cox-2表达与食管鳞状细胞癌放射敏感性的基础与临床研究
|
摘要
前言
食管癌是十大恶性肿瘤之一,我国发病率据世界首位,发病人数占全世界的50%以上。约60%以上的患者就诊时为中晚期,这些患者的治疗主要是以放射治疗为主的综合治疗,放射治疗已成为中晚期食管癌的主要治疗手段之一。中国食管癌的主要病理类型是鳞癌,属中度放射敏感性肿瘤,单纯放疗5年生存率仅10～20%,多数患者死于肿瘤局部未控和复发。寻找放疗敏感性的分子标志物,预测食管癌放射敏感性,探讨食管癌放疗抗拒性的机制,提高肿瘤局部控制率是本研究的主要目的。
研究表明Cox-2可作为评估肿瘤对放射治疗是否抵抗的一个指标,在肿瘤组织中Cox-2蛋白的表达水平越高,其放射敏感性越差。Cox-2基因表达水平的高低与放射治疗疗效之间的关系在头颈部肿瘤的治疗方面有不少报道,但在食管癌方面研究甚少。
本研究通过分析76例食管癌患者近期放疗疗效与肿瘤组织中Cox-2表达水平,探讨Cox-2表达与食管鳞状细胞癌放射抗拒性的关系；通过构建Cox-2特异性siRNA重组质粒和Cox-2表达载体,转染人食管癌EC9706细胞,分别下调和上调Cox-2基因表达,再联合不同剂量的X射线照射,观察放射结合Cox-2基因调控对细胞增殖、细胞周期、凋亡、细胞侵袭能力和放射敏感性的影响,并初步探讨沉默Cox-2基因表达的放射增敏机制；通过观测荷瘤裸鼠经放射线照射后瘤体体积的变化,从活体水平来评价双向调控Cox-2基因表达对放射线的增敏作用,为基因治疗联合放射治疗的临床应用提供理论基础和实验依据。
材料与方法
1.单纯采用放射治疗的76例食管鳞癌患者。
2.采用免疫组织化学SP法分别检测76例食管鳞癌组织中Cox-2的表达水平。并检测其中20例患者正常食管黏膜组织中Cox-2蛋白的表达。用统计学软件SPSS13.0对实验数据进行统计学处理,观察食管鳞癌组织中Cox-2蛋白表达水平与放疗抗拒性的关系。
3.针对人Cox-2基因mRNA序列,构建2个siRNA重组质粒：pRNA-U6.1-sicox214和pRNA-U6.1-sicox799,同时构建Cox-2表达载体和1个无关序列siRNA,利用脂质体转染技术转入人食管癌EC9706细胞,G418筛选得到稳定转染细胞株。同时设立转染无关序列siRNA组、空载体对照组和未转染组。
4.0Gy、2Gy、4Gy射线照射,应用荧光定量RT-PCR检测细胞中Cox-2、MMP2、Bax、Bcl-2的表达水平；Western blot检测Cox-2蛋白及AKT蛋白和磷酸化AKT蛋白(pAKT)的表达。
5.流式细胞术检测EC9706细胞周期和细胞凋亡率,比较各实验组经0、2、4Gy射线照射后细胞周期以及凋亡率的变化；
6.通过Transwell侵袭小室实验检测双向调控Cox-2表达联合射线照射对食管鳞癌细胞侵袭活性的影响。
7.CCK8实验和克隆形成实验检测不同剂量射线照射对细胞增殖能力和存活分数的影响。
8.建立稳定转染的EC9706细胞株裸鼠移植瘤模型,观测荷瘤裸鼠经20Gy射线照射后瘤体体积的变化,从体内实验进一步探讨双向调控Cox-2表达对食管鳞癌组织放射敏感性的影响。
统计学分析
采用SPSS13.0统计学软件对所有数据均进行统计学处理,两变量之间的关系分析用spearman等级相关分析表示,阳性率之间的比较用χ2检验,检验标准以α=0.05为显著性检验水准。
结果：
1.76例食管癌患者放射治疗结束后1个月做胸部增强CT扫描和食管钡餐造影检查进行疗效评价,其中放射敏感(CR+PR)55例(CR：13例；PR：42例)；NR(21例)放射抗拒。
2.免疫组织化学SP法检测证实,正常食管黏膜组织中Cox-2蛋白的表达明显低于食管鳞癌组织,放射抗拒组Cox-2蛋白的表达明显增高,且明显高于放射敏感组(p<0.05)。
3.成功构建了2个Cox-2基因siRNA重组质粒和1个Cox-2表达载体,经测序鉴定正确。利用脂质体转染技术成功导入人食管癌EC9706细胞,经G418筛选得到稳定转染的人食管癌EC9706细胞。
4.0、2、4Gy的X射线照射后,荧光定量RT-PCR和Western blot检测结果显示沉默Cox-2基因表达的EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平均显著降低,且与照射剂量呈反比。BaxmRNA表达水平显著升高,与照射剂量呈正比。而上调Cox-2表达的EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平均显著升高,BaxmRNA表达水平显著降低,与照射剂量均无明显相关性。
5.流式细胞仪检测证实Cox-2沉默组G0／G1期细胞所占比例及细胞凋亡率较Cox-2上调组及对照组均明显增高,差异有统计学意义(p<0.05)；且均有随照射剂量增加而上升的趋势。
6. Transwell侵袭小室实验结果显示,沉默Cox-2表达可以显著降低食管癌细胞EC9706的侵袭活性。
7.CCK8实验证实0、2、4Gy的射线照射,沉默Cox-2表达对细胞生长均有抑制作用,而上调Cox-2表达组细胞增殖抑制率均为负值。克隆形成实验结果显示与对照组相比沉默Cox-2表达可降低食管癌EC9706细胞集落形成能力,使细胞存活率明显下降(p<0.05)。而上调Cox-2表达相对存活率较对照组升高(p<0.05)。
8.裸鼠移植瘤实验证实接种3周后沉默Cox-2表达组的平均瘤体体积较对照组明显减低(p<0.05),上调Cox-2表达组的平均瘤体体积较对照组明显增高(p<0.05)。放射治疗20Gy后,沉默Cox-2表达组平均肿瘤体积明显减小(p<0.01),而上调组裸鼠皮下种植瘤平均体积较放疗前无明显变化(p>0.05)。
结论
1.食管鳞癌组织中Cox-2蛋白呈阳性表达,且放射抗拒组Cox-2蛋白表达率明显高于放射敏感组。
2.沉默Cox-2基因表达,EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平下降,且与照射剂量呈反比,但BaxmRNA表达水平上调,与照射剂量呈正比；而上调Cox-2表达则结果相反。
3.RNA干扰Cox-2基因表达对EC9706细胞有放射增敏作用,其增敏机制与MMP2、Bax、Bcl-2、AKT蛋白和磷酸化AKT蛋白有关。
4.裸鼠移植瘤实验显示,沉默Cox-2表达能够显著抑制瘤体生长,增加肿瘤对放射治疗的敏感性。
Introduction
