背景与目的:DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)是一种双链断裂修复蛋白,可以修复细胞的DNA双链断裂损伤。本研究通过转染DNA-PKcs反义寡核苷酸入鼻咽癌细胞株,探讨DNA-PKcs反义寡核苷酸对不同p53功能状态的鼻咽...背景与目的:DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)是一种双链断裂修复蛋白,可以修复细胞的DNA双链断裂损伤。本研究通过转染DNA-PKcs反义寡核苷酸入鼻咽癌细胞株,探讨DNA-PKcs反义寡核苷酸对不同p53功能状态的鼻咽癌细胞株的放射敏感性的影响。方法:利用LipofectamineTM2000转染DNA-PKcs反义寡核苷酸入鼻咽癌细胞株CNE-1和CNE-1-wtp53;设0、0.5、1、2、4、6、8Gy7个放射剂量点,用集落形成法分析转染反义寡核苷酸前后细胞的存活情况,并分别用线性二次模型和单击多靶模型拟合出细胞的剂量存活曲线,求出放射生物学参数α、β、α/β、SF2、D0、Dq和N值,评价细胞放射敏感性的变化。结果:转染DNA-PKcs反义寡核苷酸前,CNE-1和CNE-1-wtp53的α值分别为0.03、0.05,SF2值分别为0.73、0.50,D0值分别为2.08Gy、1.13Gy,Dq值分别为2.04Gy、1.36Gy。转染DNA-PKcs反义寡核苷酸后,CNE-1和CNE-1-wtp53的α值分别为0.04、0.26,SF2值分别为0.45、0.21,D0值分别为1.07Gy、0.83Gy,Dq值分别为1.24Gy、0.73Gy。转染DNA-PKcs反义寡核苷酸后,鼻咽癌细胞的α值增大,SF2、D0、Dq值均减小。结论:DNA-PKcs反义寡核苷酸可逆转CNE-1的辐射抗性,该逆转作用不依赖细胞的p53功能状态。展开更多
目的:探讨荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测多发性骨髓瘤(multiple myeloma,MM)、意义未明单克隆免疫球蛋白增多症(monoclonal gammopathy of undetermined significance,MGUS)及反应性浆细胞增多症(reac...目的:探讨荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测多发性骨髓瘤(multiple myeloma,MM)、意义未明单克隆免疫球蛋白增多症(monoclonal gammopathy of undetermined significance,MGUS)及反应性浆细胞增多症(reactive plasmacytosis,RP)的几种常见的异常基因,提高对几种常见浆细胞疾病的诊断及鉴别诊断水平。方法:回顾性分析2012年8月至2015年8月在中南大学湘雅医院初诊的61例MM、20例MGUS及20例RP患者的临床表现、影像学、实验室检查及FISH检测结果。结果:FISH检测61例MM患者中50例患者出现基因异常,FISH阳性检出率为81.9%,其中1q21扩增19例(31.1%)、D13S319缺失18例(29.5%)、RB1缺失10例(16.4%)、IGH易位10例(16.4%)、p53缺失7例(11.4%),出现两种或多种基因异常的阳性率为12例(19.8%);20例MGUS中FISH阳性检出率为30%,其中1q21扩增4例(20%)、IGH易位2例(10%),未发现两种或多种基因异常;而RP患者仅1例出现D13S319缺失,阳性率仅为5%。三组两两比较差异具有统计学意义(P<0.05)。结论:MM患者中FISH的阳性检出率为81.9%,明显高于MGUS及RP的患者,应用FISH可检测MM中的多种异常基因,对MM,MGUS及RP的鉴别诊断、预后判断有重要的临床参考价值。展开更多
Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathway...Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathways by which the mammalian cells respond to DSBs repairing. Our previous study suggested that CNE1 is more radioresistant than CNE2. This study was designed to answer whether the radiosensitive difference of Nasopharyngeal Carcinoma cell lines CNE1/CNE2 was related to the expression and localization of Ku70/Ku80/DNA-PKcs. Methods： Immunohistochemistry was performed to detect the subcellular localization of Ku70/Ku80/DNA-PKcs in NPC cells lines CNE1 and CNE2. Western-blot was used to determine the expression of Ku protein in total extract of CNE1 and CNE2 and semi-quantitative assay of protein expression was performed to estimate the optic density （OD） value of each band using automatic image analysis system. Results： Ku70/Ku80/DNA-PKcs primarily located in the nuclei. A