APE1/Ref-1在肿瘤放射治疗策略中的实验研究
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摘要
放射治疗随着放射肿瘤学的不断发展,在肿瘤治疗中的地位愈显重要,已成为当今治疗肿瘤最常用的方法之一。放射治疗可以治愈部分肿瘤,但对一些肿瘤,尤其是一些中晚期肿瘤的疗效并不理想,其并发症放射性损伤的发生率也较高。为了解决肿瘤细胞的放射治疗抵抗问题,进一步提高放射治疗的治愈率,临床医生和科学家们通过多项研究和努力,不断的探索出提高放射治疗疗效和降低放射性损伤的新方法。在众多研究中,放射治疗联合肿瘤基因治疗的研究有着极其重要的地位和作用。
脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1(apurinic/aprimidinic endonuclease/ redox factor-1,APE1/Ref-1)是一个双功能生物大分子,在DNA损伤修复和氧化还原作用或氧化还原信号传递过程中起着至关重要的作用。APE1/Ref-1蛋白的不正常表达、分布及功能改变不仅与细胞凋亡、肿瘤发生密切相关,而且修饰APE1/Ref-1活性的早期就可以改变细胞和机体对DNA损伤剂的反应。野生型p53基因是细胞凋亡的关键调控基因,p53基因突变与肿瘤的发生密切相关。基于对上述现状的分析研究,我们提出APE1/Ref-1不仅是肿瘤治疗的靶点而且也是防治放射性损伤的靶点之一。为进一步验证这一设想,本研究分别在体内应用重组腺病毒载体Ad5/F35- APE1/Ref-1siRNA联合人重组腺病毒rAd-p53感染肝癌细胞,通过观察体内肝癌细胞生长的抑制效果,探讨二者联合增强肝癌细胞对放射治疗敏感性的作用机制;同时,利用免疫组化和Western blot方法检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白的表达;在体内应用质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1经尾静脉注射转染小鼠肺组织,观察过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护效果,以此探讨APE1/Ref-1对放射性损伤的保护作用;为多基因治疗联合放射治疗肿瘤和防治放射性损伤提供新的实验依据。
研究目的
1.探讨双功能基因APE1/Ref-1增强肝癌细胞对放射治疗敏感性的作用及其与肿瘤抑制基因p53联合的协同作用;
2.研究双功能基因APE1/Ref-1在小鼠放射性肺损伤过程中肺组织中的表达及其与放射性损伤之间的演进关系;
3.分析小鼠肺组织过表达APE1/Ref-1蛋白后,APE1/Ref-1对放射性损伤的保护作用及其可能机制。
研究内容和方法
1.重组腺病毒载体Ad5/F35-APE1/Ref-1siRNA联合人重组腺病毒rAd-p53增强肝癌细胞对放射治疗敏感性的体内实验研究:
建立裸鼠人肝癌动物模型,观察Ad5/F35-APE1/Ref-1siRNA联合rAd-p53增强肝癌细胞对放射治疗敏感性的作用,测量肿瘤的生长,绘制肿瘤生长曲线,HE染色,光镜观察肿瘤的组织学变化,免疫组化和TUNEL法检测肿瘤细胞增殖和凋亡。
2.APE1/Ref-1在小鼠放射性肺损伤中的表达特点及其意义:
建立放射性肺损伤小鼠动物模型,采用HE染色,光镜观察小鼠放射性肺损伤的组织学变化,免疫组化和Western blot检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白表达变化特点,分析APE1/Ref-1表达变化特点与放射性肺损伤的关系,为进一步以APE1/Ref-1为靶点的基因防治放射性损伤提供实验依据。
3.APE1/Ref-1过表达对放射性肺损伤的保护作用的实验研究:
建立放射性肺损伤小鼠动物模型,观察质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1经尾静脉注射转染小鼠肺组织后,过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护效果。采用HE和Masson三联染色,光镜观察小鼠放射性肺损伤的组织学变化,免疫组化和Western blot检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白表达变化,酶联免疫吸附分析法检测血清中TGF-β1水平,分析过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护作用。
主要研究结果如下:
1.重组腺病毒载体Ad5/F35-APE1/Ref-1siRNA联合人重组腺病毒rAd-p53增强肝癌细胞对放射治疗敏感性的体内实验研究:
Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组治疗肿瘤后肿瘤的生长抑制率为93.95%,显著高于对照组和其他治疗组(p均<0.01)。与对照组肿瘤细胞相比,两种腺病毒分别有效抑制了APE1/Ref-1和提高了p53蛋白的表达水平。HE染色观察肿瘤组织学变化显示,各治疗组肿瘤内都出现程度不同的灶性坏死,其中Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组肿瘤坏死较多,而对照组未见坏死病灶。免疫组化法检测细胞增殖情况显示,Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组增殖率为(14.70±3.20)%,显著低于对照组和其他治疗组(p均<0.05)。TUNEL法检测细胞凋亡情况显示,Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组凋亡率为(28.74±1.94)%,显著高于对照组和其他治疗组(p均<0.01)。
2.APE1/Ref-1在小鼠放射性肺损伤中的表达特点及其意义:
正常小鼠肺组织上皮细胞和内皮细胞中APE1/Ref-1呈胞核表达为主,而在小鼠放射性肺损伤不同阶段肺组织上皮细胞和内皮细胞中APE1/Ref-1表达特征有所改变,呈核浆共同表达和胞浆表达为主;并且APE1/Ref-1表达呈现先增高后降低的趋势,照射后1d开始升高,3d达高峰,约是对照组2.2倍,此后随着小鼠放射性肺损伤发展逐渐降低,28d降至对照组水平,56d明显低于对照组,约是对照组0.2倍。
3.APE1/Ref-1过表达对放射性肺损伤的保护作用的实验研究:
质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1均能经尾静脉注射转染小鼠肺组织上皮细胞和内皮细胞,使其过表达APE1/Ref-1,转染后三天,小鼠肺组织上皮细胞和内皮细胞APE1/Ref-1蛋白明显增加,分别是对照组2.3倍和2.8倍。小鼠全胸单次受到致放射性肺损伤20Gy剂量的X线照射后,过表达APE1/Ref-1的小鼠肺组织病理学形态和Masson染色显示其放射性肺损伤在各个检测时间点均较单纯照射的小鼠有所改善:单纯照射组的小鼠肺组织在照射后1、3、7、14d病变以渗出为主,肺间质及肺毛细血管充血、水肿,肺泡腔及细支气管内见较多的淋巴细胞浸润,间质水肿致肺泡壁轻度增厚,部分肺泡间含水肿液;28、56d病变以慢性炎症为主,支气管和血管周围可见以淋巴细胞为主的炎症细胞浸润,肺泡壁增厚肺泡腔变小,肺泡间隔及部分肺泡腔内可见纤维素样的渗出物;纤维组织在照射后14d开始增生,28、56d肺纤维化形成,肺结构紊乱,并可见大量染成绿色的胶原纤维。而过表达APE1/Ref-1小鼠的肺组织照射后病变以渗出为主,在病变的程度、纤维组织的增生、肺纤维化的形成以及肺结构紊乱均轻于单纯照射组小鼠。血清转化生长因子TGF-β1在小鼠照射后7d含量明显增高,此后逐渐增高,56d达高峰;过表达APE1/Ref-1小鼠血清TGF-β1含量也逐渐增加,但与单纯照射组小鼠相比血清含量显著降低(p<0.05)。