Esophageal Cancer is one of malignant tumors which are threatening human health and life, and also ranks the sixth in the death rate of malignant neoplasms. Our country is a high-incidence area of esophageal cancer, where the number of patients with it is up to more than 50%of the world total. More than about 60%of patients are in the intermediate and advanced stage when treated. Such patients are given a combination treatment focused on radiation therapy, which has become one of the main therapies for intermediate and advanced esophageal cancer. The main pathological type of esophageal cancer in China is squamous cell carcinoma, which belongs to moderate radiation sensitivity tumors. The survival rate of these patients with 5 years of pure radiation is only 10%to 20%, of which most patients died of tumor local failure and recurrence. How to accurately predict the radiation sensitivity of esophageal cancer, search for the methods and mechanisms to increase the radiation sensitivity, and improve tumor local control rate, are becoming hot points for domestic and overseas scholars at present.
Researches show that Cox-2 can be used as an indicator to evaluate the resistance of the tumor to radiation therapy, as Cox-2 is higher for protein expression in tumor tissue, the radiation resistance is stronger. Such many reports as the relationship between the level of Cox-2 gene expression and the curative effect of radiation therapy are for the treatment of head and neck cancer, but so few are for esophageal researches.
This study analyzes recent radiotherapy curative effect of 76 cases and the Cox-2 expression level in tumor tissue, and discusses the relationships between Cox-2 expression and the radiation resistance to esophageal squamous cell carcinoma; transfecting esophageal EC9706 cells by constructing Cox-2 genes specific siRNA restructuring plasmid and Cox-2 expression vector, cutting and raising Cox-2 gene expression, and then combining different doses of X ray, observing the influence of radiation Cox-2 merger gene regulation on cell proliferation, cell cycle, apoptosis, cell attack ability and radiation sensitivity, and also preliminarily discussing radiotherapy sensitization mechanism of Silent Cox-2 expression; by observing the changes of tumor volume of tumor-burdened nude mice after radiation, evaluating the effect of sensitization of two-way regulating Cox-2 gene expression to radiation ray from the level of living body, which provides theoretical basis and experimental basis for clinical application of combing gene therapy with radiation therapy.
Materials and methods
(1)Only apply recent curative effect of 76 cases of patients with esophageal squamous cell carcinoma by radiation therapy.
(2)Apply immunohistochemical method SP to detect Cox-2 protein expression level in esophageal squamous cell carcinoma tissues of 76 cases. Apply statistics processing to experimental data by statistical software SPSS 13.0, observe the relationship between Cox-2 protein expression level and radiation resistance in esophageal squamous cell carcinoma tissues.
