基于克隆形态分选胃癌干细胞及其对氟尿嘧啶敏感性的检测

Isolation of gastric cancer stem cells based on colony morphology and detection of their sensitivity to 5-fluorouracll

目的研究胃癌干细胞对5．氟尿嘧啶（5．FU）的敏感性，探讨胃癌化疗耐药的细胞生物学机制。方法采用免疫组织化学染色法检测71例胃癌组织内千细胞标志物CD44和耐药蛋白胸苷酸合成酶（aS）的表达；采用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，检测其CD44和Ts的表达及自我更新能力；CCK．8法检测不同AGS细胞克隆的5一FU半数抑制浓度（IC50）。结果胃癌组织CD44和Ts表达的阳性率分别为59．2％（42／71）和56．3％（40／71），两者表达呈正相关（P〈0．01）；连续切片显示CD44阳性胃癌细胞同时表达TS。AGS细胞的克隆形态可见全克隆、次克隆、副克隆3种不同形态。全克隆高表达CD44和r11S，接种后可再次形成大量二代克隆；次克隆弱表达CD44和偈，接种后形成的二代克隆较少；而副克隆弱表达甚至不表达CD44和Ts，接种后无二代克隆形成。5-Fu作用于3个全克隆的IC50分别为（113．43±5．81）、（272．68±25．75）和（118．14±17．75）Umol／L，对1个次克隆及AGS细胞的Ic50分别为（16．97±1．01）和（27．52±0．59）la,mol／L，全克隆与次克隆和AGS细胞比较差异具有统计学意义（均P〈O．05）。结论胃癌干细胞的5-FU敏感性较低．其对化疗药耐受可能是胃癌化疗耐药的一个重要机制。

Objective To investigate the sensitivity of gastric cancer stem cells （GCSCs） to 5-fluorouracil （5-FU）, and to explore the cytobiological mechanism of drug-resistance of gastric cancer. Methods Immunohistochemical staining was employed to examine expression of stem cell marker CD44 and drug-resistance protein thymidylate synthase （TS） in 71 cases with gastric cancer. Based on morphology of cell colonies derived from AGS gastric cell line, colonies consisting of GCSCs were isolated, and expression of CD44 and ＇IS, as well as self-renewal capacity of GCSCs were detected. Sensitivity of GCSCs to 5-FU was examined through CCK-8 assay. Results The positive rates of CDdd and TS were 59.2% （42/71） and 56.3% （40/71） in gastric cancer, and expression of CD44 was associated with that of TS （~2=12.76, P〈O.O1;Kappa=0.41 ）. Serial sections indicated that CD44＋ cancer cells simultaneously expressed TS. AGS developed morpholocically diverse colonies, and the GCSCs colonies exhibited a tight and regular shape, which were called holoclone. Holoclones expressed CD44 and TS strongly, possessing capacity of robust self-renewal and forming a lot of second passage clones after incubation. Subclones expressed CD4d and TS weakly, forming less second passage clones. Paraclones almost did not express CD44 and TS, forming no second passage clone after incubation. Affected by 5-Fu, three holoclones showed less growth inhibition compared with another colony type and wild-type AGS ceils.Furthermore, IC50 of5-FU for three holoclones was （113.43±5.81）, （272.68±25.75） and （118.14± 17.75） txmol/L respectively, which were significantly higher than that of one subcolony type （16.97±1.01 ） ）xmol/L] and AGS cells [（27.52±0.59） Umol/Ll （aU P〈O.05）. Conclusions GCSCs possess lower sensitivity to 5-FU, and may play a critical role in drug-resistance of gastric cancer.