热疗对人喉癌Hep‑2顺铂耐药细胞株生物学行为的影响  

Effects of hyperthermia on biological behavior of human laryngeal cancer Hep⁃2 cisplatin⁃resistant cell line

作　　者：赵运华[1] 陆海涛[2] 陈宝刚[1] 郭有新[1] 马志红[1] 李健[1] 唐艳丽[1] 胡志红[1] Zhao Yunhua;Lu Haitao;Chen Baogang;Guo Youxin;Ma Zhihong;Li Jian;Tang Yanli;Hu Zhihong(Department of Otolaryngology,Chengde Central Hospital,Chengde 067000,China;Department of Dermatology,the Affiliated Hospital of Chengde Medical College,Chengde 067000,China)

机构地区：[1]河北省承德市中心医院耳鼻喉科,承德067000 [2]承德医学院附属医院皮肤科,承德067000

出　　处：《肿瘤研究与临床》2022年第1期26-32,共7页Cancer Research and Clinic

摘　　要：目的探讨热疗对人喉癌Hep‑2顺铂耐药(Hep‑2/CDDP)细胞株生物学行为的影响和可能机制。方法采用高浓度冲击联合浓度递增法诱导建立Hep‑2/CDDP细胞株。采用细胞计数法检测Hep‑2亲代细胞株组(未发生顺铂耐药的Hep‑2细胞,采用无顺铂的RPMI 1640培养液培养)、Hep‑2/CDDP细胞组、Hep‑2/CDDP+顺铂细胞组(采用含4 mg/L顺铂的RPMI 1640培养液培养)的细胞增殖能力。Hep‑2/CDDP细胞组和Hep‑2亲代细胞株组分别使用含有0、0.004、0.04、0.4、4、40 mg/L顺铂的培养液培养,采用四甲基偶氮唑盐(MTT)法检测Hep‑2/CDDP细胞对顺铂、长春新碱、5‑氟尿嘧啶的敏感性,计算半数抑制浓度(IC50)及耐药指数(RI)。将Hep‑2/CDDP细胞分为4组,对照组细胞于37℃继续培养24 h;热疗组细胞于43℃作用2 h后37℃继续培养22 h;顺铂组用含4 mg/L顺铂的培养液37℃继续培养24 h;热疗联合顺铂组用含4 mg/L顺铂的培养液培养,43℃作用2 h后37℃继续培养22 h。采用MTT法和流式细胞术检测热疗联合顺铂对Hep‑2/CDDP细胞增殖和早期凋亡的影响;采用析因分析法观察热疗联合顺铂对Hep‑2/CDDP细胞增殖和早期凋亡影响的交互作用;采用蛋白质印迹法检测热疗联合顺铂对Hep‑2/CDDP细胞野生型p53和PI3K表达的影响。将Hep‑2/CDDP细胞分为4组,对照组Hep‑2/CDDP细胞于37℃继续培养24 h;化疗药组采用12 mg/L长春新碱或9 mg/L 5‑氟尿嘧啶处理细胞;热疗组Hep‑2/CDDP细胞于43℃作用2 h后37℃继续培养22 h;热疗联合化疗组采用含有12 mg/L长春新碱或9 mg/L 5‑氟尿嘧啶的培养液培养,43℃作用2 h后37℃继续培养22 h。采用MTT法检测热疗分别联合长春新碱、5‑氟尿嘧啶对Hep‑2/CDDP细胞增殖的影响。结果成功建立Hep‑2/CDDP细胞株。不同时间Hep‑2/CDDP细胞组、Hep‑2亲代细胞株组和Hep‑2/CDDP+顺铂细胞组的细胞数差异均无统计学意义(均P>0.05),倍增时间分别为43.Objective To investigate the effects of hyperthermia on the biological behavior of human laryngeal cancer Hep‑2 cisplatin‑resistant(Hep‑2/CDDP)cell line and its possible mechanism.Methods Hep‑2/CDDP cell line was induced by high impact combined with increasing concentration method.Cell count method was used to detect the cell proliferation ability of Hep‑2 parental cell group(Hep‑2 cells without cisplatin‑resistance and the cells were cultured with RPMI 1640 cultured medium without cisplatin),Hep‑2/CDDP cell group and Hep‑2/CDDP+cisplatin group(using RPMI 1640 cultured medium including 4 mg/L cisplatin).Hep‑2/CDDP cell group and Hep‑2 parental cell group were treated with cultured medium including 0,0.004,0.04,0.4,4,40 mg/L cisplatin,respectively.The sensitivity of Hep‑2/CDDP cells to cisplatin,vincristine and 5‑fluorouracil was determined by using methyl thiazolyl tetrazolium(MTT)method.The half inhibitory concentration(IC50)and resistance index(RI)were also calculated.Hep‑2/CDDP cell group was divided into 4 subgroups:the cells in the control group were cultured for 24 h at 37℃;the cells in hyperthermia group were treated at 43℃for 2 h and then re‑cultured at 37℃for 22 h;the cells in cisplatin group were cultured at 37℃for 24 h in cultured medium containing 4 mg/L cisplatin.The cells in hyperthermia combined with cisplatin group were cultured in cultured medium containing 4 mg/L cisplatin,treated at 43℃for 2 h and then re‑cultured at 37℃for 22 h.The effects of hyperthermia combined with cisplatin on the proliferation and early apoptosis of Hep‑2/CDDP cells were detected by using MTT and flow cytometry.The interaction of hyperthermia combined with cisplatin on the proliferation and early apoptosis of HEP‑2/CDDP cells was observed by using factorial analysis.Western blotting was used to detect the effect of hyperthermia combined with cisplatin on the expressions of wild‑type p53 and PI3K in Hep‑2/CDDP cells.Hep‑2/CDDP cells were divided into 4 groups:the contr

关 键 词：喉肿瘤 顺铂 抗药性 肿瘤 热疗 

分 类 号：R739.65[医药卫生—肿瘤]