CAJ | 학술논문

目的探讨DNA依赖性蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)在石英致人胚肺成纤维细胞(HELF)DNA双链断裂修复中的作用.方法脂质体转染法建立DNA-PKcs的小干扰RNA(siRNA)及其阴性对照重组质粒稳定转染HELF系(简称HELF-PKcs和HELF-NC).3种方式分组及对细胞的处理:(1)25、50、100、200、300、400μg/ml浓度的石英刺激HELF 12 h;(2)200μg/ml的石英刺激HELF 0、1、2、6、12、24 h;(3)200μg/ml的石英刺激HELF-PKcs和HELF-NC 0、12、24 h.用免疫印迹法检测DNA-PKcs表达、磷酸化H2AX(H2AX)的水平.用Image-Pro plus6.0软件对条带光强度进行半定量分析;用中性彗星实验(彗尾DNA百分含量值)判断石英诱导的DNA舣链断裂损伤强度.结果不同浓度的石英刺激HELF 12 h,γH2AX水平及彗尾DNA百分含量随着石英浓度的增加逐渐升高.200 μg/ml的石英刺激HELF6 h时,彗尾DNA百分含量[(38.7±6.9)%]与对照组相比明显增加,并且在12h达峰值,24h相对12h时点明显降低,差异均有统计学意义(P<0.05).在抑制DNA-PKcs的表达的细胞,12 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平增加受抑制,彗尾DNA百分含量为(43.09±3.68)%,与石英刺激的HELF-NC相比,差异无统计学意义(P>0.05);24 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平与石英刺激的HELF-NC相比无明显筹异,差异无统计学意义(P>0.05).石英刺激的HELF-PKcs的彗尾DNA百分含量(35.79±4.26)%]明显高于石英刺激的HELF-NC,差异有统计学意义(P<0.05).结论石英可诱导DNA双链断裂损伤,DNA-PKcs是石英诱导的DNA双链断裂损伤的感受器,通过磷酸化H2AX,促进石英诱导的DNA双链断裂损伤的修复.
목적 탐토DNA의뢰성단백격매최화아단위(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)재석영치인배폐성섬유세포(HELF)DNA쌍련단렬수복중적작용.방법 지질체전염법건립DNA-PKcs적소간우RNA(siRNA)급기음성대조중조질립은정전염HELF계(간칭HELF-PKcs화HELF-NC).3충방식분조급대세포적처리:(1)25、50、100、200、300、400μg/ml농도적석영자격HELF 12 h;(2)200μg/ml적석영자격HELF 0、1、2、6、12、24 h;(3)200μg/ml적석영자격HELF-PKcs화HELF-NC 0、12、24 h.용면역인적법검측DNA-PKcs표체、린산화H2AX(H2AX)적수평.용Image-Pro plus6.0연건대조대광강도진행반정량분석;용중성혜성실험(혜미DNA백분함량치)판단석영유도적DNA의련단렬손상강도.결과 불동농도적석영자격HELF 12 h,γH2AX수평급혜미DNA백분함량수착석영농도적증가축점승고.200 μg/ml적석영자격HELF6 h시,혜미DNA백분함량[(38.7±6.9)%]여대조조상비명현증가,병차재12h체봉치,24h상대12h시점명현강저,차이균유통계학의의(P<0.05).재억제DNA-PKcs적표체적세포,12 h시,석영자격적HELF-PKcs적석영유도적γH2AX수평증가수억제,혜미DNA백분함량위(43.09±3.68)%,여석영자격적HELF-NC상비,차이무통계학의의(P>0.05);24 h시,석영자격적HELF-PKcs적석영유도적γH2AX수평여석영자격적HELF-NC상비무명현주이,차이무통계학의의(P>0.05).석영자격적HELF-PKcs적혜미DNA백분함량(35.79±4.26)%]명현고우석영자격적HELF-NC,차이유통계학의의(P<0.05).결론 석영가유도DNA쌍련단렬손상,DNA-PKcs시석영유도적DNA쌍련단렬손상적감수기,통과린산화H2AX,촉진석영유도적DNA쌍련단렬손상적수복.
Objective To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibrobtasts (HELF).Methods Two stable transfectants,HELF transfectcd with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC),were established.HELF cells were treated with 0,25,50,100,200,300 anti 400 μg/ml silica for 12 h and with 200 μg/ml silica for different times (0,1,2,6,12 and 24 h ).HELF-PKcs and HELF-NC were treated with 200 μg/ml silica for 0,12 and 24 h.The expression levels of DNA-PKcs and phosphor-H2AX(H2AX) were determined by Western blot.DNA double strand breaks were measured by neutral comet assay.Results After treatment with difterent doses of silica for 12 h,the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner.After treatment with 200 μg/ml silica for different times,the levels of H2AX increased in a time-dependent manner.The percentages of tail DNA increased sig-nificantly at 6 h,and reaching maximum at 12 h and then decreasing at 24 h.The expression level of DNA-PKcs was suppressed in HELF-PKcs.After treatment with silica at 12 h,the level of H2AX was lower in HELF-PKcs than in HELF-NC,and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells,but no significant difference was found in the percentages of tail DNA between them.The percentages of tail DNA decreased markedly in silica-treated HEI,F-NC and was sig-nificantly lower than in HELF-PKcs at 24 h (P<0.05).Conclusion Silica can induce DNA double strand breaks in human embryo lung fibroblasts.DNA-PKcs might play a major role in silica-induced DNA double strand break repair.Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.