was detected by flow cytometry. RESULTS： Small number of floating tumor spheres were isolated and expanded in serumfree conditions. These tumor spheres attached to the bottom of culture plates and began to differentiate in serum-containing medium. The proliferation and xenograft tumorigenicity of tumor sphere-forming cells （80.00% vs 10.00%, P 0.05） significantly increased compared with those cultured in serum-containing conditions. The percentage of CD15s-bearing cell population was significantly higher in tumor spheres than in the common GBC-SD cells （2.56% ± 0.38% vs 10.77% ± 0.93%, t = 18.25, P 0.05）. CONCLUSION： The cancer stem cells in GBC-SD cell line can be isolated and expanded in serum-free conditions. CD15s may be a cell surface marker for these cancer stem cells.