CAJ | 학술논문

目的研究脱氧胞苷激酶(dCK) S74位点在辐射诱导乳腺癌细胞MCF-7死亡中的作用.方法采用脂质体转染方法在MCF-7中分别构建dCK-Vector(空载体)、dCK-WT(野生型)、dCK-S74A(非磷酸化型)及dCK-S74E(过磷酸化型)4种细胞模型,均分别予以0,2,4,6和8 GyX射线照射.实时定量PCR(QPCR)及Western blot验证细胞模型,CCK-8法和集落形成实验检测细胞存活率,单丹磺酰尸胺(MDC)检测自噬发生率,流式细胞术检测细胞凋亡率.结果成功构建4种细胞模型；CCK-8结果显示,与0 Gy组相比,MCF-7和dCK-Vector经8 Gy照射后存活率下降(t=14.469和9.357,P＜0.05),dCK-WT、dCK-S74E和dCK-S74A没有改变(P＞0.05)；与dCK-Vector比较,dCK-WT和dCK-S74E照射后的总死亡率降低(x2=3.857 ～3.971和3.857～3.859,P＜0.05),dCK-S74A则没有变化(P＞0.05)；与各自0 Gy组比较,MCF7、dCK-Vector和dCK-S74A照射后凋亡发生率升高(t=-4.531、-3.688和-7.076,P＜0.05),而dCK-WT和dCK-S74E使照射诱导的凋亡率逆转了66％和68％；dCK-WT和dCK-S74E照射后自噬率分别增加22％和26％(t=-9.051和-8.411,P＜0.01),dCK-S74A、MCF-7和dCK-Vector照射后的自噬率没有明显变化(P＞0.05).结论 dCK-WT和dCK-S74E使电离辐射诱导的凋亡率增加发生逆转,同时使自噬率增加,总死亡率降低,提示dCK在Ser-74的磷酸化与乳腺癌细胞MCF-7的辐射敏感性有关.
목적 연구탈양포감격매(dCK) S74위점재복사유도유선암세포MCF-7사망중적작용.방법 채용지질체전염방법재MCF-7중분별구건dCK-Vector(공재체)、dCK-WT(야생형)、dCK-S74A(비린산화형)급dCK-S74E(과린산화형)4충세포모형,균분별여이0,2,4,6화8 GyX사선조사.실시정량PCR(QPCR)급Western blot험증세포모형,CCK-8법화집락형성실험검측세포존활솔,단단광선시알(MDC)검측자서발생솔,류식세포술검측세포조망솔.결과 성공구건4충세포모형；CCK-8결과현시,여0 Gy조상비,MCF-7화dCK-Vector경8 Gy조사후존활솔하강(t=14.469화9.357,P＜0.05),dCK-WT、dCK-S74E화dCK-S74A몰유개변(P＞0.05)；여dCK-Vector비교,dCK-WT화dCK-S74E조사후적총사망솔강저(x2=3.857 ～3.971화3.857～3.859,P＜0.05),dCK-S74A칙몰유변화(P＞0.05)；여각자0 Gy조비교,MCF7、dCK-Vector화dCK-S74A조사후조망발생솔승고(t=-4.531、-3.688화-7.076,P＜0.05),이dCK-WT화dCK-S74E사조사유도적조망솔역전료66％화68％；dCK-WT화dCK-S74E조사후자서솔분별증가22％화26％(t=-9.051화-8.411,P＜0.01),dCK-S74A、MCF-7화dCK-Vector조사후적자서솔몰유명현변화(P＞0.05).결론 dCK-WT화dCK-S74E사전리복사유도적조망솔증가발생역전,동시사자서솔증가,총사망솔강저,제시dCK재Ser-74적린산화여유선암세포MCF-7적복사민감성유관.
Objective To investigate the roles of dCK Ser-74 in radiation-induced cell death in breast cancer cells.Methods Different phenotypes of dCK plasmids were transfected into MCF-7 cells by liposome transfection,including dCK-Vector,dCK-WT (wild type),dCK-S74A (non-phosphorylation) and dCK-S74E (hyper-phosphorylation).All these cells were irradiated by 0,2,4,6,8 Gy X-rays,respectively.The transcriptional and translational level of dCK were detected with real time-PCR and Western blot,respectively.Radiosensitivity was analyzed using cell counting kit (CCK-8) and colony formation assays.Monodansylcadaverine staining (MDC) and flow cytometry were used to detect autophagy and apoptosis,respectively.Results Four phenotypes of dCK cell models were established successfully.After irradiation,the cell viabilities of MCF-7 and dCK-Vector decreased significantly as compared with mock group (t =14.469 and 9.357,P ＜ 0.05),the cell viabilities of dCK-WT,dCK-S74A and dCK-S74E showed no changes (P ＞ 0.05).The total mortalities of dCK-WT and dCK-S74E decreased significantly as compared with dCK-Vector (x2 =3.857-3.971,P ＜ 0.05),but no changes in dCK-S74A cells (P ＞0.05).The apoptosis rates in dCK-S74A,dCK-Vector and control group were up-regulated after irradiation (t =-4.531,-3.688 and-7.076,P ＜ 0.05),and the irradiation-induced apoptosis was reversed in dCK-WT and dCK-S74E (66％ and 68％ of the increase level in dCK-Vector group).The autophagy in dCK-WT and dCK-S74E increased by 22％ and 26％ (t =-9.051 and-8.411,P ＜0.01),but no changes were observed in dCK-S74A,dCK-Vector and control groups (P ＞ 0.05).Conclusions The dCK-WT and dCK-S74E could reverse the irradiation-induced apoptosis,increase the autophagy occurence,and decrease the total mortality,indicating that the phosphorylation of dCK at Ser-74 sites is related to the radiosensitivity of MCF-7 cells.