结论
1. Ad5/F35-APE1/Ref-1siRNA联合rAd-p53能增强肝癌细胞对放射治疗的敏感性,抑制肿瘤生长作用显著增强,以APE1/Ref-1和p53为靶点的多基因治疗联合放射治疗可能成为今后肝癌治疗的新方法。
2.电离辐射可以刺激小鼠肺组织上皮细胞和内皮细胞APE1/Ref-1蛋白的表达并使其表达发生由核内转向浆内和先增高后降低的特征改变,提示APE1/Ref-1可能在放射性肺损伤修复过程中起着重要作用。
3.质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1均能经尾静脉注射转染小鼠肺组织上皮细胞和内皮细胞,使其过表达APE1/Ref-1。
4. APE1/Ref-1过表达对小鼠放射性肺损伤有一定程度的保护作用,其机制可能是通过APE1/Ref-1在细胞DNA损伤修复和氧化还原调节中的特殊作用修复受损的细胞DNA,抑制活性氧产生及其活性,降低细胞因子TGF-β1的表达,减少胶原蛋白的形成,使放射性损伤病变减轻。
With the development of radiation oncology, the radiotherapy plays an important role in the tumor treatment, and has become one of the most commonly approaches. Some tumors can be cured by the radiotherapy, but the efficacy of radiotherapy on some other advanced tumors is not satisfied, meanwhile, the incidence of radiation-induced injuries is high. In order to solve the problem of radioresistance of tumor cells, improve the cure rate of radiotherapy and control the radiation-induced injury, the clinical doctors and scientists have sought out a lot of new methods. In all these research, the radiotherapy combined with gene therapy against tumor, also known as tumor gene radiotherapy, were considered as a promising stategy for future tumor treatment.
The apurinic/aprimidinic endonuclease/redox factor-1(APE1/Ref-1) is a bifunctional biomacromolecule. APE1/Ref-1 plays a vital role in DNA damage repair and oxidation reduction or redox signal transmission process. The abnormal expression, distribution and functional changes of APE1/Ref-1 are not only associated with anti-apoptosis and tumor progression, moreover, the experimental modification of its activity could change the response of cells and organism to DNA damage agents. Wild-type p53 plays a key role in cell cycle control and apoptosis, and the mutation of p53 gene is correlated with the carcinogenesis and progression of tumor. Based on these results and conclusion, we proposed that APE1/Ref-1 might be a molecular target in tumor therapy and prevention of radiation-induced injuries. In order to prove the assumption, we examined the inhibitory effect of chimeric adenoviral vector Ad5/F35 carrying human APE1/Ref-1siRNA and recombinant adenovirus carrying wild-type p53 combined with radiotherapy on human hepatocellular carcinoma cell proliferation in vivo. We also explored the action mechanism of Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells. Then we detected the expression of APE1/Ref-1 in radiation-induced lung injury by immunohistochemistry and western blot. Mice lung tissues were transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 via the tail vein by injection to overexpress APE1/Ref-1, and we investigated the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model. The study will present a new strategy for the combination of radiotherapy and gene therapy against tumor and the control of radiation-induced injury.
Objective
1. To investigate the effects of Ad5/F35-APE1/Ref-1siRNA on radiosensitivity of human hepatocellular carcinoma and its synergistic effect with rAd-p53 in nude mice model.
2. To investigate the expression of APE1/Ref-1 in mice model of radiation-induced lung injury and explore its correlation with radiation-induced lung injury.
3. To observe the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via tail vein and investigate the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model.
