胃癌CD133阳性细胞的纯化及其生物学特性研究

Study on Biological Characteristics of CD133 Positive Population in Gastric Cancer Cells by Magnetic Activated Cell Sorting In Vitro

目的检测人胃癌KATO-Ⅲ、SGC7901、AGS及MKN-45细胞株中CD133基因及蛋白的表达,研究CD133阳性(CD133+)细胞与CD133阴性(CD133-)细胞的生物学特性的差异。方法采用半定量聚合酶链反应及免疫蛋白印迹法分别检测KATO-Ⅲ、SGC7901、AGS及MKN-45细胞株中CD133基因和蛋白的表达;免疫磁珠细胞技术将KATO-Ⅲ细胞株中CD133+和CD133-细胞分离纯化,对比两者生长形态特点、体外增殖能力及分化能力的差异;采用CCK-8法分析CD133+和CD133-细胞对不同浓度(0.05、0.10、0.20、0.50、1.00mg/ml)化疗药物5-氟尿嘧啶(5-FU)敏感性的差别。结果 KATO-Ⅲ细胞中CD133基因及蛋白表达量均明显高于SGC7901、AGS及MKN-45细胞(P<0.05)。CD133+细胞在无血清培养基中呈球形克隆悬浮生长,相比CD133-细胞具有更强的体外增殖能力及潜在分化能力。5-FU耐药性实验中,CD133+细胞在0.50mg/ml浓度下的抑制率较CD133-细胞明显降低(P<0.05),但其他浓度时二者比较差异无统计学意义(P>0.05)。结论人胃癌细胞株KATO-Ⅲ中的CD133+细胞具有一定增殖、分化及耐药潜能,CD133可作为胃肿瘤起始细胞亚群的表面标志之一。

Objective To detect the expressions of CD133 in four gastric cancer cell lines（KATO-Ⅲ,SGC7901,AGS,and MKN-45） and investigate the different biological characteristics of CD133 positive（CD133＋） cells and CD133 negative（CD133-） cells in the KATO-Ⅲ cell lines. Methods The CD133 gene and protein expressions of four gastric cancer cell lines were evaluated by semi-quantitative RT-PCR and Western blot,respectively.CD133＋ cells in KATO-Ⅲ cell lines were isolated by magnetic activated cell sorting（MACS） and examined in morphology,growth characteristics,proliferation,and differentiation in vitro.The sensitivity of different concentrations（0.05,0.10,0.20,0.50,and 1.00 mg/ml） 5-fluoropyrimidinedione（5-FU） were contrasted by drug sensitivity testing in vitro（Cell Counting Kit 8-assay） between CD133＋ cells and CD133-cells. Results The expressions of CD133 gene and protein in the KATO-Ⅲ cell lines were significantly higher than those in the other three cell lines（P〈0.05）.CD133＋ cells produced spheroid colonies in serum-free medium culture and were stronger abilities of proliferation and differentiation than those of CD133-cells in vitro.The inhibitor rate of the CD133＋ cells at concentration of 0.50 mg/ml was lower than that of CD133-cells（P〈0.05）. Conclusions Cell population with CD133＋ in the KATO-Ⅲ cell lines have strong ability of cloning,better capability of proliferation and differentiation,as well as anticancer drug resistance to 5-FU.CD133 can be applied as one of surface markers for the detection to gastric cancer initiator cells.