【摘要】 目的 基于TGF-β/Smad信号通路探究HSF1基因对CAL-27细胞放疗抵抗作用机制。方法 用人口腔鳞癌细胞CAL-27建立放疗抵抗模型，随机分为抵抗组（放射抵抗CAL-27细胞）、NC组（放射抵抗CAL-27细胞转染空载体）、HSF1组（放射抵抗CAL-27细胞转染HSF1shRNA干扰慢病毒载体）。18只SD裸鼠分为口腔癌抵抗组（注射放射抵抗CAL-27细胞）、沉默HSF1组（注射HSF1shRNA干扰慢病毒载体放射抵抗CAL-27细胞），每组各9只。RT-PCR与Western blot检测HSF1、TGF-β、Smad2、Smad3、Smad7基因及蛋白水平。流式细胞仪检测各组口腔癌细胞凋亡情况。双荧光素酶报告基因分析HSF1与TGF-β/Smad2/Smad3靶向关系，游标卡尺测量瘤体并计算瘤体体积。结果 放疗前，CAL-27口腔癌细胞形态无变化。放疗后，放疗抵抗口腔癌细胞株排列更加紊乱且细胞数目明显增加。抵抗组CAL-27口腔癌细胞中HSF1、TGF-β、Smad2、Smad3、Smad7基因与蛋白表达水平与NC组比较无显著差异（P>0.05）。与NC组相比，HSF1组CAL-27口腔癌细胞中HSF1、TGF-β、Smad2、Smad3基因与蛋白表达水平降低，Smad7基因与蛋白表达水平升高（P<0.05）,HSF1与TGF-β、Smad2及Smad3表达水平呈现正相关性（P<0.05），与Smad7表达水平呈现负相关性（P<0.05）。抵抗组CAL-27口腔癌细胞凋亡率与NC组比较无显著差异（t=0.891,P=0.394）,NC组细胞凋亡率低于HSF1组，组间比较差异显著（t=24.110,P<0.001）。双荧光素酶报告基因实验发现，与pcDNA3.1相比，转染pcDNA3.1-HSF1后野生型TGF-β/Smad2/Smad3活性升高。口腔癌抵抗组裸鼠瘤体体积明显高于沉默HSF1组（t=7.379,P<0.001）。结论 沉默HSF1表达可促进口腔癌放疗抵抗细胞凋亡，降低裸鼠瘤体体积，其作用机制可能与调控TGF-β/Smad有关，可提高口腔癌放疗敏感性。更多还原

【Abstract】 Objective Exploring the mechanism of HSF1gene on radiotherapy resistance of CAL-27cells based on TGF-β/Smad signaling pathway.Methods Human oral squamous cell carcinoma cells CAL-27established a radiotherapy resistance model,and were randomly divided into resistance group(radio-resistant CAL-27cells),NC group(radioresistant CAL-27cells transfected with empty vector),and HSF1group(radio-resistant CAL-27cells)Transfection of HSF1shRNA interference lentiviral vector).Eighteen SD nude mice were divided into oral cancer resistance group(injection of radio-resistant CAL-27cells)and silent HSF1group(injection of HSF1shRNA interference lentiviral vector radio-resistant CAL-27cells),with 9mice in each group.RT-PCR and Western blot detection of HSF1,TGF-β,Smad2,Smad3,Smad7gene and protein levels.Flow cytometry detected the apoptosis of oral cancer cells in each group.Dual luciferase reporter gene analysis HSF1and TGF-β/Smad2/Smad3targeting relationship,The vernier caliper measures the tumor and calculates the volume of the tumor.Results Before radiotherapy,there was no change in the morphology of CAL-27oral cancer cells.After radiotherapy,the arrangement of radiotherapy-resistant oral cancer cell lines became more disordered and the number of cells increased significantly.The expression levels of HSF1,TGF-β,Smad2,Smad3,Smad7genes and protein in CAL-27oral cancer cells in the resistance group were not significantly different from those in the NC group(P>0.05).Compared with the NC group,HSF1,TGF-β,Smad2,Smad3gene and protein expression levels in CAL-27oral cancer cells in HSF1group decreased,and Smad7gene and protein expression levels increased(P<0.05).HSF1was positively correlated with TGF-β,Smad2and Smad3expression levels(P<0.05),and negatively correlated with Smad7expression levels(P<0.05).The apoptosis rate of CAL-27oral cancer cells in the resistance group was not significantly different from that of the NC group(t=0.891,P=0.394).The apoptosis rate of the NC group was lower than that of the HSF1group,and the difference between the groups was significant(t=24.110,P<0.001).The double luciferase reporter gene experiment found that compared with pcDNA3.1,the activity of wild-type TGF-β/Smad2/Smad3increased after transfection with pcDNA3.1-HSF1.The tumor volume of nude mice in the oral cancer resistance group was significantly higher than that in the silent HSF1group(t=7.379,P<0.001).Conclusion Silencing HSF1expression can promote oral cancer radiotherapy-resistant cell apoptosis and reduce the tumor volume in nude mice.Its mechanism may be related to the regulation of TGF-β/Smad and improve the sensitivity of oral cancer radiotherapy.更多还原