(3)Aimed at Cox-2 gene mRNA sequence of human being, constructing two siRNA restructuring plasmid:pRNA-U6.1-siCox214 and pRNA-U6.1-siCox799, and also build Cox-2 expression vector and one irrelevant sequence siRNA, using liposome technology to convert into esophageal EC9706 cells of human, acquiring stable transfecting cell line by G418 screening. At the same time setting up transfecting irrelevant sequence siRNA group, empty carrier group and non-transfecting group
(4)Through 0 Gy,2 Gy,4 Gy ray radiation, apply quantitative fluorescence RT-PCR to detect expression level of Cox-2, MMP2, Bax, Bcl-2 in cells; apply Western blot to test the expression of Cox-2 protein, AKT protein and phosphorylation protein AKT (pAKT).
(5)Detect EC9706 cell cycle and cell apoptosis rate by flow cytometry, compare the changes of cell cycle and apoptosis rate after 0,2,4Gy ray among experimental groups.
(6) Detect the influence of two-way regulating Cox-2 expression with radiation on metastatic activity of esophageal cancer cells through Transwell invasion assay.
(7)Detect the influence of cell proliferation ability and live scores under different doses of radiation by experiment CCK8 and Cloning form experiment.
(8)Establish a stable transfecting implant tumor model in nude mice with EC 9706 cell lines, observe the changes of tumor volume of tumor-burdened nude mice after 20Gy radiation, explore the influence of two-way regulating Cox-2 gene expression to radiation ray from the level of living body on radiation sensitivity of esophageal squamous cell carcinoma organization.
Results:
(1)76 cases of esophageal patients are given efficacy evaluation by chest CT enhanced scanning and esophagus barium meal X-ray examination in one month after radiotherapy, in which 55 cases(CR:13 cases; PR:42 cases) of radiation sensitivity(CR+PR);NR(21 cases) radiation resistance.
(2)The test of immunohistochemical SP method proves that, if Cox-2 expression in esophageal squamous cell carcinoma tissues is higher than the expression in normal esophageal mucosal tissues, Cox-2 expression in radiation resistance group obviously increases higher, and also obviously higher than in radiation sensitivity group.
(3)Successfully construct two Cox-2 gene siRNA restructuring plasmid and 1 cox-2 expression vector, which is right through sequencing appraisal. Use liposome transfecting technology to induct successfully to EC9706 esophageal cells, and get steady transfecting esophageal EC9706 cells through G418 screening.
(4)After the X ray radiation of 0,2,4 Gy, fluorescence quantitative RT-PCR and Western blot tests showed that the expression level of MMP2mRNA, AKT protein and phosphorylation AKT are significantly lower, and with the illuminate dose is inverse ratio. BaxmRNA significantly increased the expression level, and radiation doses than positively. And Cox-2 express raised EC9706 cells MMP2mRNA, Bcl-2 mRNA, AKT protein and AKT phosphorylation of expression level were significantly increased, BaxmRNA expression were significantly reduced, and radiation dose were not significantly correlation.
(5) Flow cytometric detect proof Cox-2 silent group GO/G1 phase cells proportion and apoptosis rate is Cox-2 group were significantly raised control and increased, the difference was statistically significant (p< 0.05); And all have radiation dose increase with the rising trend.
(6) Transwell hit small room the experiment results, silent Cox-2 expression can significantly reduce esophageal EC9706 cells of metastatic activity.
(7) CCK8 experiments 0,2,4 Gy rays, silence Cox-2 expression of cell growth has inhibitory effect, and raised cox-2 express group cell proliferation inhibition rate are a negative value. Cloning form the results show that compared with the control silence Cox-2 expression can reduce esophageal EC9706 cells colony formation ability, making the cell survival rate decreased obviously (p< 0.05). And raise Cox-2 expression, the relative survival rate is higher than control group (p< 0.05).
(8)The experiment of transplanting tumor in nude mice proves, after 3 weeks of vaccination, average volume in silence Cox-2 expression group is significantly lower than in control group (p< 0.05), raise average volume in Cox-2 expression group is evidently higher than in control group (p< 0.05). After 20Gy radiation therapy, average tumor volume in silence Cox-2 expression group is obviously lower (p< 0.01), and average volume of subcutaneous tumor of nude mice in raise group has no significant change which related to the volume before radiotherapy (p> 0.05).
Conclusions:
(1) Cox-2 protein expression in Esophageal squamous cell carcinoma tissues is positive, and Cox-2 protein expression rate in radiation resistence group is significantly higher than in radiation sensitity group.
(2) Silence Cox-2 gene expression can regulate down the expression level of MMP2mRNA, Bcl-2 mRNA, AKT protein and AKT phosphorylation in EC9706 cells, and their illuminate dose is inverse ratio. Raising the expression level of BaxmRNA correlates to radiation doses positively. And raise Cox-2 expression, which raise Expression level of MMP2mRNA, Bcl-2 mRNA, AKT protein and phosphorylation AKT in EC9706 cells, and regulate down BaxmRNA expression level, which was not significantly correlation with the radiation dose.
(3)RNA interference to Cox-2 gene expression EC 9706 cells have radiotherapy sensitization effect, its sensitization mechanism may be relevant to MMP2, Bax, Bcl-2, AKT protein and protein phosphorylation AKT.
