CAJ | 학술논문

目的建立研究旁效应DNA损伤的新的实验模型,探究低剂量α粒子辐射产生的旁效应DNA双链断裂(DSB)和修复的效应特征.方法利用发红色荧光的HsBrk1-RFP融合蛋白标记其中一个细胞系,达到在共培养的条件下将直接受照细胞和非照射旁细胞区分开来,建立旁效应实验模型,α粒子照射细胞,γH2AX免疫荧光杂交和激光共聚焦显微观察检测DNA DSB损伤和修复动力学.结果建立了HFS-RFP细胞和HFS细胞共培养的旁效应研究实验模型.无论是0.1Gy还是0.2 Gyα粒子照射,直接受照细胞HFS-RFP和旁效应细胞HFS中的γH2AX簇集点数随时间变化的趋势相似,均在照后1h达到高峰,随即减少,但在照射后8h又出现一个高峰值,显示第二次DNA DSB损伤.旁效应二次DSB损伤要明显弱于直接受照细胞的二次损伤.结论建立的细胞共培养旁效应实验模型能够成功地应用于低剂量的α粒子诱导的旁效应研究,并且低剂量的α粒子照射产生旁效应DSB损伤与修复的变化趋势与直接照射细胞的损伤相似,旁效应二次DSB损伤弱于直接照射细胞的二次损伤,可能与DNA簇集复杂损伤发生比例有关.
목적 건립연구방효응DNA손상적신적실험모형,탐구저제량α입자복사산생적방효응DNA쌍련단렬(DSB)화수복적효응특정.방법 이용발홍색형광적HsBrk1-RFP융합단백표기기중일개세포계,체도재공배양적조건하장직접수조세포화비조사방세포구분개래,건립방효응실험모형,α입자조사세포,γH2AX면역형광잡교화격광공취초현미관찰검측DNA DSB손상화수복동역학.결과 건립료HFS-RFP세포화HFS세포공배양적방효응연구실험모형.무론시0.1Gy환시0.2 Gyα입자조사,직접수조세포HFS-RFP화방효응세포HFS중적γH2AX족집점수수시간변화적추세상사,균재조후1h체도고봉,수즉감소,단재조사후8h우출현일개고봉치,현시제이차DNA DSB손상.방효응이차DSB손상요명현약우직접수조세포적이차손상.결론 건립적세포공배양방효응실험모형능구성공지응용우저제량적α입자유도적방효응연구,병차저제량적α입자조사산생방효응DSB손상여수복적변화추세여직접조사세포적손상상사,방효응이차DSB손상약우직접조사세포적이차손상,가능여DNA족집복잡손상발생비례유관.
Objective To establish an experimental model for the study of α-particle-induced bystander effect of DNA damage and investigate the characteristics of bystander DNA double-strand break (DSB).Methods The red fluorescence fusion protein of HsBrkl-RFP was used to mark the cytoplasm of one cell line to distinguish the irradiated target cells (HFS-RFP) and the non-irradiated bystander cells (HFS) in the co-culture cellular model.After α-particle irradiation,cellular DSB and its repair kinetics were analyzed by the immunofluorescence staining of γH2AX and laser confocal microscope observation.Results A bystander studying model was established by co-culturing human HFS-RFP cells with its partner HSF cells.After 0.1 Gy or 0.2 Gy α-particle irradiation,the similar kinetics of γH2AX foci production and abatement were observed in both irradiated HFS-RFP cells and non-irradiated bystander HFS cells,in which the highest level of γH2AX foci was detected at 1 h post-irradiation.The second peak of γH2AX foci formation appeared at 8 h post-irradiation,which possibly indicates the occurrence of secondary DSB.However,the production of secondary DSB in the bystander cells was weaker than that in the irradiated cells.Conclusions The cell co-culture model can be used for bystander effect investigation.Bystander DSB can be effectively induce by irradiation and the secondary breakage of DNA DSB in the bystander cells may relative to the consequential biochemical processing of clustered DNA damage.