CD44＋/CD24＋宫颈癌细胞对X射线照射诱导凋亡的抗拒性

Resistance of CD44＋/CD24＋ expressing cervical cancer cells on apoptosis induced by X-ray irradiation

目的 探讨CD44 ＋/CD24＋宫颈癌细胞的放射抗拒性机制.方法 体外培养人宫颈鳞癌（Siha）细胞,6MVX射线给予8、16、30 Gy照射,流式细胞仪分选出CD44 ＋/CD24＋细胞.采用克隆形成实验拟合细胞存活曲线,比较细胞的放射敏感性;Hochest 33258荧光染色和透射电镜观察细胞形态变化;流式细胞术（FCM）分析细胞凋亡率;凝胶电泳观察凋亡细胞的DNA条带;反转录-聚合酶链反应（RT-PCR）法检测细胞凋亡相关基因mRNA的表达.结果 随着剂量的增加,CD44 ＋/CD24＋宫颈癌细胞比例逐渐增加;CD44 ＋/CD24＋细胞放射敏感性下降（t=93.99 ～400.45,P＜0.05）;CD44 ＋/CD24＋细胞中bcl-2、survivin、Oct4基因表达水平较亲代Siha细胞升高（t=221.35、941.65、82.27,P＜0.01）;Hochest 33258、透射电镜、DNA片段凝胶电泳及流式细胞定量研究显示,Siha细胞出现明显细胞凋亡改变,而Siha CD44 ＋/CD24＋细胞则未出现.结论 CD44＋/CD24＋宫颈癌细胞抵抗放疗的机制是抗拒射线诱导的细胞凋亡.

Objective To explore whether CD44 ＋/CD24 ＋ expressing cervical cancer cells are resistant to X-ray irradiation and investigate the underlying mechanism.Methods Cervical cancer cell line （Siha） was cultured in vitro and the CD44 ＋/CD24 ＋ expressing cells were sorted with a flow cytometer.The cells were irradiated with 8,16 and 30 Gy of 6 MV X-rays.Colony formation test was used to evaluate the radiosensitivity of CD44 ＋/CD24 ＋ expressing cervical cancer cells.Cell morphology was observed by electronmicroscopy,cell apoptosis was analyzed with a flow cytometer and also verified with a DNA ＆quot;ladder＆quot; assay.Gene expression was determined by RT-PCR.Results After radiation,the ratio of CD44 ＋/CD24 ＋ cells significantly increased.Compared to Siha cells,the radiosensitivity of CD44 ＋/ CD24 ＋ cells decreased （t =93.99-400.45,P 〈0.05）,and the expressions of bcl-2,survivin and Oct4 mRNA increased in CD44 ＋/CD24 ＋ cells （t =221.35,941.65,82.27,P 〈0.01）.Both apoptotic body and specific DNA ＆quot;ladder＆quot; pattern were observed in cells but not in the CD44 ＋/CD24 ＋ Sihacells which had no obvious morphological changes of apoptosis.Conclusions The CD44 ＋/CD24 ＋ expressing cervical cancer cells are resistant to X-rays due to expression of anti-apoptosis factors.