part of nucleolus in CNE1 and CNE2 showed positive dyeing of DNA-PKcs. Protein expression of Ku70/Ku80/DNA-PKcs was detected in CNE1 and CNE2, and the integral optical density （IOD） of Ku70 protein was 22.03 ± 7.56 and 19.98 ± 6.04 respectively （t=0.021, P〉0.05）, while the IODs of Ku80 protein in the two cell lines were 33.44 ± 12.87 and 28.98 ± 9.24 respectively （t=0.24, P〉0.05）, and the IODs of DNA-PKcs protein were 45.03 ± 1.77 and 40.87 ± 4.19 （t=1.58, P〉0.05）. The above results suggested that the basic expression of Ku70/Ku80/DNA-PKcs had no statistic difference between the different radiosensitive NPC cell lines CNE1 and CNE2. Conclusion： The variation of radiosensitivity in NPC cell lines CNE1 and CNE2 has no obviously correlation with the subcellular localization and basic expression of DNA-PK protein. So we presumed that the difference of radiosensitivity between CNE1 and CNE2 may be on account of some other factors than subcellular localization and basic expression of DNA-PK.展开更多
基金This work was supported by the National Natural Science Foundation of China(No.30070237)National High Technology Research and Development Program of China(No.2006AA02Z4B4).
文摘本文土要研究DNA依赖的蛋白激酶(DNA-dependent protein kinase,DNA-PK)与鼻咽癌细胞放射敏感性之间的关系。克隆形成实验分析鼻咽癌细胞CNE1/CNE2的剂量存活曲线,Signa TECT DNA-PK试剂盒检测DNA-PK活性,免疫荧光及激光显微共聚焦分析放疗前及放疗后15min、1h、6h、12h和24h CNE1/CNE2细胞中Kus及DNA-PKcs的亚细胞定位,Western blot分析两株细胞中Kus蛋白的表达。结果显示:CNE1细胞在每个剂量点的存活分数均高于CNE2细胞;同时发现放疗前后CNE1细胞中的DNA-PK活性也均高于CNE2细胞,但两株细胞中Ku70/Ku80蛋白表达无明显差异;放疗可使DNA- PK活性增加,且各个检测时间点CNE1细胞增加的幅度大于CNE2细胞;DNA-PK亚基可同时定位于胞浆和胞核,但主要位于胞核,细胞照射后Ku70、Ku80和DNA-PKcs从胞浆转运到胞核。结果表明:DNA-PK活性更高可能足CNE1细胞较CNE2细胞更能抵抗放射的原因之一;放疗所致DNA-PK活性增高可能与DNA-PK亚基从胞浆转运到胞核有关,而与Ku蛋白表达的总量无关。
文摘背景与目的:DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)是一种双链断裂修复蛋白,可以修复细胞的DNA双链断裂损伤。本研究通过转染DNA-PKcs反义寡核苷酸入鼻咽癌细胞株,探讨DNA-PKcs反义寡核苷酸对不同p53功能状态的鼻咽癌细胞株的放射敏感性的影响。方法:利用LipofectamineTM2000转染DNA-PKcs反义寡核苷酸入鼻咽癌细胞株CNE-1和CNE-1-wtp53;设0、0.5、1、2、4、6、8Gy7个放射剂量点,用集落形成法分析转染反义寡核苷酸前后细胞的存活情况,并分别用线性二次模型和单击多靶模型拟合出细胞的剂量存活曲线,求出放射生物学参数α、β、α/β、SF2、D0、Dq和N值,评价细胞放射敏感性的变化。结果:转染DNA-PKcs反义寡核苷酸前,CNE-1和CNE-1-wtp53的α值分别为0.03、0.05,SF2值分别为0.73、0.50,D0值分别为2.08Gy、1.13Gy,Dq值分别为2.04Gy、1.36Gy。转染DNA-PKcs反义寡核苷酸后,CNE-1和CNE-1-wtp53的α值分别为0.04、0.26,SF2值分别为0.45、0.21,D0值分别为1.07Gy、0.83Gy,Dq值分别为1.24Gy、0.73Gy。转染DNA-PKcs反义寡核苷酸后,鼻咽癌细胞的α值增大,SF2、D0、Dq值均减小。结论:DNA-PKcs反义寡核苷酸可逆转CNE-1的辐射抗性,该逆转作用不依赖细胞的p53功能状态。