(4)Apply X-ray radiotherapy to transplant tumor in nude mice, silence Cox-2 expression can significantly inhibit the growth of tumor volume, and increase the sensitivity of the tumor for radiation therapy.
食管癌是十大恶性肿瘤之一,我国发病率据世界首位,发病人数占全世界的50%以上。约60%以上的患者就诊时为中晚期,这些患者的治疗主要是以放射治疗为主的综合治疗,放射治疗已成为中晚期食管癌的主要治疗手段之一。中国食管癌的主要病理类型是鳞癌,属中度放射敏感性肿瘤,单纯放疗5年生存率仅10～20%,多数患者死于肿瘤局部未控和复发。寻找放疗敏感性的分子标志物,预测食管癌放射敏感性,探讨食管癌放疗抗拒性的机制,提高肿瘤局部控制率是本研究的主要目的。
研究表明Cox-2可作为评估肿瘤对放射治疗是否抵抗的一个指标,在肿瘤组织中Cox-2蛋白的表达水平越高,其放射敏感性越差。Cox-2基因表达水平的高低与放射治疗疗效之间的关系在头颈部肿瘤的治疗方面有不少报道,但在食管癌方面研究甚少。
本研究通过分析76例食管癌患者近期放疗疗效与肿瘤组织中Cox-2表达水平,探讨Cox-2表达与食管鳞状细胞癌放射抗拒性的关系；通过构建Cox-2特异性siRNA重组质粒和Cox-2表达载体,转染人食管癌EC9706细胞,分别下调和上调Cox-2基因表达,再联合不同剂量的X射线照射,观察放射结合Cox-2基因调控对细胞增殖、细胞周期、凋亡、细胞侵袭能力和放射敏感性的影响,并初步探讨沉默Cox-2基因表达的放射增敏机制；通过观测荷瘤裸鼠经放射线照射后瘤体体积的变化,从活体水平来评价双向调控Cox-2基因表达对放射线的增敏作用,为基因治疗联合放射治疗的临床应用提供理论基础和实验依据。
材料与方法
1.单纯采用放射治疗的76例食管鳞癌患者。
2.采用免疫组织化学SP法分别检测76例食管鳞癌组织中Cox-2的表达水平。并检测其中20例患者正常食管黏膜组织中Cox-2蛋白的表达。用统计学软件SPSS13.0对实验数据进行统计学处理,观察食管鳞癌组织中Cox-2蛋白表达水平与放疗抗拒性的关系。
3.针对人Cox-2基因mRNA序列,构建2个siRNA重组质粒：pRNA-U6.1-sicox214和pRNA-U6.1-sicox799,同时构建Cox-2表达载体和1个无关序列siRNA,利用脂质体转染技术转入人食管癌EC9706细胞,G418筛选得到稳定转染细胞株。同时设立转染无关序列siRNA组、空载体对照组和未转染组。
4.0Gy、2Gy、4Gy射线照射,应用荧光定量RT-PCR检测细胞中Cox-2、MMP2、Bax、Bcl-2的表达水平；Western blot检测Cox-2蛋白及AKT蛋白和磷酸化AKT蛋白(pAKT)的表达。
5.流式细胞术检测EC9706细胞周期和细胞凋亡率,比较各实验组经0、2、4Gy射线照射后细胞周期以及凋亡率的变化；
6.通过Transwell侵袭小室实验检测双向调控Cox-2表达联合射线照射对食管鳞癌细胞侵袭活性的影响。
7.CCK8实验和克隆形成实验检测不同剂量射线照射对细胞增殖能力和存活分数的影响。
8.建立稳定转染的EC9706细胞株裸鼠移植瘤模型,观测荷瘤裸鼠经20Gy射线照射后瘤体体积的变化,从体内实验进一步探讨双向调控Cox-2表达对食管鳞癌组织放射敏感性的影响。
统计学分析
采用SPSS13.0统计学软件对所有数据均进行统计学处理,两变量之间的关系分析用spearman等级相关分析表示,阳性率之间的比较用χ2检验,检验标准以α=0.05为显著性检验水准。
结果：
1.76例食管癌患者放射治疗结束后1个月做胸部增强CT扫描和食管钡餐造影检查进行疗效评价,其中放射敏感(CR+PR)55例(CR：13例；PR：42例)；NR(21例)放射抗拒。
2.免疫组织化学SP法检测证实,正常食管黏膜组织中Cox-2蛋白的表达明显低于食管鳞癌组织,放射抗拒组Cox-2蛋白的表达明显增高,且明显高于放射敏感组(p<0.05)。
3.成功构建了2个Cox-2基因siRNA重组质粒和1个Cox-2表达载体,经测序鉴定正确。利用脂质体转染技术成功导入人食管癌EC9706细胞,经G418筛选得到稳定转染的人食管癌EC9706细胞。
4.0、2、4Gy的X射线照射后,荧光定量RT-PCR和Western blot检测结果显示沉默Cox-2基因表达的EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平均显著降低,且与照射剂量呈反比。BaxmRNA表达水平显著升高,与照射剂量呈正比。而上调Cox-2表达的EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平均显著升高,BaxmRNA表达水平显著降低,与照射剂量均无明显相关性。
5.流式细胞仪检测证实Cox-2沉默组G0／G1期细胞所占比例及细胞凋亡率较Cox-2上调组及对照组均明显增高,差异有统计学意义(p<0.05)；且均有随照射剂量增加而上升的趋势。
6. Transwell侵袭小室实验结果显示,沉默Cox-2表达可以显著降低食管癌细胞EC9706的侵袭活性。
7.CCK8实验证实0、2、4Gy的射线照射,沉默Cox-2表达对细胞生长均有抑制作用,而上调Cox-2表达组细胞增殖抑制率均为负值。克隆形成实验结果显示与对照组相比沉默Cox-2表达可降低食管癌EC9706细胞集落形成能力,使细胞存活率明显下降(p<0.05)。而上调Cox-2表达相对存活率较对照组升高(p<0.05)。
8.裸鼠移植瘤实验证实接种3周后沉默Cox-2表达组的平均瘤体体积较对照组明显减低(p<0.05),上调Cox-2表达组的平均瘤体体积较对照组明显增高(p<0.05)。放射治疗20Gy后,沉默Cox-2表达组平均肿瘤体积明显减小(p<0.01),而上调组裸鼠皮下种植瘤平均体积较放疗前无明显变化(p>0.05)。
结论
1.食管鳞癌组织中Cox-2蛋白呈阳性表达,且放射抗拒组Cox-2蛋白表达率明显高于放射敏感组。
2.沉默Cox-2基因表达,EC9706细胞中MMP2mRNA、Bcl-2mRNA、AKT蛋白和磷酸化AKT的表达水平下降,且与照射剂量呈反比,但BaxmRNA表达水平上调,与照射剂量呈正比；而上调Cox-2表达则结果相反。
3.RNA干扰Cox-2基因表达对EC9706细胞有放射增敏作用,其增敏机制与MMP2、Bax、Bcl-2、AKT蛋白和磷酸化AKT蛋白有关。
4.裸鼠移植瘤实验显示,沉默Cox-2表达能够显著抑制瘤体生长,增加肿瘤对放射治疗的敏感性。
Introduction
