出 处:《中南大学学报(医学版)》2021年第2期113-120,共8页Journal of Central South University :Medical Science
基 金:国家自然科学基金(81902794)。
摘 要:目的:放射治疗(以下简称放疗)是结直肠癌治疗的主要手段之一,但放疗抵抗往往导致治疗失败。为了改善放疗抵抗,本研究探索H+-ATP合酶抑制剂寡霉素A对结直肠癌HT29细胞放疗敏感性的影响及其机制。方法:采用MTT和糖酵解实验检测不同浓度的寡霉素A在不同时间点对结直肠癌HT29细胞存活率和糖酵解的影响。在体外合成siRNA-PFK1并将之转染到HT29细胞中,采用MTT和克隆形成实验检测寡霉素A对结直肠癌细胞放射敏感性的作用;采用蛋白质印迹法检测寡霉素A对糖酵解酶磷酸果糖激酶1(phosphofructokinase 1,PFK1)表达的影响,比较单纯siRNA-PFK1转染和寡霉素A联合siRNA-PFK1转染对细胞生存和糖酵解的影响。用4 Gy X线照射后,对比寡霉素A联合siRNA-PFK1转染与单纯siRNA-PFK1转染对细胞生存的影响。结果:与0μmol/L寡霉素A组比较,4μmol/L寡霉素A处理的HT29细胞存活率显著增高(P<0.05),细胞/葡萄糖摄取及乳酸和ATP的产生显著增加(均P<0.01)。给予不同剂量(0,2,4,6和8 Gy)的X线照射后,4μmol/L寡霉素A组的克隆形成率及细胞存活率均较0μmol/L寡霉素A组显著增高(均P<0.01)。计算得出寡霉素A对HT29细胞的放射增敏比为0.4886。4μmol/L寡霉素A处理组细胞PFK1的表达水平较0μmol/L寡霉素A组明显升高(P<0.001)。与正常对照组比较,糖酵解水平、克隆形成率、细胞存活率均在HT29细胞转染siRNA-PFK1后降低(均P<0.05),而寡霉素A联合siRNA-PFK1转染组均较siRNA-PFK1组升高(P<0.001)。用4 Gy X线照射后,siRNA-PFK1转染组与单纯照射组比较,克隆形成率和细胞存活率降低(P<0.01或P<0.001),而在寡霉素A联合siRNA-PFK1转染组均较siRNA-PFK1转染组升高(均P<0.001)。结论:寡霉素A可增加结直肠癌HT29细胞放疗抵抗,其机制可能与上调PFK1表达、增强糖酵解有关。Objective:Radiotherapy is one of the main therapies for colorectal cancer,but radioresistance often leads to radiotherapy failure.To improve the radioresistance,we explore the effect of oligomycin A,the H+-ATP synthase inhibitor,on the sensitivity of HT29 colorectal cancer cells to irradiation and its underlying mechanisms.Methods:The effects of different concentrations of oligomycin A on the survival rate and glycolysis of HT29 colorectal cancer cells at different time points were investigated via MTT and glycolysis assay.siRNA-PFK1 was synthesized in vitro and transfected into HT29 cells.The effects of oligomycin A on radiosensitivity of HT29 colorectal cancer cells were measured via MTT and colony formation assay.Western blotting was used to detect the effect of oligomycin A on the expression of glycolytic enzyme PFK1.We compared difference between the effects of siRNA-PFK1 group and oligomycin A combined with siRNA-PFK1 group on cell survival and glycolysis.After 4 Gy X-ray irradiation,the effects of cell survival and glycolysis between the siRNA-PFK1 group and the oligomycin A combined with siRNA-PFK1 group were compared.Results:Compared with the 0μmol/L oligomycin A group,the cell survival rate of HT29 cells treated with 4μmol/L oligomycin A was significantly increased(P<0.05),and the glucose uptake,the lactic acid,and the ATP production were also significantly increased(all P<0.01).After X-ray irradiation at different doses(0,2,4,6,and 8 Gy),the colony formation rate and cell survival rate of the 4μmol/L oligomycin A treated group were significantly higher than those in the 0μmol/L oligomycin A group(both P<0.01).The sensitization enhancement ratio of oligomycin A on HT29 colorectal cancer cells was 0.4886.The expression of PFK1 in the 4μmol/L oligomycin A group was significantly higher than that in the 0μmol/L oligomycin A group(P<0.001).The glycolysis level,colony formation rate,and cell survival rate of the siRNA-PFK1 HT29 cells group were significantly lower than those in the 0μmol/L oligomycin
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