BACKGROUND:Fluorouracil is a basic chemotherapy drug for gastric cancer, and its drug resistance is commonly seen in clinic. Cancer stem cels have low sensitivity to chemotherapy drugs, which may be an important cause of tumor recurrence and progression folowing chemotherpay. OBJECTIVE: To explore the sensitivity of gastric cancer stem cels to 5-fluorouracil and related biology mechanism underlying multidrug resistance. METHODS: Stem cel marker CD44 and multidrug resistance protein thymidylate synthase in 69 cases of gastric cancer tissues were tested by immunohistochemical staining. The selection strategy was based on the clonal morphology, and gastric cancer stem cels were isolated from human gastric cancer cel line AGS to detect the expression of CD44 and thymidylate synthase and the self renewing ability. Inhibitory concentration 50 (IC50) of 5-fluorouracil in different AGS cel clone was detected using cel counting kit-8. RESULTS AND CONCLUSION:In 69 cases of gastric cancer tissues, the positive expression rates of thymidylate synthase and CD44 were 57% (39/69) and 61% (42/69), respectively, and there was a positive correlation between the expression of CD44 and thymidylate synthase (Kappa=0.41,χ2=11.59,P < 0.05). Cloning efficiency of the AGS cel line was 39% (29/69), wherein, vice cloning, secondary cloning, and ful clone proportions were 17% (5/29), 69% (20/29), 14% (4/29), respectively. After sub-cloning, the clone was digested using trysin folowed by low-density passage; vice clones were unable to passage, secondary clones could only passage a few, and ful clones could serialy passage. After treatment with different concentrations of 5-fluorouracil, the ful clone growth inhibition rate was significantly lower than that of secondary clones and AGS cels (P < 0.05). These findings show that gastric cancer stem cels are less sensitive to 5-fluorouracil, indicating the cels have a resistance to chemotherapy drugs, which may be one of the chemotherapy resistance mechanisms in the clinical treatment of gastric cancer.%背景:氟尿嘧啶是胃癌化疗的基础药物,临床上耐药现象较为常见.肿瘤干细胞对化疗药敏感性较低,可能是导致化疗后肿瘤复发进展的重要原因.目的:探讨胃癌干细胞对氟尿嘧啶敏感性,从细胞生物学角度分析胃癌的化疗耐药机制.方法:利用免疫组织化学染色法检测69例胃癌组织内干细胞标志物CD44和耐药蛋白胸苷酸合成酶表达情况;基于克隆形态的分选策略,从AGS胃癌细胞系内分离胃癌干细胞克隆,检测CD44和胸苷酸合成酶的表达和自我更新能力;利用CCK-8法检测不同AGS细胞克隆的5-氟尿嘧啶半数抑制浓度(IC50).结果与结论:69例胃癌组织标本中,胸苷酸合成酶和CD44的表达阳性率分别为57%(39/69)和61%(42/69), CD44与胸苷酸合成酶的表达之间呈正相关关系(Kappa=0.41,χ2=11.59,P < 0.05).AGS细胞系的克隆形成率为39%(29/69),其中,副克隆、次克隆、全克隆所占比例分别为 17%(5/29)、69%(20/29)、14%(4/29).二代克隆形成后,采用胰酶消化克隆并再次低密度接种传代,副克隆均无法传代,次克隆仅少数可连续传代,全克隆均可连续传代.不同浓度5-氟尿嘧啶作用之后,全克隆的生长抑制率均显著低于次克隆和AGS细胞,经比较差异均有显著性意义(P < 0.05).以上结果表明胃癌干细胞对5-氟尿嘧啶敏感性较低,其对化疗药物耐受,可能是临床胃癌化疗耐药的产生机制之一.
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