文摘目的:探讨荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测多发性骨髓瘤(multiple myeloma,MM)、意义未明单克隆免疫球蛋白增多症(monoclonal gammopathy of undetermined significance,MGUS)及反应性浆细胞增多症(reactive plasmacytosis,RP)的几种常见的异常基因,提高对几种常见浆细胞疾病的诊断及鉴别诊断水平。方法:回顾性分析2012年8月至2015年8月在中南大学湘雅医院初诊的61例MM、20例MGUS及20例RP患者的临床表现、影像学、实验室检查及FISH检测结果。结果:FISH检测61例MM患者中50例患者出现基因异常,FISH阳性检出率为81.9%,其中1q21扩增19例(31.1%)、D13S319缺失18例(29.5%)、RB1缺失10例(16.4%)、IGH易位10例(16.4%)、p53缺失7例(11.4%),出现两种或多种基因异常的阳性率为12例(19.8%);20例MGUS中FISH阳性检出率为30%,其中1q21扩增4例(20%)、IGH易位2例(10%),未发现两种或多种基因异常;而RP患者仅1例出现D13S319缺失,阳性率仅为5%。三组两两比较差异具有统计学意义(P<0.05)。结论:MM患者中FISH的阳性检出率为81.9%,明显高于MGUS及RP的患者,应用FISH可检测MM中的多种异常基因,对MM,MGUS及RP的鉴别诊断、预后判断有重要的临床参考价值。
基金This work was supported by a grant fromthe National Natural Science Foundation of China (No.30070237).
文摘Objective： Radiosensitivity is mainly determined by the number of DNA double-strand breaks （DSBs） induced by ionizing radiation and the extent of its repair. The DNA-PK complex formation is one of the major pathways by which the mammalian cells respond to DSBs repairing. Our previous study suggested that CNE1 is more radioresistant than CNE2. This study was designed to answer whether the radiosensitive difference of Nasopharyngeal Carcinoma cell lines CNE1/CNE2 was related to the expression and localization of Ku70/Ku80/DNA-PKcs. Methods： Immunohistochemistry was performed to detect the subcellular localization of Ku70/Ku80/DNA-PKcs in NPC cells lines CNE1 and CNE2. Western-blot was used to determine the expression of Ku protein in total extract of CNE1 and CNE2 and semi-quantitative assay of protein expression was performed to estimate the optic density （OD） value of each band using automatic image analysis system. Results： Ku70/Ku80/DNA-PKcs primarily located in the nuclei. A part of nucleolus in CNE1 and CNE2 showed positive dyeing of DNA-PKcs. Protein expression of Ku70/Ku80/DNA-PKcs was detected in CNE1 and CNE2, and the integral optical density （IOD） of Ku70 protein was 22.03 ± 7.56 and 19.98 ± 6.04 respectively （t=0.021, P〉0.05）, while the IODs of Ku80 protein in the two cell lines were 33.44 ± 12.87 and 28.98 ± 9.24 respectively （t=0.24, P〉0.05）, and the IODs of DNA-PKcs protein were 45.03 ± 1.77 and 40.87 ± 4.19 （t=1.58, P〉0.05）. The above results suggested that the basic expression of Ku70/Ku80/DNA-PKcs had no statistic difference between the different radiosensitive NPC cell lines CNE1 and CNE2. Conclusion： The variation of radiosensitivity in NPC cell lines CNE1 and CNE2 has no obviously correlation with the subcellular localization and basic expression of DNA-PK protein. So we presumed that the difference of radiosensitivity between CNE1 and CNE2 may be on account of some other factors than subcellular localization and basic expression of DNA-PK.
