食管癌细胞株中肿瘤干性细胞群的放射生物学特性

Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

目的探讨人食管癌细胞株KYSEl50和TEl中肿瘤干性细胞群与放射抵抗的关系。方法采用无血清悬浮培养法富集食管癌细胞株中富含肿瘤干性细胞群的球囊，逆转录聚合酶链反应（RT-PCR）法检测其干细胞特性基因表达；采用克隆形成实验检测细胞球囊与亲本细胞放射敏感性的差异，并检测不同细胞群在不同剂量照射后的成球数量；采用流式细胞仪检测放疗前后细胞周期和细胞表面分子标记CD44^＋CD271^＋的表达。结果无血清悬浮培养法能富集食管癌KYSEl50和TEl细胞株中具有肿瘤干性的细胞球囊，并且随着传代代数的增加细胞球囊数量增加，第1、2、3代KYSEl50细胞球囊的数量分别为（25±2）个、（37±2）个和（47±3）个，第1、2、3代TEl细胞球囊的数量分别为（15．4±3）个、（24±3）个和（36±4）个，一定的放射剂量可使KYSEl50和TEl细胞成球率增加。KYSEl50细胞球囊和亲本细胞的2Gy时细胞存活分数（sF2Cy）分别为0．81±0．03和0．69±-0．04，TEl细胞球囊和亲本细胞的SF2Cy分别为0．87±0．01和0．80±0．03，差异均有统计学意义（均P〈0．05）。KYSEl50和TEl亲本细胞照射后出现明显的G2期阻滞。同一放射剂量下，放射对食管肿瘤干性细胞的增殖抑制作用低于食管癌亲本细胞（P〈0．05）。在0、4、8Gy照射下，KYSEl50亲本细胞的CD44^＋CD271^＋细胞比例分别为（1．08±O．03）％、（1．29±0．07）％和（1．11±0．09）％，TEl亲本细胞的CD44’CD271’细胞比例分别为（1．16±0．11）％、（0．97±0．08）％和（1．45±0．35）％，差异无统计学意义（均P〉0．05）；KYSEl50细胞球囊的CIM4^＋CD271^＋细胞比例分别为（35．83±1．23）％、（44．90±1．67）％和（57．77±1．88）％，TEl细胞球囊的CD44^＋CD271^＋细胞比例分别为（16．07±O．91）％、（22．67±1．12）％和（33．27±1．07）％。KYSEl50和TEl细胞球囊的4Gy照射组和8Gy照射组与0Gy照射组比较，差异均有统计学意义（均P〈0．05）。结论食管癌KYSEl50、TEl细胞球囊含有的肿瘤干性细胞群较亲本细胞更具放射抗拒性，食管癌肿瘤干性细胞群与放射抗拒性产生机制有关。

Objective To study the cancer stem cell populations in esophageal cancer cell lines KYSE150 and TEl and identify whether resulting stem-like cell spheres display radiation resistance characteristics. Methods Serum-free medium （SFM） suspension was used to culture the esophageal cancer stem cell lines and enrich the esophageal stem-like cell spheres. RT-PCR assay was used to detect the stem cell gene expression in the sphere cells. Radiosensitivity of the sphere cells and parental cells were evaluated by clone formation assay. Different cells after irradiation at different doses were tested to evaluate the changes of sphere formation, and cell cycle and CD44 ^＋ CD271 ^＋ expression of the sphere cells were also analyzed by flow eytometry before and after irradiation. Results Cancer stem-like cell spheres were generated from KYSE150 and TEl cells and enriched by culture in serum-free medium, and the number of spheres was increasing alone with the increase of cell passages. The numbers of spheres formed from the 1st, 2nd and 3rd generations of KYSE150 cells were 25± 2, 37 ±2 and 47 ± 3, respectively. The numbers of spheres formed from the 1st, 2nd and 3rd generations of TEl cells were 15 ±3, 24 ±3 and 36 ±4, respectively. Certain doses of radiation increased the sphere formation rate. The average survival fraction （SF2） of the suspension-cultured KYSE150 stem-like cell spheres after 2 Gy irradiation were 0.81 ± 0.03 and 0.69 ± 0.04, while that of TEl parental cells were 0.87 ±0.01 and 0.80 ±0.03 （P 〈0.05 for all）. In the esophageal parental KYSE150 and TEl cells, arrest at G2 phase was induced after irradiation. After the same dose of irradiation, the inhibition of proliferation of the cancer stem cells was lower than that of the parent cells （ P 〈 0.05 ）. After 0, 4 and 8 Gy irradiation, the CD44^＋ CD271 ^＋ cell percentage of KYSE150 parental ceils were （1.08 ±0.03）%, （1.29 ±0.07）% and （1.11 ±0.09）% ; the CD44^＋ CD271 ^＋ cell percentage of TEl parental cells were （1.16 ±0.11）%, （0.97 ±0.08）% and （1.45 ±0.35）% （P 〉0.05 for all）. After 0, 4 and 8 Gy irradiation, the percentage of CD44 ^＋ CD271 ^＋ cells of KYSE150 stem cell-like spheres were （35.83 ± 1.23）%, （44.90 ± 1.67）% and （57.77 ± 1.88）%, and that of TEl stem cell-like spheres were （16.07 ±0.91）%, （22.67 ± 1.12）% and （33.27±1.07）%. Compared the4 Gy and 8 Gy irradiated KYSE150 and TEl stem-like cell spheres with the 0 Gy irradiated spheres, the differences were statistically significant （P 〈 0.05 for all）. Conclusions The cancer stem cells in KYSE150 and TEl spheres are more radio-resistant than their parental ceils. It may suggest that cancer stem cell populations in the esophageal cancer cells are related to the mechanism of occurrence of radioresistance.