DNA-PKcs基因敲除及其抑制剂影响DNA双链断裂非同源重组末端连接修复的研究

DNA double strand breakage (DSB) is the most serious DNA damage within cells caused by ionizing radiation, leading to cellular apoptosis, cell cycle arrest and DSB repair, which is also the foundation of radiation biology. We have applied 2 specific cell lines with knockdown of the two important component of heterodimer Ku70 and/or Ku80 which is involved in DSB repair pathway, non homologous end joining (NHEJ) (Ku70-/-, Ku80-/-) through genetic engineering technology. Our researches have demonstrated that Ku70-/- and/or Ku80-/- cells are more sensitive to ionizing radiation that their wild type cells. Cells that are infected with adenovirus containing Ku70 antisense RNA activated by heat shock, or dominant negative Ku70 which was changed the NH2 terminal structure of Ku70 through gene therapy have been shown pronounced radiosensitivity through suppression of DSB repair pathway under normoxic or even hypoxic circumstance. .In this project we will (1) establish knockdown cell line which will be inactivated with DNA dependent protein kinase activity (DNA PKcs-/-) through gene engineering technique; (2) elucidate the DSB NHEJ repair mechanisms caused by ionizing radiation induced DSB, its related signaling pathways, and factors involved in NHEJ pathway；（3）explore the difference in DSB and NHEJ repair pathway caused by ionizing radiation under normoxic and hypoxic circumstances; (4)corroborate the difference in cellular apoptosis, cell cycle arrest and radiosensitivity with application of DNA PKcs inhibitor among MEF wild type cells, DNA PKcs-/- cells, and Ku70-/- cells. By doing this we are expecting to explore specific repair factor(s) as potential target therapy involved in the DSB repair especially on NHEJ pathway, to achieve the purpose of individualized cancer treatment, and break through the obstacle in radiation oncology.

DNA双链断裂（DSB）是细胞受到电离辐射（IR）最严重的DNA损伤，导致细胞凋亡、细胞周期阻滞和DSB修复。我们利用Ku70反义RNA或显性负性效应(DNKu70)进行基因治疗，比较与敲除DSB修复之非同源重组末端连接（NHEJ）途径Ku基因（Ku70-/-）细胞的放射敏感性，研究显示其放射敏感性与Ku70-/-细胞相似，可抑制肿瘤细胞DSB NHEJ修复能力，增强富氧和乏氧下的细胞杀伤。拟通过基因工程技术敲除DNA依赖蛋白激酶（DNA-PKcs）基因，建立DNA-PKcs-/-细胞,阐明IR致DSB NHEJ修复机制、相关信号通路以及影响DSB NHEJ修复的因素；探讨乏氧处理以及应用DNA-PKcs抑制剂对IR致DSB、NHEJ修复和放射敏感性之间的差异，细胞凋亡、细胞周期阻滞和相关基因表达改变，探索以DSB 修复通路上特定修复因子为潜在靶点，增强肿瘤放疗敏感性，实现肿瘤个体化治疗。

DNA双链断裂（DSB）是细胞受到电离辐射（IR）最严重的DNA损伤，导致细胞凋亡、细胞周期阻滞和DSB修复。DSB主要修复方式包括非同源末端连接（NHEJ）修复和同源重组修复（HR）。DNA-PKcs是NHEJ修复通路中的重要因子。.利用构建载体将PGK-neo基因通过同源取代方法取代DNA-PKcs基因上外显子3的3’末端片段和部分内含子3片段，转染至CJ7 ES细胞，将其植入雌性C57Bl/6小鼠孕育子代小鼠，通过PCR技术鉴定并获得DNA-PKcs-/-小鼠。从13.5天的野生型和DNA-PKcs-/-小鼠胚胎细胞中分离成纤维细胞（MEF），建立稳定的MEF/野生型和MEF/DNA-PKcs-/-细胞株。与MEF/野生型细胞相比，MEF/DNA-PKcs-/-细胞株对IR更加敏感，其放射敏感性显著增加的机制包括，DSB修复延迟、细胞周期阻滞、细胞周期检查点激活并在DSB损伤区域聚集等。应用NHEJ修复通路抑制剂，能够显著增加MEF/野生型细胞的放射敏感性，其机制是受细胞周期检查点调节。.通过比较DNA依赖蛋白激酶催化亚基（DNA-PKcs+/+）和DNA-PKcs-/- 小鼠胚胎成纤维细胞（MEF）中电离辐射（IR）诱导γH2AX焦点形成数量的差异；对鼻咽癌SUNE-1细胞进行IR致DNA双链断裂（DSB）的应用研究。研究显示利用细胞免疫荧光动态检测放疗后致γH2AX焦点形成，为动态定量研究DSB损伤及修复提供了新的思路。.肿瘤乏氧微环境减弱IR诱导DSB损伤，是肿瘤对放化疗抗拒和患者预后不良的重要因素。鼻咽癌在我国南方地区高发，临床观察鼻咽癌乏氧现象极为常见。目前对应用放疗联合DSB修复抑制剂改善肿瘤微环境、抑制乏氧状态下DSB损伤修复并促进肿瘤细胞凋亡的研究很少。利用可经乏氧调控生物标志融合基因（HSV1-TK/GFP）表达的人鼻咽癌乏氧研究模型，探讨NHEJ修复抑制剂研究在常氧/乏氧增加鼻咽癌SUNE1/HSV1-TK/GFP细胞放射敏感性的机制。体内外研究显示，IR联合NHEJ修复抑制剂能够显著延迟SUNE1/HSV1-TK/GFP移植瘤生长，影响肿瘤乏氧区域分布并促进肿瘤细胞凋亡。.本项目的完成有望为今后开展鼻咽癌个体化治疗，即乏氧影像引导下的精准放疗和靶向治疗提供科学依据。