Materials and Methods
1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model: Nude mice hepatocarcinoma xenograft model was established with SMMC-7721 human hepatocellular carcinoma cell line. When the tumor volumes reached 40~50mm3 on the seventh to tenth day after inoculation of SMMC-7721 cells, 28 mice were randomly divided into seven groups: control group, the radiotherapy group, Ad5/F35- APE1/Ref-1siRNA group, Ad5/F35-APE1/Ref-1siRNA plus radiotherapy group, rAd-p53 group, rAd-p53 plus radiotherapy group and Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group. The tumor volumes were measured and the tumor growth curves were drawn. The histology of specimens was examined by HE stain. The expression of Ki-67 protein was detected by immunohistochemistry. Apoptosis index was detected by TUNEL technique.
2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injury
The mice model of radiation-induced lung injury was established. The histology of specimens was examined by HE stain. The expression of APE1/Ref-1 in the lung tissues of mice of radiation lung injury was detected by immunohistochemistry and western blot methods. We also explored the correlation of APE1/Ref-1 with the radiation-induced lung injury. The study will present a new strategy for the control of radiation-induced injury by gene therapy.
3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:
The mice omni-thorax irradiation pulmonary model was established. We observed the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via the tail vein and investigated the protective effect of APE1/Ref-1 over-expression in mice model of radiation-induced lung injury. The histology and collagen of lungs was examined by HE stain and Masson-trichrome separately, the expression of APE1/Ref-1 protein was detected by immunohistochemistry and western blot. Enzyme linked-immuno-sorbent assay (ELISA) was applied to detect the TGF-β1 in serum.
Results
1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model:
Ad5/F35-APE1/Ref-1siRNA suppressed significantly the expression of APE1/Ref-1 protein, and rAd-p53 enhanced significantly the expression of p53 protein in vivo. The tumor growth inhibition ratio, PI and AI in Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group were significantly different, compared with the control group and other therapy groups (p<0.01).
2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injury
The high expression of APE1/Ref-1 was detected in nuclei of epithelial cells and endothelial cells in normal mice lung tissues. During the process of radiation-induced lung injury in mice model, the changes of three types of APE1/Ref-1 positive staining were observed; the expression of APE1/Ref-1 protein was detected in nuclear, cytoplasmic and mixed type. The expression of APE1/Ref-1 protein began to increase 1d after irradiation, reached its peak value on 3d, it was 2.2-fold compared with control group, and then decreased at 7d~56d. The expression of APE1/Ref-1 protein was significantly increased after 3d post-irradiation, and then was significantly decreased at 56d, and was 0.2-fold compared with control group.
3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:
After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased. It was 2.3-fold and 2.8-fold compared with lung tissue epithelial cells and endothelial cells of untransfected mice, separately. After single irradiation at omni-thorax of the mice with 20 Gy of 8 MV X irradiation, the pathological study showed marked lung injury in the radiation group while only slight hyperemia, hemorrhage, exudation and the forming of collagen in prevention and treatment groups by HE stain and Masson-trichrome. The levels of TGF-β1 in the serum increased 7d after irradiation, and then increased at 14d~56d, it reached its peak value at 56d, there was significant difference comparaed with the control group(P<0.01), and also reduced in two prevention and treatment groups and the radiation group(p<0.05). However, there was no significant difference between two prevention and treatment groups (p>0.05).
Conclusion
1. Ad5/F35-APE1/Ref-1siRNA and rAd-p53 combined with radiotherapy can significantly inhibit the growth and enhance radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model. Therefore, the gene therapy of Ad5/F35-APE1siRNA combined with rAd-p53 may be a promising approach to treat human hepatocellular carcinoma in the future.
2. Irradiation can up-regulate the expression of APE1/Ref-1 protein, while APE1/Ref-1 has the characteristic translocation from nucleus to cytoplasma. The expression of APE1/Ref-1 protein was increased at first and then decreased, which suggests that the APE1/Ref-1 may play a very important role in the development of radiation-induced lung injury.
3. After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased.
4. APE1/Ref-1 could show protective effects on the radiation-induced lung injury and the mechanism may be through repairing DNA lesions, blocking the production of reactive oxygen species and inhibiting the activity of redox regulation, decreasing the expression of TGF-β1, reducing the forming of collagen so as to ameliorate the radiation-induced lung injury.
脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1(apurinic/aprimidinic endonuclease/ redox factor-1,APE1/Ref-1)是一个双功能生物大分子,在DNA损伤修复和氧化还原作用或氧化还原信号传递过程中起着至关重要的作用。APE1/Ref-1蛋白的不正常表达、分布及功能改变不仅与细胞凋亡、肿瘤发生密切相关,而且修饰APE1/Ref-1活性的早期就可以改变细胞和机体对DNA损伤剂的反应。野生型p53基因是细胞凋亡的关键调控基因,p53基因突变与肿瘤的发生密切相关。基于对上述现状的分析研究,我们提出APE1/Ref-1不仅是肿瘤治疗的靶点而且也是防治放射性损伤的靶点之一。为进一步验证这一设想,本研究分别在体内应用重组腺病毒载体Ad5/F35- APE1/Ref-1siRNA联合人重组腺病毒rAd-p53感染肝癌细胞,通过观察体内肝癌细胞生长的抑制效果,探讨二者联合增强肝癌细胞对放射治疗敏感性的作用机制;同时,利用免疫组化和Western blot方法检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白的表达;在体内应用质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1经尾静脉注射转染小鼠肺组织,观察过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护效果,以此探讨APE1/Ref-1对放射性损伤的保护作用;为多基因治疗联合放射治疗肿瘤和防治放射性损伤提供新的实验依据。
研究目的
1.探讨双功能基因APE1/Ref-1增强肝癌细胞对放射治疗敏感性的作用及其与肿瘤抑制基因p53联合的协同作用;
2.研究双功能基因APE1/Ref-1在小鼠放射性肺损伤过程中肺组织中的表达及其与放射性损伤之间的演进关系;
3.分析小鼠肺组织过表达APE1/Ref-1蛋白后,APE1/Ref-1对放射性损伤的保护作用及其可能机制。
研究内容和方法
1.重组腺病毒载体Ad5/F35-APE1/Ref-1siRNA联合人重组腺病毒rAd-p53增强肝癌细胞对放射治疗敏感性的体内实验研究:
建立裸鼠人肝癌动物模型,观察Ad5/F35-APE1/Ref-1siRNA联合rAd-p53增强肝癌细胞对放射治疗敏感性的作用,测量肿瘤的生长,绘制肿瘤生长曲线,HE染色,光镜观察肿瘤的组织学变化,免疫组化和TUNEL法检测肿瘤细胞增殖和凋亡。
2.APE1/Ref-1在小鼠放射性肺损伤中的表达特点及其意义:
建立放射性肺损伤小鼠动物模型,采用HE染色,光镜观察小鼠放射性肺损伤的组织学变化,免疫组化和Western blot检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白表达变化特点,分析APE1/Ref-1表达变化特点与放射性肺损伤的关系,为进一步以APE1/Ref-1为靶点的基因防治放射性损伤提供实验依据。
3.APE1/Ref-1过表达对放射性肺损伤的保护作用的实验研究:
建立放射性肺损伤小鼠动物模型,观察质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1经尾静脉注射转染小鼠肺组织后,过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护效果。采用HE和Masson三联染色,光镜观察小鼠放射性肺损伤的组织学变化,免疫组化和Western blot检测小鼠放射性肺损伤过程中APE1/Ref-1蛋白表达变化,酶联免疫吸附分析法检测血清中TGF-β1水平,分析过表达APE1/Ref-1蛋白对小鼠放射性肺损伤的保护作用。
主要研究结果如下:
1.重组腺病毒载体Ad5/F35-APE1/Ref-1siRNA联合人重组腺病毒rAd-p53增强肝癌细胞对放射治疗敏感性的体内实验研究:
Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组治疗肿瘤后肿瘤的生长抑制率为93.95%,显著高于对照组和其他治疗组(p均<0.01)。与对照组肿瘤细胞相比,两种腺病毒分别有效抑制了APE1/Ref-1和提高了p53蛋白的表达水平。HE染色观察肿瘤组织学变化显示,各治疗组肿瘤内都出现程度不同的灶性坏死,其中Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组肿瘤坏死较多,而对照组未见坏死病灶。免疫组化法检测细胞增殖情况显示,Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组增殖率为(14.70±3.20)%,显著低于对照组和其他治疗组(p均<0.05)。TUNEL法检测细胞凋亡情况显示,Ad5/F35-APE1/Ref-1siRNA+rAd-p53+IR联合治疗组凋亡率为(28.74±1.94)%,显著高于对照组和其他治疗组(p均<0.01)。
2.APE1/Ref-1在小鼠放射性肺损伤中的表达特点及其意义:
正常小鼠肺组织上皮细胞和内皮细胞中APE1/Ref-1呈胞核表达为主,而在小鼠放射性肺损伤不同阶段肺组织上皮细胞和内皮细胞中APE1/Ref-1表达特征有所改变,呈核浆共同表达和胞浆表达为主;并且APE1/Ref-1表达呈现先增高后降低的趋势,照射后1d开始升高,3d达高峰,约是对照组2.2倍,此后随着小鼠放射性肺损伤发展逐渐降低,28d降至对照组水平,56d明显低于对照组,约是对照组0.2倍。
3.APE1/Ref-1过表达对放射性肺损伤的保护作用的实验研究:
质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1均能经尾静脉注射转染小鼠肺组织上皮细胞和内皮细胞,使其过表达APE1/Ref-1,转染后三天,小鼠肺组织上皮细胞和内皮细胞APE1/Ref-1蛋白明显增加,分别是对照组2.3倍和2.8倍。小鼠全胸单次受到致放射性肺损伤20Gy剂量的X线照射后,过表达APE1/Ref-1的小鼠肺组织病理学形态和Masson染色显示其放射性肺损伤在各个检测时间点均较单纯照射的小鼠有所改善:单纯照射组的小鼠肺组织在照射后1、3、7、14d病变以渗出为主,肺间质及肺毛细血管充血、水肿,肺泡腔及细支气管内见较多的淋巴细胞浸润,间质水肿致肺泡壁轻度增厚,部分肺泡间含水肿液;28、56d病变以慢性炎症为主,支气管和血管周围可见以淋巴细胞为主的炎症细胞浸润,肺泡壁增厚肺泡腔变小,肺泡间隔及部分肺泡腔内可见纤维素样的渗出物;纤维组织在照射后14d开始增生,28、56d肺纤维化形成,肺结构紊乱,并可见大量染成绿色的胶原纤维。而过表达APE1/Ref-1小鼠的肺组织照射后病变以渗出为主,在病变的程度、纤维组织的增生、肺纤维化的形成以及肺结构紊乱均轻于单纯照射组小鼠。血清转化生长因子TGF-β1在小鼠照射后7d含量明显增高,此后逐渐增高,56d达高峰;过表达APE1/Ref-1小鼠血清TGF-β1含量也逐渐增加,但与单纯照射组小鼠相比血清含量显著降低(p<0.05)。
结论
1. Ad5/F35-APE1/Ref-1siRNA联合rAd-p53能增强肝癌细胞对放射治疗的敏感性,抑制肿瘤生长作用显著增强,以APE1/Ref-1和p53为靶点的多基因治疗联合放射治疗可能成为今后肝癌治疗的新方法。
2.电离辐射可以刺激小鼠肺组织上皮细胞和内皮细胞APE1/Ref-1蛋白的表达并使其表达发生由核内转向浆内和先增高后降低的特征改变,提示APE1/Ref-1可能在放射性肺损伤修复过程中起着重要作用。
3.质粒pcDNA3.1/flAPE1/Ref-1和pcDNA3.1/mtAPE1/Ref-1均能经尾静脉注射转染小鼠肺组织上皮细胞和内皮细胞,使其过表达APE1/Ref-1。
4. APE1/Ref-1过表达对小鼠放射性肺损伤有一定程度的保护作用,其机制可能是通过APE1/Ref-1在细胞DNA损伤修复和氧化还原调节中的特殊作用修复受损的细胞DNA,抑制活性氧产生及其活性,降低细胞因子TGF-β1的表达,减少胶原蛋白的形成,使放射性损伤病变减轻。
With the development of radiation oncology, the radiotherapy plays an important role in the tumor treatment, and has become one of the most commonly approaches. Some tumors can be cured by the radiotherapy, but the efficacy of radiotherapy on some other advanced tumors is not satisfied, meanwhile, the incidence of radiation-induced injuries is high. In order to solve the problem of radioresistance of tumor cells, improve the cure rate of radiotherapy and control the radiation-induced injury, the clinical doctors and scientists have sought out a lot of new methods. In all these research, the radiotherapy combined with gene therapy against tumor, also known as tumor gene radiotherapy, were considered as a promising stategy for future tumor treatment.
