铜吸收转运蛋白介导放射性肠损伤的作用及机制研究  

Mechanisms of copper transporter 1 gene in regulating radiation induced intestinal injury

作　　者：王议贤 刘立[1] 莫韦 朱巍[1] 冯亚辉 焦旸[1] 曹建平[1] Wang Yixian;Liu Li;Mo Wei;Zhu Wei;Feng Yahui;Jiao Yang;Cao Jianping(School of Radiation Medicine and Protection,State Key Laboratory of Radiation Medicine and Protection,Soochow University,Suzhou 215123,China;Second Affiliated Hospital of Chengdu Medical College,China National Nuclear Corporation No.416 Hospital,Chengdu 610051,China)

机构地区：[1]苏州大学放射医学与防护学院,放射医学与辐射防护国家重点实验室,苏州215123 [2]成都医学院第二附属医院核工业四一六医院,成都610051

出　　处：《中华放射医学与防护杂志》2023年第6期401-408,共8页Chinese Journal of Radiological Medicine and Protection

基　　金：国家自然科学基金(U1967220,81872552);国家重点研发计划项目(2022YFC2503700,2022YFC2503703)。

摘　　要：目的探讨铜吸收转运蛋白(CTR1)在辐射诱导的小肠细胞放射损伤中发挥的作用及机制。方法采用人小肠上皮细胞(HIEC)和大鼠小肠隐窝上皮细胞(IEC⁃6),分别经2、4、6、8 Gy和5、10、15、20 Gy X射线照射,构建放射损伤细胞模型。照后2、4、8、24、48 h收集细胞,通过Western blot检测CTR1蛋白对辐射的时间⁃剂量响应。将CTR1 shRNA转染入HIEC和IEC⁃6细胞后进行X射线照射,采用电感耦合等离子体质谱仪(ICP⁃MS)检测细胞内铜水平。通过克隆形成实验确定CTR1对上述细胞辐射敏感性作用,检测活性氧(ROS)水平和DNA损伤来进一步探讨相关机制。Western blot检测X射线照射以及沉默CTR1对抗氧化蛋白Nrf2、HO⁃1,铜死亡相关蛋白DLAT、LIAS和FDX1表达的影响,初步确定CTR1的调控机制。结果Western blot表明两株细胞经不同剂量的X射线照射后CTR1表达均显著上调,并且呈显著的时间剂量响应关系(t=3.53、3.45、6.37、11.11、11.13,P<0.05)。沉默CTR1后的两株细胞放射敏感性均低于对照,增敏比分别为1.146、1.201。沉默CTR1缓解了辐射诱导IEC⁃6细胞内的铜蓄积(t=3.10,P<0.05)。两株细胞照射沉默组ROS产量显著低于照射对照组(t=5.23、2.96,P<0.05);并且照射沉默组γ⁃H2AX蛋白表达较照射对照组有所减少(t=7.50、4.29,P<0.05)。此外,X射线诱导Nrf2、HO⁃1蛋白表达上调;沉默CTR1后Nrf2和HO⁃1的表达会进一步增加。电离辐射导致铜死亡标志物DLAT以及铁硫簇蛋白LIAS、FDX1丢失,而沉默CTR1能促进上述蛋白表达水平恢复正常。结论CTR1沉默后的小肠细胞HIEC和IEC⁃6辐射抗性增强,涉及的机制可能与氧化应激和铜死亡途径有关。Objective To investigate the effects and mechanisms of copper transporter 1(CTR1)in radiation induced intestinal injury in vitro.Methods Human small intestinal epithelial cells(HIEC)were irradiated with 2,4,6,8 Gy of X⁃rays and rat intestinal epithelial cells(IEC⁃6)were irradiated with 5,10,15,20 Gy of X⁃rays.At 2,4,8,24,and 48 h after irradiation,the expression of CTR1 was detected by Western blot assay.In some experiments,HIEC and IEC⁃6 cells were transfected with CTR1 shRNA and then exposed to X⁃rays.Copper levels were detected by Inductively Coupled Plasma Mass Spectrometry(ICP⁃MS).The radiosensitivity of cells was verified by colonogenic assay,the cellular reactive oxygen species(ROS)level and DNA damage were detected to further explore the related mechanism.In addition,Western blot was applied to detect the expressions of antioxidants and cuproptosis associated proteins in enterocytes after silencing CTR1 or irradiation.Results The expression of CTR1 was increased by X⁃ray irradiation in a dose⁃dependent manner(t=3.53,3.45,6.37,11.11,11.13,P<0.05).CTR1 expression was successfully diminished by CTR1 shRNA adenovirus vectors.According to the survival curves,the enhancement ratios of the radiosensitivity of HIEC and IEC⁃6 cells with CTR1 knocking⁃down were 1.146 and 1.201,respectively.Radiation⁃induced copper accumulation was alleviated after CTR1 silencing in IEC⁃6 cells(t=3.10,P<0.05).At 0.5 h after irradiation,the ROS production in the CTR1 knockdown group was significantly lower than that in the control group(t=5.23,2.96,P<0.05).At 1 h after irradiation,the protein expression ofγ⁃H2AX in the CTR1 knockdown group was obviously lower than that in the control group(t=7.50,4.29,P<0.05).The expressions of Nrf2 and HO⁃1 were increased after irradiation,which could be further increased after CTR1 silencing.In addition,cuproptosis associated protein DLAT,LIAS and FDX1 were reduced post⁃irradiation,which were recovered after CTR1 silencing.Conclusions The radioresistance of HIEC and I

关 键 词：电离辐射 放射性肠损伤 铜吸收转运蛋白 铜死亡 

分 类 号：R818[医药卫生—放射医学]