【作者】 王芹；

【导师】 樊飞跃；

【作者基本信息】 北京协和医学院 ， 放射医学， 2014， 博士

【摘要】 大肠癌,包括结肠癌和直肠癌,是威胁人类生命健康的常见消化道恶性肿瘤。放射治疗是大肠癌的主要治疗手段之一,但放射治疗大肠癌的辐射耐受现象严重影响大肠癌病人的疗效,放疗抵抗性成为大肠癌放疗面临严峻且迫切需要解决的难题。电离辐射后细胞DNA损伤的修复是肿瘤放疗效果不佳的主要原因之一。X射线修复交叉互补(X-ray repair cross complementing, XRCC)基因家族(XRCC1～XRCC11)对电离辐射诱导的DNA损伤修复发挥重要作用。DNA损伤通过碱基切除修复、核苷酸切除修复、错配修复、同源重组修复(homologous recombination,HR)和非同源末端连接(non-homologous end joining, NHEJ)等多种方式进行修复,从而维持生物体基因组的完整性和抑制肿瘤的发生。XRCC2是重要的参与HR途径的基因之一,其高表达与增加辐射诱导的DNA损伤抵抗有关。XRCC2基因修复缺陷表现出对电离辐射的敏感性增高,而XRCC2蛋白过表达则对放射线耐受。提示通过抑制肿瘤细胞XRCC2的表达,有可能提高临床肿瘤放射治疗的敏感性。目前尚未见到关于大肠癌中XRCC2表达水平以及XRCC2与放射敏感性关系的研究报道。降低XRCC2的表达是否可以改变大肠癌细胞的放射敏感性,XRCC2是否可以预测大肠癌放射治疗的疗效,目前在国内外未见相关的研究报道。目的：本实验通过大肠癌体外细胞模型和体内动物模型,探讨shRNA介导的XRCC2基因沉默是否影响大肠癌细胞的放疗敏感性及其疗效,阐明XRCC2在大肠癌放疗敏感性中的关键作用和初步相关机制。方法：(1)体外细胞实验：将shRNA-XRCC2转染人大肠癌T84细胞以沉默XRCC2基因表达,采用蛋白免疫印迹法和实时定量PCR法检测沉默XRCC2基因的效率；采用MTT法检测T84细胞的增殖。经X-射线照射后,采用克隆形成法检测T84细胞的放射敏感性；采用碱性“彗星”电泳法测定T84细胞的DNA损伤修复；流式细胞术检测T84细胞的细胞周期；Annexin V-FITC/PI双染法检测T84细胞的细胞凋亡率。(2)体内细胞实验：同时将shRNA-XRCC2转染的大肠癌T84细胞接种于BALB/c裸鼠建立移植瘤模型,进行X-射线照射,检测肿瘤的体积和重量变化,并对肿瘤组织进行病理分析。结果：(1)在体外细胞实验中,shRNA-XRCC2转染有效抑制了T84细胞中XRCC2蛋白和mRNA的表达。经嘌呤酶素筛选,得到了稳定的XRCC2基因沉默的大肠癌T84细胞系。细胞生长曲线表明,沉默XRCC2表达明显抑制了T84细胞的增殖。克隆形成实验显示,XRCC2基因沉默的T84细胞经X-射线照射后,克隆形成数目显著减少,表明XRCC2基因沉默提高了T84细胞的放射敏感性。彗星实验表明,沉默XRCC2表达的T84细胞DNA损伤增多,DNA损伤修复能力下降。流式细胞术检测显示,XRCC2基因沉默显著诱导了辐射导致的细胞凋亡和细胞阻滞在G2/M期。(2)在体内细胞实验中,转染shRNA-XRCC2的裸鼠种植瘤生长缓慢,肿瘤体积和重量明显减少。肿瘤病理组织学分析表明,转染shRNA-XRCC2的肿瘤组织核分裂相减少,多见大小不等的坏死区。说明沉默XRCC2表达提高了裸鼠大肠癌对辐射的敏感性,肿瘤生长受到明显的抑制作用。结论：shRNA介导的XRCC2基因沉默有效抑制了体外大肠癌细胞和体内裸鼠大肠癌肿瘤的生长,沉默XRCC2表达对体外和体内大肠癌细胞对X射线的反应具有一致性,即均提高了大肠癌对放射的敏感性。提示XRCC2有希望在大肠癌的临床放射治疗敏感性中作为一重要的靶向基因。更多还原