The apurinic/aprimidinic endonuclease/redox factor-1(APE1/Ref-1) is a bifunctional biomacromolecule. APE1/Ref-1 plays a vital role in DNA damage repair and oxidation reduction or redox signal transmission process. The abnormal expression, distribution and functional changes of APE1/Ref-1 are not only associated with anti-apoptosis and tumor progression, moreover, the experimental modification of its activity could change the response of cells and organism to DNA damage agents. Wild-type p53 plays a key role in cell cycle control and apoptosis, and the mutation of p53 gene is correlated with the carcinogenesis and progression of tumor. Based on these results and conclusion, we proposed that APE1/Ref-1 might be a molecular target in tumor therapy and prevention of radiation-induced injuries. In order to prove the assumption, we examined the inhibitory effect of chimeric adenoviral vector Ad5/F35 carrying human APE1/Ref-1siRNA and recombinant adenovirus carrying wild-type p53 combined with radiotherapy on human hepatocellular carcinoma cell proliferation in vivo. We also explored the action mechanism of Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells. Then we detected the expression of APE1/Ref-1 in radiation-induced lung injury by immunohistochemistry and western blot. Mice lung tissues were transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 via the tail vein by injection to overexpress APE1/Ref-1, and we investigated the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model. The study will present a new strategy for the combination of radiotherapy and gene therapy against tumor and the control of radiation-induced injury.
Objective
1. To investigate the effects of Ad5/F35-APE1/Ref-1siRNA on radiosensitivity of human hepatocellular carcinoma and its synergistic effect with rAd-p53 in nude mice model.
2. To investigate the expression of APE1/Ref-1 in mice model of radiation-induced lung injury and explore its correlation with radiation-induced lung injury.
3. To observe the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via tail vein and investigate the protective effect of APE1/Ref-1 on radiation-induced lung injury in mice model.
Materials and Methods
1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model: Nude mice hepatocarcinoma xenograft model was established with SMMC-7721 human hepatocellular carcinoma cell line. When the tumor volumes reached 40~50mm3 on the seventh to tenth day after inoculation of SMMC-7721 cells, 28 mice were randomly divided into seven groups: control group, the radiotherapy group, Ad5/F35- APE1/Ref-1siRNA group, Ad5/F35-APE1/Ref-1siRNA plus radiotherapy group, rAd-p53 group, rAd-p53 plus radiotherapy group and Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group. The tumor volumes were measured and the tumor growth curves were drawn. The histology of specimens was examined by HE stain. The expression of Ki-67 protein was detected by immunohistochemistry. Apoptosis index was detected by TUNEL technique.
2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injury
The mice model of radiation-induced lung injury was established. The histology of specimens was examined by HE stain. The expression of APE1/Ref-1 in the lung tissues of mice of radiation lung injury was detected by immunohistochemistry and western blot methods. We also explored the correlation of APE1/Ref-1 with the radiation-induced lung injury. The study will present a new strategy for the control of radiation-induced injury by gene therapy.
3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:
The mice omni-thorax irradiation pulmonary model was established. We observed the lung tissues of mice transferred by the plasmid pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 injected via the tail vein and investigated the protective effect of APE1/Ref-1 over-expression in mice model of radiation-induced lung injury. The histology and collagen of lungs was examined by HE stain and Masson-trichrome separately, the expression of APE1/Ref-1 protein was detected by immunohistochemistry and western blot. Enzyme linked-immuno-sorbent assay (ELISA) was applied to detect the TGF-β1 in serum.
