CAJ | 학술논문

目的观察曲妥珠单抗对乳腺癌SKBR3细胞中Her-2的入核过程和核内DNA损伤修复的影响,探讨曲妥珠单抗的放疗增敏机制.方法克隆形成实验检测曲妥珠单抗对辐射后细胞存活分数(SF)的影响,荧光共聚焦观察曲妥珠单抗对DNA双链断裂(DSB)损伤标志物γ H2AX表达和Her-2核转运过程的影响,免疫印迹法检测曲妥珠单抗对辐射诱导细胞的胞核内Her-2蛋白、DNA依赖蛋白激酶( DNA-PK)功能亚单位DNA-PKcs的表达最的改变.结果细胞克隆形成实验中,曲妥珠单抗干预组接受2 Gy时SF( SF2)为0.321±0.022,单纯照射组为0.547±0.046(P=0.000)；辐射后γ H2AX的荧光灰度表达提高,曲妥珠单抗干预组为85.40±25.63,单纯照射组为18.53±44.32( P=0.000)；Her-2蛋白入核灰度表达降低,单纯照射组为52.80±19.74,曲妥珠单抗组为21.41±10.55( P=0.000)；免疫印迹实验表明曲妥珠单抗干预组辐射后早期核内DNA-PKcs及Her-2的表达下降.结论曲妥珠单抗能够抑制辐射诱导的Her-2入核过程,并减少核内Her-2和DNA-PKcs的表达,从而可能提高辐射后早期的DSB损伤.
목적 관찰곡타주단항대유선암SKBR3세포중Her-2적입핵과정화핵내DNA손상수복적영향,탐토곡타주단항적방료증민궤제.방법 극륭형성실험검측곡타주단항대복사후세포존활분수(SF)적영향,형광공취초관찰곡타주단항대DNA쌍련단렬(DSB)손상표지물γ H2AX표체화Her-2핵전운과정적영향,면역인적법검측곡타주단항대복사유도세포적포핵내Her-2단백、DNA의뢰단백격매( DNA-PK)공능아단위DNA-PKcs적표체최적개변.결과 세포극륭형성실험중,곡타주단항간예조접수2 Gy시SF( SF2)위0.321±0.022,단순조사조위0.547±0.046(P=0.000)；복사후γ H2AX적형광회도표체제고,곡타주단항간예조위85.40±25.63,단순조사조위18.53±44.32( P=0.000)；Her-2단백입핵회도표체강저,단순조사조위52.80±19.74,곡타주단항조위21.41±10.55( P=0.000)；면역인적실험표명곡타주단항간예조복사후조기핵내DNA-PKcs급Her-2적표체하강.결론 곡타주단항능구억제복사유도적Her-2입핵과정,병감소핵내Her-2화DNA-PKcs적표체,종이가능제고복사후조기적DSB손상.
Objective To observe the influence of trastuzumab on DNA break repair and Her-2 nuclear import after radiation in breast cancer cell line SKBR3,and discuss the radiosensitivity mechanism of trastuzumab.Methods Clone formation assay was used to analyze the difference of survival fractions between radiation group and radiation plus trastuzumab group.Confocal microscopy was applied to observe the influence of trastuzumab in the nuclear import process of Her-2 and the expression of γH2AX after radiation,which is considered as the marker of DNA double strand break.Western blotting was used to detect the expression of Her-2 and DNA-PKcs in nuclei after radiation.Results The result of clone formation assayshowed that the SF2 in radiation group was 0.547±0.046 and 0.321±0.022 in the radiation plus trastuzumab group were significantly decreased,the results of confocal microscopy showed that trastuzumab postponed the nuclear import process of Her-2 (52.80±19.74 in radiation group,21.41±10.55 in the radiation group),and increased expression of γH2AX after radiation (85.40±25.63 in radiation group,18.53±44.32 in the radiation group),and western blotting revealed trastuzumab reduced the expression of Her-2,DNA-PKcs in nuclei.Conclusion Trastuzumab can inhibit the radiation induced nuclear import of Her-2,and decrease Her-2,DNA-PKcs in nuclei to increase the DSB on early stage after radiation.