CDK4/6抑制剂帕博西尼通过抑制RB磷酸化防护肠上皮细胞放射损伤  

CDK4/6 inhibitor palbociclib protects intestinal epithelial cells from radiation injury by inhibiting RB phosphorylation

作　　者：纪奥强 王训 张雪文 乔子肢 邢爽 申星 余祖胤 JI Aoqiang;WANG Xun;ZHANG Xuewen;QIAO Zizhi;XING Shuang;SHEN Xing;YU Zuyin(School of Life Sciences,Anhui Medical University,Hefei 230032,China;Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]安徽医科大学生命科学学院,合肥230032 [2]军事科学院军事医学研究院,北京100850

出　　处：《军事医学》2024年第11期809-814,共6页Military Medical Sciences

摘　　要：目的 探究CDK4/6抑制剂帕博西尼对大鼠小肠隐窝上皮细胞系(IEC-6)放射损伤的防护作用及其机制。方法 (1)应用CCK-8比色法、集落形成实验、PI染色法、Annexin V-FITC/PI双标法、Western印迹检测法,检测帕博西尼对照后IEC-6细胞增殖、细胞周期、细胞凋亡、DNA损伤及Rb-780、795位点磷酸化的影响;(2)建立IEC-6 Rb过表达细胞系,应用CCK-8比色法、PI染色法、Annexin V-PE/7-AAD双标法、Western印迹检测法,检测Rb过表达对IEC-6细胞系的增殖、周期、凋亡的影响,并在Rb过表达IEC-6细胞系上验证Rb过表达对帕博西尼辐射损伤防护作用的影响。结果 (1)帕博西尼提高10Gy X线照后IEC-6细胞的增殖活力、增加4~8 Gy X线照后集落形成能力,同时降低10 Gy照后G_(2)/M期占比,减少照后IEC-6细胞凋亡。蛋白表达结果表明,帕博西尼抑制Rb-780、795位点磷酸化并减少IEC-6细胞照后γ-H2AX的产生;(2)IEC-6细胞经视网膜母细胞瘤蛋白(Rb)过表达慢病毒转染后,Rb总蛋白表达显著增加,Rb-780、795位点磷酸化水平显著提高,细胞增殖加快、细胞周期中S期占比增加、10 Gy X线照后凋亡率显著增加;(3)较低剂量帕博西尼(0.05μmol/L)处理后,能抑制Rb过表达IEC-6细胞的Rb-780、795位点磷酸化,减少10 Gy X线照射IEC-6 Rb过表达细胞的凋亡,但不抑制IEC-6空载体细胞Rb-780、795位点磷酸化,且对其照射损伤无防护作用。结论 CDK4/6抑制剂帕博西尼对放射引起的大鼠小肠隐窝上皮细胞系损伤具有显著的防护作用,Rb蛋白可能是其发挥放射防护作用的关键分子。Objective To investigate the protective effect and mechanism of CDK4/6 inhibitor palbociclib against radiation-induced injury in rats'small intestinal crypt epithelial cell line(IEC-6).Methods(1)Using CCK-8 colorimetric assay,colony formation assay,the PI staining method,Annexin V-FITC/PI dual staining and Western blot,the impact of palbociclib on proliferation,cell cycle dynamics,apoptosis,DNA damage,as well as phosphorylation events at Rb-780 and 795 sites in irradiated IEC-6 cells was investigated.(2)An IEC-6 cell line overexpressing Rb was established,and the effects of Rb overexpression on proliferation,cell cycle,and apoptosis in the IEC-6 cell line were assessed using the CCK-8 colorimetric assay,PI staining method,Annexin V-PE/7-AAD dual staining and Western blot.Additionally,the impact of Rb overexpression on the protective effect of palbociclib against radiation damage was verified in the Rb-overexpressing IEC-6 cell line.Results(1)Administration of palbociclib was demonstrated to enhance the proliferative capacity of IEC-6 cells following exposure to 10 Gy X-ray irradiation,increase colony formation ability post 4-8 Gy X-ray exposure while concomitantly reducing the proportion of cells in the G_(2)/M phase after 10 Gy irradiation and diminishing apoptosis in irradiated IEC-6 cells.Protein expression analysis revealed that palbociclib inhibited phosphorylation at Rb-780 and 795 sites and attenuated the production ofγ-H2AX in IEC-6 cells following irradiation.(2)After transfection of IEC-6 cells with Rb overexpression lentivirus,there was a significant increase in the total protein expression of Rb.The phosphorylation levels at Rb-780 and 795 sites were markedly elevated.Consequently,cell proliferation was accelerated,the proportion of cells in the S phase of the cell cycle increased,and the apoptotic rate significantly rose after exposure to 10 Gy X-ray irradiation.(3)Treatment with a lower dose of palbociclib(0.05μmol/L)was found to suppress the phosphorylation of Rb at 780 and 795 sites in IEC-6 cel

关 键 词：帕博西尼 电离辐射 IEC-6细胞 集落形成 凋亡 细胞周期 Rb蛋白磷酸化 

分 类 号：R818.052[医药卫生—放射医学]