Results
1. Study of the effects of Ad5/F35-APE1/Ref-1siRNA and rAd-p53 on radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model:
Ad5/F35-APE1/Ref-1siRNA suppressed significantly the expression of APE1/Ref-1 protein, and rAd-p53 enhanced significantly the expression of p53 protein in vivo. The tumor growth inhibition ratio, PI and AI in Ad5/F35-APE1/Ref-1siRNA combined with rAd-p53 plus radiotherapy group were significantly different, compared with the control group and other therapy groups (p<0.01).
2. Expression of APE1/Ref-1 and its significance in mice model of radiation-induced lung injury
The high expression of APE1/Ref-1 was detected in nuclei of epithelial cells and endothelial cells in normal mice lung tissues. During the process of radiation-induced lung injury in mice model, the changes of three types of APE1/Ref-1 positive staining were observed; the expression of APE1/Ref-1 protein was detected in nuclear, cytoplasmic and mixed type. The expression of APE1/Ref-1 protein began to increase 1d after irradiation, reached its peak value on 3d, it was 2.2-fold compared with control group, and then decreased at 7d~56d. The expression of APE1/Ref-1 protein was significantly increased after 3d post-irradiation, and then was significantly decreased at 56d, and was 0.2-fold compared with control group.
3. To investigate the protective effect of APE1/Ref-1 over expression in mice model of radiation-induced lung injury:
After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased. It was 2.3-fold and 2.8-fold compared with lung tissue epithelial cells and endothelial cells of untransfected mice, separately. After single irradiation at omni-thorax of the mice with 20 Gy of 8 MV X irradiation, the pathological study showed marked lung injury in the radiation group while only slight hyperemia, hemorrhage, exudation and the forming of collagen in prevention and treatment groups by HE stain and Masson-trichrome. The levels of TGF-β1 in the serum increased 7d after irradiation, and then increased at 14d~56d, it reached its peak value at 56d, there was significant difference comparaed with the control group(P<0.01), and also reduced in two prevention and treatment groups and the radiation group(p<0.05). However, there was no significant difference between two prevention and treatment groups (p>0.05).
Conclusion
1. Ad5/F35-APE1/Ref-1siRNA and rAd-p53 combined with radiotherapy can significantly inhibit the growth and enhance radiosensitivity of human hepatocellular carcinoma cells in tumor-bearing nude mice model. Therefore, the gene therapy of Ad5/F35-APE1siRNA combined with rAd-p53 may be a promising approach to treat human hepatocellular carcinoma in the future.
2. Irradiation can up-regulate the expression of APE1/Ref-1 protein, while APE1/Ref-1 has the characteristic translocation from nucleus to cytoplasma. The expression of APE1/Ref-1 protein was increased at first and then decreased, which suggests that the APE1/Ref-1 may play a very important role in the development of radiation-induced lung injury.
3. After transfection of pcDNA3.1/flAPE1/Ref-1 and pcDNA3.1/mtAPE1/Ref-1 in vivo, the expression of APE1/Ref-1 protein in epithelial cells and endothelial cells of mice lung tissue was increased.
4. APE1/Ref-1 could show protective effects on the radiation-induced lung injury and the mechanism may be through repairing DNA lesions, blocking the production of reactive oxygen species and inhibiting the activity of redox regulation, decreasing the expression of TGF-β1, reducing the forming of collagen so as to ameliorate the radiation-induced lung injury.
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2. Wood RD, Mitchell M, Lindahl T. Human DNA repair genes[J]. Science, 2005, 577(1-2):275-283.
3.谷志远,赵亚力.现代医学分子生物学[M].北京:人民军医出版社, 2004:53-69.
4. Jaiswal M, LaRusso NF, Nishioka N, et al. Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide[J]. Cancer Res, 2001, 61(17):6388-6393.
5. Asagoshi K, Yamada T, Terato H, et al. Distinct repair activities of human
7,8-dihydro-8-oxoguanine DNA glycosylase for formamidopyridine and7,8-dihydro-8-oxoguanine[J]. J Biol Chem, 2000, 275 (7):4956-4964.
6. Dantzer F, Bj?r?s M, Luna L, et al. Comparative analysis of 8-oxoG:C, 8-oxoG:A, A:C and C:C DNA repair in extracts from wild type or 8-oxoG DNA glycosylase deficient mammalian and bacterial cells[J]. DNA Repair (Amst). 2003, 2(6):707-718.
7. Cong WM, Bakker A, Swalsky PA, et al. Multiple genetic alterations involved in the tumorigenesis of human cholangiocarcinoma: a molecular genetic and clinicopathological study[J]. J cancer Res Clin Oncol, 2001, 127(3):187-192.
8. Kannan S, Pang H, Foster DC, et al. Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity. Cell Death Differ J T-Cell death and differentiation. England, 2006, (2):311-323.
9. Bravard A, Vacher M, Gouget B, et al. Redox regulation of human OGG1 activity in response to cellular oxidative stress. Mol Cell Biol, 2006, 26(20):7430-7436.
10. Akiyama K, Seki S, Oshida T, et al. Structure, promoter analysis and chromosomal assignment of the human APEX gene[J]. Biochim Biophys Acta, 1994, 1219(1):15-25.
11. Gorman M A, Morera S, Rothwell D G, et al. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites[J]. EMBO J, 1997, 16(21):6548-6558.
12. Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1[J]. Mutat Res, 2000, 461(2): 83-108.
13. Robertson KA, Bullock HA, Xu Y, et al. Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation[J]. Cancer Res, 2001, 61(5):2220-2225.
14. Fishel ML, Kelley MR. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target. Mol Aspects Med. 2007, 28(3-4):375-95.