【Abstract】 Colorectal cancer, including colon cancer and rectal cancer, is one of the most common tumors of the digestive tract threatening the human life and health seriously. Although radiotherapy is one of methods of colorectal cancer treatment, the radioresistance in radiotherapy seriously affects the curative effect of colorectal cancer patients. DNA damage’s repair after exposure to ionizing radiation is one of reasons of tumors resistance to radiotherapy. X-ray repair cross complementing gene family (XRCC1-XRCC11) plays an important role in repairing DNA damages induced by ionizing radiation. DNA damages are repaired by various mechanisms such as base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining pathways to maintain the integrity of the genetic information and inhibit the formation of tumors. XRCC2is the key protein of DNA homologous recombination repair pathway, and its high expression is associated with enhanced resistance to DNA damage induced by ionizing radiation. XRCC2gene defects in some cell lines show an increased sensitivity to radiation, whereas the abnormal upregulation of XRCC2gene expression renders tumor cells resistance to radiation. Thus, we propose that the inhibiting of XRCC2expression in tumor cells may enhance their radiosensitivity. However, the studies on XRCC2expression in colorectal cancer and its association with sensitivity to radiation were not found. Until now, it is not yet known whether lowering XRCC2expression can affect the sensitivity of radiotherapy for colorectal cancer or XRCC2can predict the efficacy of colorectal cancer radiotherapy.Objective:The goal of the project is to study the effects of XRCC2gene silencing mediated by shRNA on radiosensitivity of colorectal cancer cells in vitro and in vivo and to elucidate the relationship between XRCC2role and the mechanism of colorectal cancer radiotherapy.Methods:The vector-based shRNA plasmid (shRNA-XRCC2) was transfected into colorectal cancer T84cell line to silence XRCC2gene expression. The efficiency of XRCC2silencing was determined by western blot and real-time PCR analyses. The growth curve of T84cells in vitro was examined by MTT assay. The effect of XRCC2suppression on T84cells’radiosensitivity to X-radiation was examined by colony formation assay. DNA damage’s repair of T84cells was determined by alkaline comet assay. The relationship between the sensitivity of T84cells to radiation and the cell cycle distribution or cell apoptosis was performed by flow cytometric analysis. Colorectal cancer T84cells transfected shRNA-XRCC2were transfered into BALB/c nude mice to establish a xenograft model in vivo.The curative effect and pathological analysis of xenografts were investigated after xenograft received radiotherapy.Results:(1) In vitro:XRCC2protein and mRNA expression of colorectal cancer T84cells was effectively silenced by shRNA-XRCC2transfection. Colorectal cancer T84cell line silencing XRCC2gene expression stablely was achieved successfully through selecting with purine enzyme. Knockdown of XRCC2expression by shRNA inhibited cell growth of T84cells as evaluated by MTT assay. The number of colonies formed in shRNA-XRCC2cells was significantly decreased, which showed that shRNA-mediated XRCC2suppression rendered T84tumor cells more sensitive to radiation treatment. DNA damages were increased and the capability of DNA damage’s repair was decreased in T84cells as examined by comet assay. Suppression of XRCC2expression resulted in an elevation of cell apoptosis and cell cycle arrested in G2/M phase induced by radiation through flow cytometric analysis.(2)In vivo:Tumor xenograft transfected with shRNA-XRCC2in nude mice grew slowly and the tumor volume and the tumor weight were decreased significantly. Pathological analysis showed that karyokinesis was decreased and small areas of necrosis were found in tumor xenograft treated by shRNA-XRCC2transfection. The data suggested that knockdown of XRCC2expression enhanced the tumor’s sensitivity to radiation in nude mice and tumor xenograft’s growth was retarded.Conclusion:XRCC2gene silencing mediated by shRNA inhibited the growth of colorectal tumor cells in vitro and in vivo and has radiosensitization effects on colorectal tumor cells in vitro and in vivo, ie. increased tumor’s sensitivity to radiation. These data strongly suggested that XRCC2may be further developed as a promising therapeutic target for the treatment of radioresistant human colorectal cancer.更多还原