X线反复照射人食管鳞癌细胞系KYSE-150的生物效应研究

摘要

目的 建立人食管鳞癌放射抗拒性细胞系,并检测肿瘤干细胞标志物的表达,以期了解肿瘤干细胞与放射抗拒性形成之间的关系.方法 分次照射建立人食管鳞癌放射抗拒性细胞系KYSE-150R,在相差显微镜下观察细胞形态学差异,用G显带法进行染色体分析.用成克隆实验验证KYSE-150和KYSE-150R细胞的放射敏感性差异,流式细胞术检测它们的细胞周期分布,Western Blotting法检测它们中肿瘤干细胞(CSCs)标志物β-catenin和Integrin-β_1的表达.结果 KYSE-150R细胞的群体倍增时间长于亲代细胞KYSE-150[(25.90±0.55)h:(23.62±0.23)h,t=6.62,P=0.00].KYSE-150R细胞的染色体数目增加,并观察到染色体畸变.KYSE-150R细胞SF_2、D_0、D_q及N值均高于KYSE-150细胞,Dn值比为1.21.流式细胞仪分析显示KYSE-150细胞照射后S期比例增加(45.35%±4.03%:55.09%±1.70%,t=-3.86,P=0.02),G_2+M期比例下降(9.91%±3.83%:1.15%±0.32%,t=3.95,P=0.02),而KYSE-150R则变化不大.Western Blotting结果 显示KYSE-150R中的β-catenin和Integin-β_1的表达量约为KYSE-150细胞的2倍.结论 新细胞系KYSE-150B比其亲本更具放射抗拒性,其含有的CSCs比例也较其亲本高,证明CSCs与放射抗拒性产生机制有关.%Objective To establish a radioresistant human cell line from esophageal squamous car-cinoma cells and detect the marker expression of cancer stem cells (CSCs). Methods A radioresistant hu-man esophageal squamous carcinoma cell line KYSE-150R was established by fractionated irradiation. Mor-phological changes from KYSE-150 to KYSE-150R were observed by phase- contrast microscopy. Karyotype analysis was performed by G-banding. The radiosensitivity of these two cell lines was assessed by colony for-marion assays. The cell cycle distribution was assayed by flow cytometry. CSCs markers of β-catenin and In-tegrin-β_1 were measured by Western Blot. Results The population doubling time of KYSE-150 and KYSE-150R were (23.62±0.23) hrand (25.90±0.55) hr, respectively (t =6.62,P=0.00). The numbers of chromosomes in KYSE-150R cells were increased and chromosome aberrations were observed. The SF_2, D_0, D_q and N values of KYSE-150R were all higher than those of KYSE-150. The ratio of D_0 values was 1.21. After irradiation, the number of S-phase cells of KYSE-150 increased from 45.35%±4.03% to 55.09%± 1.70% (t = -3.86,P=0.02) and G2/M phase cells decreased from 9.91%±3.83% to 1.15%±0.32% (t = 3.95, P = 0.02). However, no apparent change of cell cycle distribution for KYSE- 150R was observed. The expression levels of CSCs markers, β-catenin and Integrin-β_1 in KYSE-150R were about 2 times of those in KYSE-150. Conclusions The new cell line KYSE-150R is more radioresistant than its parental cell line KYSE-150. The CSCs in KYSE-150R is more than those in KYSE-150, which may suggest that CSCs is re-lated with the radioresistance.