Esophageal Cancer is one of malignant tumors which are threatening human health and life, and also ranks the sixth in the death rate of malignant neoplasms. Our country is a high-incidence area of esophageal cancer, where the number of patients with it is up to more than 50%of the world total. More than about 60%of patients are in the intermediate and advanced stage when treated. Such patients are given a combination treatment focused on radiation therapy, which has become one of the main therapies for intermediate and advanced esophageal cancer. The main pathological type of esophageal cancer in China is squamous cell carcinoma, which belongs to moderate radiation sensitivity tumors. The survival rate of these patients with 5 years of pure radiation is only 10%to 20%, of which most patients died of tumor local failure and recurrence. How to accurately predict the radiation sensitivity of esophageal cancer, search for the methods and mechanisms to increase the radiation sensitivity, and improve tumor local control rate, are becoming hot points for domestic and overseas scholars at present.
Researches show that Cox-2 can be used as an indicator to evaluate the resistance of the tumor to radiation therapy, as Cox-2 is higher for protein expression in tumor tissue, the radiation resistance is stronger. Such many reports as the relationship between the level of Cox-2 gene expression and the curative effect of radiation therapy are for the treatment of head and neck cancer, but so few are for esophageal researches.
This study analyzes recent radiotherapy curative effect of 76 cases and the Cox-2 expression level in tumor tissue, and discusses the relationships between Cox-2 expression and the radiation resistance to esophageal squamous cell carcinoma; transfecting esophageal EC9706 cells by constructing Cox-2 genes specific siRNA restructuring plasmid and Cox-2 expression vector, cutting and raising Cox-2 gene expression, and then combining different doses of X ray, observing the influence of radiation Cox-2 merger gene regulation on cell proliferation, cell cycle, apoptosis, cell attack ability and radiation sensitivity, and also preliminarily discussing radiotherapy sensitization mechanism of Silent Cox-2 expression; by observing the changes of tumor volume of tumor-burdened nude mice after radiation, evaluating the effect of sensitization of two-way regulating Cox-2 gene expression to radiation ray from the level of living body, which provides theoretical basis and experimental basis for clinical application of combing gene therapy with radiation therapy.
Materials and methods
(1)Only apply recent curative effect of 76 cases of patients with esophageal squamous cell carcinoma by radiation therapy.
(2)Apply immunohistochemical method SP to detect Cox-2 protein expression level in esophageal squamous cell carcinoma tissues of 76 cases. Apply statistics processing to experimental data by statistical software SPSS 13.0, observe the relationship between Cox-2 protein expression level and radiation resistance in esophageal squamous cell carcinoma tissues.
