CAJ | 학술논문

目的研究耐辐射球菌pprI原核基因的真核表达载体的构建及其转染对离体真核细胞急性放射损伤的防护作用.方法应用DNA重组技术构建pEGFP-c1-pprI质粒;采用脂质体转染技术分别将空载体质粒pEGFP-c1和重组质粒pEGFP-c1-pprI转入人肺上皮细胞系Beas-2B细胞,筛选稳定转染细胞系;应用集落形成实验检测受照转染细胞的生存能力;流式细胞仪检测细胞周期和凋亡率的变化;荧光显微镜观察受照细胞ROS荧光强度;免疫荧光实验观察受照细胞不同时间γ-H2AX焦点数目.结果成功地构建了pprI原核基因的真核表达载体,并在Beas-2B细胞中表达了PprI融合蛋白,建立了稳定转染细胞系.与空白对照组和空载体组相比,细胞克隆生存曲线显示转染了pprI基因的Beas-2B细胞经不同剂量辐射,其存活分数SF增加,该曲线参数D0、Dq、N增高;转染了pprI基因的Beas-2B细胞受照后的ROS的荧光强度和不同时间(1、2、4 h) γ-H2AX的焦点数均显著减低(F=16.73、19.47、6.94,P＜0.05);此外,该细胞G2期阻滞(F=139.73、237.92,P＜0.05)和凋亡率显著减低(F=626.02、2 052,P＜0.05).结论耐辐射球菌pprI原核基因可在离体真核细胞中稳定表达,并且显著提高受照真核细胞的辐射抗性.
목적 연구내복사구균pprI원핵기인적진핵표체재체적구건급기전염대리체진핵세포급성방사손상적방호작용.방법 응용DNA중조기술구건pEGFP-c1-pprI질립;채용지질체전염기술분별장공재체질립pEGFP-c1화중조질립pEGFP-c1-pprI전입인폐상피세포계Beas-2B세포,사선은정전염세포계;응용집락형성실험검측수조전염세포적생존능력;류식세포의검측세포주기화조망솔적변화;형광현미경관찰수조세포ROS형광강도;면역형광실험관찰수조세포불동시간γ-H2AX초점수목.결과 성공지구건료pprI원핵기인적진핵표체재체,병재Beas-2B세포중표체료PprI융합단백,건립료은정전염세포계.여공백대조조화공재체조상비,세포극륭생존곡선현시전염료pprI기인적Beas-2B세포경불동제량복사,기존활분수SF증가,해곡선삼수D0、Dq、N증고;전염료pprI기인적Beas-2B세포수조후적ROS적형광강도화불동시간(1、2、4 h) γ-H2AX적초점수균현저감저(F=16.73、19.47、6.94,P＜0.05);차외,해세포G2기조체(F=139.73、237.92,P＜0.05)화조망솔현저감저(F=626.02、2 052,P＜0.05).결론 내복사구균pprI원핵기인가재리체진핵세포중은정표체,병차현저제고수조진핵세포적복사항성.
Objective To construct the eukaryotic expression vector of pprI gene from Deinococcus radiodurans R1 and investigate its radioresistant effects in eukaryotic cells.Methods A recombinant vector pEGFP-c1-pprI was constructed by DNA recombinant technique.The empty vector pEGFP-c1 and the pEGFP-c1-pprI were transferred into human lung epithelial cells Beas-2B by LipofectamineTM 2000,respectively.Then the infected cells were screened in order to develop a cell line with stable expression of pprI gene.Cell survival rate was tested by clone-forming assay.Cell cycle distribution and apoptosis were detected by a flow cytometry.The fluorescence intensity of reactive oxygen species (ROS) was observed by a fluorescent microscope.γ-H2AX foci in the irradiated cell was detected by immunofluorescence.Results The eukaryotic expression plasmid of pprI prokaryotic gene was constructed and PprI fusion protein was expressed in human lung epithelial cells successfully,and the cell line (2BG) with a stable pprI gene expression was established.After irradiation,the cell survival fraction of 2BG cells was significantly higher than Beas-2B cells so that the value of D0 、Dq and N of the survival curve were increased.Moreover,the fluorescence intensity of ROS and the number of γ-H2AX foci in 2BG cells were also lower than those of B eas-2B cells(F =16.73,19.47,6.94,P ＜ 0.05).Between these two cell lines,the apoptosis rate and cell cycle G2 arrest also had significant difference (F =139.73,237.92,P ＜ 0.05).Conclusions The pprI gene from Deinococcus radiodurans RI can be stably expressed in the eukaryotic cells and it allows the transferred cells to have a radioresistant function.