15.谭永红,王东,肖桃元等.大鼠半胸照射致肺纤维化模型的病理学观察[J].第三军医大学学报,2006, 28(2):104-106.
16. Seo YR, Kelley MR, Smith ML. Selenomethionine regulation of p53 by a ref1-dependent redox mechanism[J]. Proc Natl Acad Sci U S A, 2002,99(22):14548-14553.
17. Huang LE, Arany Z, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit[J]. J Biol Chem, 1996, 271(50):32253-32359.
18. Angkeow P, Deshpande SS, Qi B, et al. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis[J]. Cell Death Differ, 2002, 9(7):717-725.
19. Tell G, Damante G, Caldwell D, et al. The intracellular localization of APE1/Ref-1: more than a passive phenomenon? [J]. Antioxid Redox Siqnal, 2005, 7(3-4):367-384.
20. Thompson L H, Brookman KW, Jones NJ, et al. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange[J]. Mol Cell Biol, 1990, 10(12):6160-6171.
21. Kubota Y, Nash RA, Klungland A, et al. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein[J]. EMBO J, 1996, 15(23):6662-6670.
22. Thornton K, Forstner M, Shen MR, et al. Purification, characterization and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein[J]. Protein Expr Purif, 1999, 16(2):236-242.
23. Schreiber V, AméJC, DolléP, et al. Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1[J]. J Biol Chem, 2002, 277(25):23028-23036.
24. Gryk MR, Marintchev A, Mullen GP. Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group1, with DNA polymerase beta[J]. Nucleic Acids Res, 2003, 31(2):580-588.
25.宋春英,谭文,林东昕.中国人DNA修复基因XRCC1单核苷酸多态及其与食管癌风险的关系[J].癌症,2001, 20(1):28-31.
26.王良群,吴旭梅,范雪云等. DNA修复基因XRCC1单核苷酸多态与辐射损伤易感性相关性研究[J].环境与职业医学,2007, 24(1):36-39.
27.吴旭梅,王良群,范雪云等. XRCC1基因多态性与辐射致染色体损伤易感性的关系[J].中国职业医学,2007, 34(1):7-10.
28. Shinohara A, Ogawa H, Matsuda Y, et al. Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA[J]. Nat Genet, 1993, 4(3):239-243.
29. Schmutte C, Tombline G, Rhiem K, et, al. Characterization of the human Rad51 genomic locus and examination of tumors with 15q14-15 loss of heterozygosity (LOH)[J]. Cancer Res, 1999, 59(18):4564-4569.
30. Stürzbecher HW, Donzelmann B, Henning W, et al. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction[J]. EMBO J, 1996, 15(8):1992-2002.
31. Scully R, Chen J, Plug A, et al. Association of BRCA1 with Rad51 in mitotic and meiotic cells[J]. Cell, 1997, 88(2):265-275.
32. Zhang H, Tombline G, Weber BL. BRCA1, BRCA2, and DNA damage response: collision or collusion?[J]. Cell, 1998, 92(4):433-436.
33. Bertrand P, Lambert S, Joubert C, Lopez B S. Overexpression of mammalian Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome f agmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells[J ]. Oncogene, 2003, 22(4):7587-7592.
34. Wick W, Petersen I, Schmutzler RK, et al. Evidence for a novel tumor suppressor gene on chromosome 15 associated with progression to a metastatic stage in breast cancer[J]. Oncogene, 1996, 12(5):973-978.
35. Maacke H, Jost K, Optiz S, et al. DNA repair and recombination factor Rad51 is over-expressed in human pancreatic adenocarcinoma[J]. Oncogene, 2000, 19(23):2791-2795.
36. Maacke H, Optiz S, Jost K, et al. Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer[J]. Int J Cancer, 2000, 88(6):907-913.
37.易俊林,蔡微田,凭岡真宽.RNAi干扰技术失活Rad51基因增加肿瘤细胞放射敏感性的研究[J].中华放射肿瘤学杂志,2006, 15(6):473-476.
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2. Shen JC, Loeb LA. Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site. J Biol Chem, 2003, 278(47):46994- 47001.
3. Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1. Mutat Res, 2000, 461(2):83-108.
4. Tell G, Pines A, Paron I, et al. Redox effector factor-1 regulates the activity of thyroid transcription factor 1 by controlling the redox state of the N transcriptional activation domain. J Biol Chem, 2002, 277(17):14564-14574.
5. Tell G, Damante G, Caldwell D, et al. The intracellular localization of APE1/Ref-1: more than a passive phenomenon? Antioxid Redox Signal, 2005, 7(3-4):367-384.
6. Gros L, Ishchenko AA, Ide H, et al. The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway. Nucleic Acids Res, 2004, 32(1):73-81.
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55. Anscher MS, Murase T, prescotl DM. Changes in plasma TGF-beta levels during pulmonary radiotherapy as a predictor of the risk of developing radiation pneumonitis. Int J Radiat Oncol Biol Phys. 1994, 30(3): 671- 676.
1.药立波.医学分子生物学[M].北京:人民卫生出版社, 2004:50-64.
2. Wood RD, Mitchell M, Lindahl T. Human DNA repair genes[J]. Science, 2005, 577(1-2):275-283.
3.谷志远,赵亚力.现代医学分子生物学[M].北京:人民军医出版社, 2004:53-69.
4. Jaiswal M, LaRusso NF, Nishioka N, et al. Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide[J]. Cancer Res, 2001, 61(17):6388-6393.