(3)Aimed at Cox-2 gene mRNA sequence of human being, constructing two siRNA restructuring plasmid:pRNA-U6.1-siCox214 and pRNA-U6.1-siCox799, and also build Cox-2 expression vector and one irrelevant sequence siRNA, using liposome technology to convert into esophageal EC9706 cells of human, acquiring stable transfecting cell line by G418 screening. At the same time setting up transfecting irrelevant sequence siRNA group, empty carrier group and non-transfecting group
(4)Through 0 Gy,2 Gy,4 Gy ray radiation, apply quantitative fluorescence RT-PCR to detect expression level of Cox-2, MMP2, Bax, Bcl-2 in cells; apply Western blot to test the expression of Cox-2 protein, AKT protein and phosphorylation protein AKT (pAKT).
(5)Detect EC9706 cell cycle and cell apoptosis rate by flow cytometry, compare the changes of cell cycle and apoptosis rate after 0,2,4Gy ray among experimental groups.
(6) Detect the influence of two-way regulating Cox-2 expression with radiation on metastatic activity of esophageal cancer cells through Transwell invasion assay.
(7)Detect the influence of cell proliferation ability and live scores under different doses of radiation by experiment CCK8 and Cloning form experiment.
(8)Establish a stable transfecting implant tumor model in nude mice with EC 9706 cell lines, observe the changes of tumor volume of tumor-burdened nude mice after 20Gy radiation, explore the influence of two-way regulating Cox-2 gene expression to radiation ray from the level of living body on radiation sensitivity of esophageal squamous cell carcinoma organization.
Results:
(1)76 cases of esophageal patients are given efficacy evaluation by chest CT enhanced scanning and esophagus barium meal X-ray examination in one month after radiotherapy, in which 55 cases(CR:13 cases; PR:42 cases) of radiation sensitivity(CR+PR);NR(21 cases) radiation resistance.
(2)The test of immunohistochemical SP method proves that, if Cox-2 expression in esophageal squamous cell carcinoma tissues is higher than the expression in normal esophageal mucosal tissues, Cox-2 expression in radiation resistance group obviously increases higher, and also obviously higher than in radiation sensitivity group.
(3)Successfully construct two Cox-2 gene siRNA restructuring plasmid and 1 cox-2 expression vector, which is right through sequencing appraisal. Use liposome transfecting technology to induct successfully to EC9706 esophageal cells, and get steady transfecting esophageal EC9706 cells through G418 screening.
(4)After the X ray radiation of 0,2,4 Gy, fluorescence quantitative RT-PCR and Western blot tests showed that the expression level of MMP2mRNA, AKT protein and phosphorylation AKT are significantly lower, and with the illuminate dose is inverse ratio. BaxmRNA significantly increased the expression level, and radiation doses than positively. And Cox-2 express raised EC9706 cells MMP2mRNA, Bcl-2 mRNA, AKT protein and AKT phosphorylation of expression level were significantly increased, BaxmRNA expression were significantly reduced, and radiation dose were not significantly correlation.
(5) Flow cytometric detect proof Cox-2 silent group GO/G1 phase cells proportion and apoptosis rate is Cox-2 group were significantly raised control and increased, the difference was statistically significant (p< 0.05); And all have radiation dose increase with the rising trend.
(6) Transwell hit small room the experiment results, silent Cox-2 expression can significantly reduce esophageal EC9706 cells of metastatic activity.
(7) CCK8 experiments 0,2,4 Gy rays, silence Cox-2 expression of cell growth has inhibitory effect, and raised cox-2 express group cell proliferation inhibition rate are a negative value. Cloning form the results show that compared with the control silence Cox-2 expression can reduce esophageal EC9706 cells colony formation ability, making the cell survival rate decreased obviously (p< 0.05). And raise Cox-2 expression, the relative survival rate is higher than control group (p< 0.05).
(8)The experiment of transplanting tumor in nude mice proves, after 3 weeks of vaccination, average volume in silence Cox-2 expression group is significantly lower than in control group (p< 0.05), raise average volume in Cox-2 expression group is evidently higher than in control group (p< 0.05). After 20Gy radiation therapy, average tumor volume in silence Cox-2 expression group is obviously lower (p< 0.01), and average volume of subcutaneous tumor of nude mice in raise group has no significant change which related to the volume before radiotherapy (p> 0.05).
Conclusions:
(1) Cox-2 protein expression in Esophageal squamous cell carcinoma tissues is positive, and Cox-2 protein expression rate in radiation resistence group is significantly higher than in radiation sensitity group.
(2) Silence Cox-2 gene expression can regulate down the expression level of MMP2mRNA, Bcl-2 mRNA, AKT protein and AKT phosphorylation in EC9706 cells, and their illuminate dose is inverse ratio. Raising the expression level of BaxmRNA correlates to radiation doses positively. And raise Cox-2 expression, which raise Expression level of MMP2mRNA, Bcl-2 mRNA, AKT protein and phosphorylation AKT in EC9706 cells, and regulate down BaxmRNA expression level, which was not significantly correlation with the radiation dose.
