核因子-κB抑制剂对胶质瘤干细胞放疗增敏的作用  

Pyrrolidine dithiocarbamate radiosensitizing glioma stem - like cells through inhibition of nuclear factor- kappa B pathway

作　　者：刘俊峰 谢涛 刘羽 罗龙军 徐成 沈璐 雷霆[1] 叶飞[1] Liu Junfeng;Xie Tao;Liu Yu;Lno Longjun;Xu Cheng;Shen Lu;Lei Ting;Ye Fei(Department of Neurosurgery,Tong,]i Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,Chin;Department of Obstetrics and Gynecology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院神经外科,武汉430030 [2]华中科技大学同济医学院附属同济医院妇产科,武汉430030

出　　处：《中华实验外科杂志》2018年第9期1684-1686,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81272426、81602203）

摘　　要：目的探讨核因子-κB（NF-κB）抑制剂四氢化吡咯二硫代氨基甲酸酯（PDTC）对胶质瘤干细胞（GSLC）放疗增敏的作用和机制。方法用无血清悬浮培养方法培养胶质瘤干细胞U87-sph，并用实时定量聚合酶链反应（Real-timePCR）测定干细胞标志物的表达。用Westernblot比较放疗后不同时间点U87与U87-sph中NF-κB及p-NF-κB表达。用膜联蛋白V（AnnexinV）-异硫氰酸荧光素（FITC）／碘化丙锭（PI）双染方法检测单独PDTC、单独放疗、PDTC联合放疗等不同分组的U87和U87-sph的凋亡率。采用Westernblot检测U87-sph在PDTC联合放疗时凋亡相关蛋白及NF-κB通路蛋白的表达。结果RT-PCR检测U87-sph中干细胞标志物的表达较其亲本细胞株增加。Westernblot结果显示x射线照射后不同时间点，U87-sph中p-NF-κB／p65表达明显增加（P=0．001），且呈时间依赖性。而在U87细胞中变化不明显（P=0．059）。U87-sph的单独PDTC处理组，单独放疗组和PDTC联合放疗组的凋亡率与对照组比较依次增加（1．10±O．07）倍（P=0．651）、（1．49±0．12）倍（P=0．009）、（3．20±0．19）倍（P=0．005）。PDTC联合放疗组的凋亡率与单独PDTC组、单独放疗组比较均明显增加（P=0．003，P=0．004）。Westernblot结果显示U87-sph的PDTC联合放疗组中剪切型半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-8与剪切型Caspase-3表达均明显增加；U87．sph在单独PDTC作用时，p-NF-κB、P-抑制因子KB（p-κB）表达均降低，单独放疗时，两者均增强，而在PDTC联合放疗时，两者表达是降低的；U87-sph中细胞FLICE抑制蛋白（cFLIP）的表达趋势与p-NF-κB相同。结论放疗后GSLC中NF-κB通路明显激活。PDTC可以通过抑制放疗后GSLC中NF-κB通路的激活从而诱导GSLC凋亡。Objective To investigate the effect and the mechanism of pyrrolidine dithiocarbamate （PDTC） on radiosensitization of glioma stem - like cells （GSLCs）. Methods GSLCs, U87 - sph, were cultured from U87 cells using serum free medium. The expression of stem cell markers were analyzed using real -time quantitative polymerase chain reaction （Real -time PCR）. The expression of nuclear factor - kappa B （ NF - κB） and p - NF - κB of U87 and U87 - sph at different time point after irradiation （IR） were detected using Western blotting. Cells were divided into four groups： control group, PDTC alone group, IR alone group and PDTC combined IR group. The apoptotic rates of the four groups of U87 and U87 -sph were analyzed using annexin V -fluoresceine isothiocyanate （FITC）/propidium iodide （PI） kit. The expression of apoptosis - related proteins and NF - κB pathway proteins of U87 - sph in the above four groups was analyzed using Western blotting. Results The expression of stem cell markers were in- creased in U87 - sph compared with the parental glioma cells. Western blotting results showed that the pro-tein levels of p - NF - κB/p65 were increased time dependently in U87 - sph at different time points after irradiation （P = 0. 001 ） , while not changed significantly in U87 cells （P = 0. 059）. The apoptotic rates of U87 -sph in PDTC alone group, IR alone group and PDTC combined IR group were increased （1.10±0.07） times （P=0.651）, （1.49±0. 12） times （P=0. 009） and （3.20±0. 19） times （P=0. 005）, respectively, compared with control group. The apoptotic rates of PDTC combined IR group were significantly increased compared with PDTC alone group （ P = 0. 003 ） and IR alone group （ P = 0. 004 ）. Western blotting results showed that the expressions of cleaved cysteinyl aspartate - specific protease （Caspase） - 8 and cleaved Caspase -3 were both increased in PDTC combined IR group. The protein levels of p - NF - κB, p - inhibitor KB （p -κB） and cellu

关 键 词：胶质瘤 肿瘤干细胞 四氢化吡咯二硫代氨基甲酸酯 核因子-ΚB 放疗增敏 

分 类 号：R739.41[医药卫生—肿瘤]