5. Asagoshi K, Yamada T, Terato H, et al. Distinct repair activities of human
7,8-dihydro-8-oxoguanine DNA glycosylase for formamidopyridine and7,8-dihydro-8-oxoguanine[J]. J Biol Chem, 2000, 275 (7):4956-4964.
6. Dantzer F, Bj?r?s M, Luna L, et al. Comparative analysis of 8-oxoG:C, 8-oxoG:A, A:C and C:C DNA repair in extracts from wild type or 8-oxoG DNA glycosylase deficient mammalian and bacterial cells[J]. DNA Repair (Amst). 2003, 2(6):707-718.
7. Cong WM, Bakker A, Swalsky PA, et al. Multiple genetic alterations involved in the tumorigenesis of human cholangiocarcinoma: a molecular genetic and clinicopathological study[J]. J cancer Res Clin Oncol, 2001, 127(3):187-192.
8. Kannan S, Pang H, Foster DC, et al. Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity. Cell Death Differ J T-Cell death and differentiation. England, 2006, (2):311-323.
9. Bravard A, Vacher M, Gouget B, et al. Redox regulation of human OGG1 activity in response to cellular oxidative stress. Mol Cell Biol, 2006, 26(20):7430-7436.
10. Akiyama K, Seki S, Oshida T, et al. Structure, promoter analysis and chromosomal assignment of the human APEX gene[J]. Biochim Biophys Acta, 1994, 1219(1):15-25.
11. Gorman M A, Morera S, Rothwell D G, et al. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites[J]. EMBO J, 1997, 16(21):6548-6558.
12. Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1[J]. Mutat Res, 2000, 461(2): 83-108.
13. Robertson KA, Bullock HA, Xu Y, et al. Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation[J]. Cancer Res, 2001, 61(5):2220-2225.
14. Fishel ML, Kelley MR. The DNA base excision repair protein Ape1/Ref-1 as a therapeutic and chemopreventive target. Mol Aspects Med. 2007, 28(3-4):375-95.
15.谭永红,王东,肖桃元等.大鼠半胸照射致肺纤维化模型的病理学观察[J].第三军医大学学报,2006, 28(2):104-106.
16. Seo YR, Kelley MR, Smith ML. Selenomethionine regulation of p53 by a ref1-dependent redox mechanism[J]. Proc Natl Acad Sci U S A, 2002,99(22):14548-14553.
17. Huang LE, Arany Z, Livingston DM, et al. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit[J]. J Biol Chem, 1996, 271(50):32253-32359.
18. Angkeow P, Deshpande SS, Qi B, et al. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis[J]. Cell Death Differ, 2002, 9(7):717-725.
19. Tell G, Damante G, Caldwell D, et al. The intracellular localization of APE1/Ref-1: more than a passive phenomenon? [J]. Antioxid Redox Siqnal, 2005, 7(3-4):367-384.
20. Thompson L H, Brookman KW, Jones NJ, et al. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange[J]. Mol Cell Biol, 1990, 10(12):6160-6171.
21. Kubota Y, Nash RA, Klungland A, et al. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein[J]. EMBO J, 1996, 15(23):6662-6670.
22. Thornton K, Forstner M, Shen MR, et al. Purification, characterization and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein[J]. Protein Expr Purif, 1999, 16(2):236-242.
23. Schreiber V, AméJC, DolléP, et al. Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1[J]. J Biol Chem, 2002, 277(25):23028-23036.
24. Gryk MR, Marintchev A, Mullen GP. Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group1, with DNA polymerase beta[J]. Nucleic Acids Res, 2003, 31(2):580-588.
25.宋春英,谭文,林东昕.中国人DNA修复基因XRCC1单核苷酸多态及其与食管癌风险的关系[J].癌症,2001, 20(1):28-31.
26.王良群,吴旭梅,范雪云等. DNA修复基因XRCC1单核苷酸多态与辐射损伤易感性相关性研究[J].环境与职业医学,2007, 24(1):36-39.
27.吴旭梅,王良群,范雪云等. XRCC1基因多态性与辐射致染色体损伤易感性的关系[J].中国职业医学,2007, 34(1):7-10.
28. Shinohara A, Ogawa H, Matsuda Y, et al. Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA[J]. Nat Genet, 1993, 4(3):239-243.
29. Schmutte C, Tombline G, Rhiem K, et, al. Characterization of the human Rad51 genomic locus and examination of tumors with 15q14-15 loss of heterozygosity (LOH)[J]. Cancer Res, 1999, 59(18):4564-4569.
30. Stürzbecher HW, Donzelmann B, Henning W, et al. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction[J]. EMBO J, 1996, 15(8):1992-2002.
31. Scully R, Chen J, Plug A, et al. Association of BRCA1 with Rad51 in mitotic and meiotic cells[J]. Cell, 1997, 88(2):265-275.
32. Zhang H, Tombline G, Weber BL. BRCA1, BRCA2, and DNA damage response: collision or collusion?[J]. Cell, 1998, 92(4):433-436.
33. Bertrand P, Lambert S, Joubert C, Lopez B S. Overexpression of mammalian Rad51 does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome f agmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells[J ]. Oncogene, 2003, 22(4):7587-7592.
34. Wick W, Petersen I, Schmutzler RK, et al. Evidence for a novel tumor suppressor gene on chromosome 15 associated with progression to a metastatic stage in breast cancer[J]. Oncogene, 1996, 12(5):973-978.
35. Maacke H, Jost K, Optiz S, et al. DNA repair and recombination factor Rad51 is over-expressed in human pancreatic adenocarcinoma[J]. Oncogene, 2000, 19(23):2791-2795.
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