(3)RNA interference to Cox-2 gene expression EC 9706 cells have radiotherapy sensitization effect, its sensitization mechanism may be relevant to MMP2, Bax, Bcl-2, AKT protein and protein phosphorylation AKT.
(4)Apply X-ray radiotherapy to transplant tumor in nude mice, silence Cox-2 expression can significantly inhibit the growth of tumor volume, and increase the sensitivity of the tumor for radiation therapy.
引文
[1]Fearlay F, Bray P, Pisani, et al. Globocan 2000:cancer incidence, mortality and prevalence worldwide [M].Lyon France:IARC press,2001:581.
[2]邹小农.食管癌流行病学[J].中华肿瘤防治杂志,2006,13(18)：18-21.
[3]韩企夏,施学辉.食管癌[A].见汤钊猷.现代肿瘤学[M].第二版.上海：上海医科大学出版社,2000：691.
[4]蔡凯,胡联民,陈刚,等.后程加速超分割三维适形放疗食管癌的临床研究[J].实用肿瘤杂志,2011,26(6)：631-634.
[5]殷慰伯.食管癌的放射治疗进展[J].实用肿瘤杂志,2006,21(2)：99-108.
[6]肖泽芬,食管癌[A].见殷蔚伯,谷铣之.肿瘤放射治疗学[M].第三版.北京：中国协和医科大学出版社,2002：610.
[7]KARIM A, MCCARTHY K, JAWAHAR A, et al. Differential cycloooxygenase-2 enzyme expression in radiosensitive versus radioresistant glioblastoma multiforme cell line[J]. AnticancerRes,2005,25:675-679.
[8]NIX P, LIND M, GREENMAN J, et al. Expression of cox-2 protein in radioresistant laryngeal cancer[J].Ann Oncol,2004,15:797-801.
[9]TERAKADO N, SHINTANI S, YANO J, et al. Overexpression of cyclooxygenasse-2 is associated with radioresistance in oral squamous cell carcinoma [J].Oral Oncol,2004,40:383-389.
[10]Liu Y, Zhao S, Liu G, et al. The value of cyclooxygenasse-2 to predict the effect of radiotherapy in nasopharyngeal carcinoma [J]. Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2007,21 (5):199-202.
[11]谢传高,王兴鹏.环氧合酶—2与消化系统肿瘤[J].国外医学.消化系疾病分册,2002,22(1)：20-23.
[12]Karim A, McCarthy K, Jawahar, et al. Differential eyelooxygenas-2 enzyme expression in radiosensitive Versus radioresistant glioblastoma multiform cell lines[J].Antieaneer Res,2005,25 (1B):675-679.
[13]Nix P, Lind M, Greenman J, et al. Expression of COX-2 protein in radiosensistant laryngeal cancer [J].Ann Oncol,2004,15(5):797-801.
[14]Terakado N, Shintani S, Yano J, et al. Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma [J]. Oral Oncol,2004,40(4):383-389.
[15]Huang WZ, Fu JH, Wang DK, et al. Overexpress of cyclooxygenase-2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients [J]. Dis Esophagus 2008,21(8):679-684.
[16]殷蔚伯,谷铣之.肿瘤放射治疗学.[M].北京：中国协和医科大学出版社,2002 610-611.
[17]刘彤华,夏求洁,刘复生等.消化道疾病.白希清.病理学(第二版)[M]北京：科学出版社，1992：215-217.
[18]Williams CS, Dubois RN. Prostaglandin endoperoxide synthase:why two isoform? [J]Am J physiol,1996,270(3):393-400.
[19]Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth [J] J ClinInvest,2000,105(11):1589-1594.
[20]PENG JP, CHANG HC, HWANG CF, et al. Overepression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis[J].Oral Oncol,2005,41:903-908.
[21]KARIM A, MCCARTHY K, JAWAHAR A, et al. Differential cycloooxygenase-2 enzyme expression in radiosensitive versus radioresistant glioblastoma multiforme cell line[J]. Anticancer Res,2005,25:675-679.
[22]Kuipers GK, Radiosensitization of human glioma cells by cyelooxygenase-2 inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration [J].Int J Radiat Bio,2007,183 (10):677-685.
[23]朱辉,王慧娟,贺春雨,等.食管鳞状细胞癌组织中COX-2和MGMT蛋白的表达.[J].郑州大学学报：医学版,2011,46(1)：125-128.
[24]Lagorec C, Paraf F, Vidaud D, et al. cyelooxygenase-2 is expressed frequently and early in Barrett's esophagus and assoiated adenocarcinomar [J]. His topathology,2003,42 (5): 457-460.
[25]燕春艳,涂玉林,钟苗,等Bcl-2siRNA的设计合成及其对HL-60 bcl-2基因表达的干扰作用[J].白血病·淋巴瘤,2006,15(5)：323-326.
[26]Hao JH, Gu QL, Liu BY, et al. Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA [J]. Chin Med J(Engl),2007,120(23): 2105-2111.
