X线反复照射人食管鳞癌细胞系KYSE-150的生物效应研究

Biological response of human esophageal squamous carcinoma cell line KYSE-150 to fractionated xray irradiation in vitro

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—β1的表达。结果KYSE-150R细胞的群体倍增时间长于亲代细胞KYSE-1501（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SF2、D0、Dq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），G2＋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。WesternB lotting结果显示KYSE-150R中的β—catenin和Integfin—β1的表达量约为KYSE-150细胞的2倍。结论新细胞系KYSE-150R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 carcinoma cells and detect the marker expression of cancer stem cells （CSCs）. Methods A radioresistant human esophageal squamous carcinoma cell line KYSE-150R was established by fractionated irradiation. Morphological 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 formarion assays. The cell cycle distribution was assayed by flow cytometry. CSCs markers of β-catenin and Integrin-β1 were measured by Western Blot. Results The population doubling time of KYSE-150 and KYSE- 150R were （ 23.62 ± 0.23 ） hr and （ 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 SF2, Do, Dq and N values of KYSE-150R were all higher than those of KYSE-150. The ratio of Do values was 1.21. After irradiation, the number of S-phase ceils 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-β1 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 related with the radioresistance.