摘要
目的探讨谷胱甘S-转移酶P1(GSTP1)与放射性肺损伤关系及相关机制。方法应用人正常肺上皮细胞系(BEAS-2B)细胞,针对GSTP1 mRNA序列设计筛选两条有效的RNA干扰;转染后为实验组(siRNA-1、siRNA-2),转染阴性对照siRNA者为阴性对照组(NC)。蛋白免疫印迹法(WB)检测GSTP1蛋白表达;1-氯-2,4-二硝基苯(CDNB)检测GST酶的活性;DCFH-DA探针孵育,利用流式细胞术检测活性氧平均荧光强度(MFI);Annexin-v/PI染料孵育,利用流式细胞术检测细胞凋亡;MTT法检测细胞活性,并绘制生长曲线;WB检测上皮间充质转化(EMT)相关指标。结果照射后活性氧检测MFI实验组较阴性对照组高(6774.66±399.60∶8759.00±256.96∶9 967.67±735.11,P<0.05)。实验组凋亡细胞比例较阴性对照组高(12.3±1.16∶17.38±1.65∶22.88±1.20,P<0.05);MTT法测吸光度(A)值实验组较阴性对照组低;实验组EMT程度高于对照组细胞。结论干涉肺上皮细胞GSTP1蛋白表达,照射后会提高细胞内活性氧水平、凋亡细胞比例,并进一步降低细胞增殖速率,此外肺上皮细胞的EMT发生更加显著。GSTP1蛋白含量下降可能导致辐射诱导肺细胞损伤加重,并促进EMT发生。
Objective To investigate the association and mechanism between glutathione S-transferase P1(GSTP1) and radiation-induced lung injury. Methods Two effective GSTP1 siRNAs were designed and synthesized. The normal lung epithelial cell line BEAS-2B cells were transfected with GSTP1 siRNA (experimental group,siRNA-1,siRNA-2) and negative control siRNA (negative control group,NC).Western blot was performed to detect the expression levels of GSTP1 protein and EMT-related proteins. CDNB was adopted to evaluate the activity of GSTs. DCFH-DA probe was used for incubation. Flow cytometry was conducted to detect the median fluorescence intensity (MFI) and cellular apoptosis. Annexin-v/PI staining was utilized for incubation. MTT assay was performed to measure the proliferation of BEAS-2B,and the growth curve was drawn based on the results. Results After radiation,compared with the NC group,the ROS level and MFI were significantly higher in experimental group (6774.66±399.60 vs.8759.00±256.96 vs.9967.67±735.11,P<0.05).In the experimental group,the percentage of cellular apoptosis was remarkably higher than that in the NC group (12.3±1.16 vs.17.38±1.65 vs.22.88±1.20,P<0.05).MTT assay demonstrated that the OD values in the experimental group were significantly lower than that in the NC group everyday. Further more,the level of EMT process is higher in the experimental group. Conclusions Interfering with the GSTP1 expression in lung epithelial cells can increase the intracellular ROS level,increase the percentage of cellular apoptosis,and reduce the cell proliferation rate following γ-radiation. Besides,it can also promote the epithelial mesenchymal transition in lung epithelial cells. The down-regulation of GSTP1 protein expression level probably aggravates the radiation-induced lung cell injury and promotes the epithelial mesenchymal transition.
作者
贺琦多
马娜
杜乐辉
杨陟华
王易龙
孙泽文
俞伟
黄祥
朱茂祥
曲宝林
He Qiduo;Ma Na;Du Lehui;Yang Zhihua;Wang Yilong;Sun Zewen;Yu Wei;Huang Xiang;Zhu Maoxiang;Qu Baolin(Department of Radiation Oncology,PLA General Hospital,Beijing 100853,China;PLA Medical School,Beijing 100853,China;Academy of Military Medical Sciences,Beijing 100850,China;School of Physics and Nuclear Energy Engineering,Beihang University,Beijing 100191,China;Advanced Innovation Center for Precision Medicine,Beihang University,Beijing 100083,China)
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2019年第5期385-388,共4页
Chinese Journal of Radiation Oncology
关键词
肺上皮细胞系/照射
氧化应激
凋亡
上皮间充质转化
Lung epithelial cell line/irradiation
Oxidative stress
Apoptosis
Epithelial mesenchymal transition
