人喉癌干细胞的培养鉴定及其增殖的抑制研究

The Indentification and Suppression Effection of Proliferation on Human Laryngeal Cancer Stem Cell

喉癌发病率逐年上升,常规化疗药物如顺铂(cisplatin, DDP)和盐酸阿霉素(doxorubicin hydrochloride, DOX)在杀伤喉癌细胞、减少远处转移和提高患者生存率方面至关重要,但对肿瘤干细胞的杀伤效果欠佳。靶向性或选择性杀伤肿瘤干细胞,有望提高化放疗敏感性,减少癌症转移及复发。该文主要通过体外悬浮培养人喉癌干细胞(Hep2 sphere),运用qRT-PCR、Western blot等实验方法检测Hep2 sphere相关因子的表达、细胞周期和耐药性等数据。结果显示,其在一定程度上具备肿瘤干细胞样特征。克隆形成实验和细胞计数法检测得到的人喉癌干细胞增殖能力弱,多处于静息状态G0/G1期。q RT-PCR结果显示,在Hep2 sphere中CD44、ALDH、p-AKT、bcl2和p21表达显著上调, bax和AKT表达下调。Western blot也证实,在Hep2 sphere中bax和AKT蛋白表达下调, bcl2、p21蛋白表达显著上调。MTT实验检测表明,在不同化疗药物处理下, Hep2 sphere的细胞存活率明显高于Hep2;在腺病毒ZD55-Trail处理下, Hep2的细胞存活率则明显高于Hep2 sphere。研究证明,悬浮培养的人喉癌干细胞具有喉癌干细胞特性;腺病毒ZD55-Trail对人喉癌干细胞具有较强杀伤效果,对分化的喉癌细胞影响较弱, DDP、Dox和5-Fu则相反。

The incidence of laryngeal cancer is increasing year by year.Traditional chemotherapy drugs such as cisplatin,doxorubicin hydrochloride,are essential in killing laryngeal cancer cells,reducing laryngeal cancer distant metastasis and improving patient survival,but they are not effective in killing cancer stem cells(CSCs).Targeting or selectively killing CSCs is expected to improve the sensitivity of chemoradiation,reduce cancer metastasis and recurrence.In this paper,laryngeal cancer stem cells(Hep2 sphere)were obtained by suspension culture of Hep2 in vitro.The expressions of Hep2 sphere related factors,cell cycle and resistance were detected by qRT-PCR and Western blot,indicating that they have tumor stem cells characteristic to some extent.Cloning formation experiment and cell counting show that many Hep2 sphere are in the resting stste G0/G1 phase and their proliferation ability is weak.qRT-PCR results indicated Hep2 sphere upregulated CD44,ALDH1,p-AKT,bcl2 as well as p21 gene and downregulated bax,AKT gene campared to Hep2 cells.Western blot results confirmed that bax and AKT protein expression was downregulted in Hep2 sphere,bcl2 and p21 protein expression were significantly upregulated.MTT assay showed that the cell viability of Hep2 sphere was significantly higher than that of Hep2 under different drug treatments.Under the treatment of adenovirus ZD55-Trail,the cell survival rate of Hep2 was significantly higher than that of Hep2 sphere.This study have shown that suspension cultured laryngeal cancer stem cells have stem cell characteristics;adenovirus ZD55-Trail has stronger killing effect on laryngeal cancer stem cells,and weaker on differentiated Hep2,but DDP,Dox and 5-Fu are on the contrary.