[27]袁月秀,王言奎,罗兵等.化学合成siRNA对宫颈癌细胞SiHa中E6基因表达的抑制作用[J].中国癌症杂志,2007,12(17)：914-919.
[28]Tchurikov NA, Chistyakova LG, Zavilgelsky GB, et al. Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli. J.Biol.Chem,2000,275 (34):26523-26529.
[29]Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001,409(6818):363-366.
[30]Kihara A, Kabeya Y, Ohsumi Y, et al. Beclinphosphatidy linositool 3-kinase complex functions at the trans-Golgi network. EMBO Rep,2001,2:330-335.
[31]Siddhesh D, David G, Diane J. DNA-based therapeutics and DNA delivery systems:A comprehensive review [J].The AAPS J,2005,7(1):E61-E77.
[32]Castanotto D, Li H, Rossi JJ. Function siRNA expression from transfected PCR products. RNA,2002,8(11):1454-1460.
[33]PENG JP, CHANG HC, HWANG CF, et al. Over expression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis [J]. Oral Oncol,2005, 41(9):903-908.
[34]DAISUKE S.DANIEL V,HIDEKAZU K, et al. Increasing cyclooxygenase-2 (cox-2) gene expression in progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2[J]. Int J Cancer,2006,119(4):765-770.
[35]ChantrainCF, Henriet P, Jodele S, et al..Mechanisms of Pericyte recruitment in tumor angiogenesis:a new rolefor metalloproteinases[J].Eur J Cancer,2006,42(3):310-318.
[36]Son KN> Hwang J, Kwon BS, et al. Human CC chemokine CCL23 enhances expression of matrixmetalloproteinase-2 and invasion of vascular endothelial cells [J]. Biochem Biophys Res Cornrnun,2006,340(2):498-504.
[37]Edwards JG, Mclaren J, Jonea JL,et al. Matrix metalloproteinases-2and9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura[J].Br J Caneer,2003,88(10):1553-1559.
[38]Mook OR,Freder Iksw M,Van Noorden CJ. The role of gelatinases in colorectal cancer progression and metastasis[J].Biochim BiophysActa,2004,1705(2):69-89.
[39]Patel BR,Shan PM, Rawal UM,et al. Activation of MMP2 and MMP9 in patients with oral squamous cell carcinoma[J].Surg Oncol,2005,90(3):81-88.
[40]John A, Tuszynski GThe role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis[J].Pathol Oncol Res,2001,7(1):14-23.
[41]Yu XF, Han ZC. Matrix metalloproteinases in bone mmarrow:roles of gelatinases in physiological hematopoiesis and hematopoietic malignancies [J]. Histopathol,2006,21(5): 519-531.
[42]Adams JM,Cory S.The bcl-2 protein family:arbiters of cell survival. Science,1998,281 (5381):1322-1326.
[43]O'Neill J, Manion M, Schwartz P, et al. Promises and challenges of targeting Bcl-2 anti-apoptotic proteins for cancer therapy. Biochim Biophys Acta,2004,1705(l):43-51.
[44]Leung RK, Whittaker PA. RNA interference:from gene silencing to gene-specific therapeutics. Pharmacol Ther,2005,107(2)222-239.
[45]Kirkin V, Joos S, Zornig M. The role of Bcl-2 falllily members in tumor genesis. Biochim Biophys Acta,2004,1644(2-3):229-249.
[46]McCabe ML, Dlamini Z. The molecular mechanisms of oesophageal cancer. Int Immunopharmacol,2005,5(7-8):1113-1130.
[47]刘军叶,郭鹞,郭国祯.Bcl-2基因特异性小干涉RNA增强食管癌细胞辐射敏感性的实验研究[J].国际放射医学核医学杂志,2006,30(2)：110-113.
[48]Hao JH, Gu QL, Liu BY, et al. Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA. Chin Med J,2007,120(23):2105-2111.
[49]Hara T, Omura-Minamisawa M, Chao C, et al. Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 over expressing radioresistant tumor cell. Int J Radiat Oncol Phy,2005,61:517-528.
[50]Scopa CD, Vagianos C, Kardamakis D, et al.Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with rectal cancer. Appl Immunohistochem Mol Morphol 2001，9(4):329-334.
[51]艾义国，饶旭光.马立民，等.食管癌组织环氧化酶-2和Bcl-2蛋白的表达与临床病理的关系研究[J].汕头大学医学院学报,2009.1(22)：21-24.
[52]Kim D, Kim S, KoH, et al.Akt/PKB promotes cancer cell invasion via increased motility and metalbproteinase production [J]. FASEB J 2001,15(11):1953-1956.
[53]Zhong H, Chiles K, Feldser D,et al. Modulation of hypoxia inducble factor 1 alpha expression by the epidermal growth factor/phosphatidy linositol3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells implications for tumor angiogenesis and therapeutics[J].cance Res,2000,60(6):1541-1544.
[54]冯学泉,王金环,徐新女,等.碱性成纤维细胞生长因子小干扰RNA与人类免疫缺陷病毒1型病毒蛋白R基因对裸鼠移胶质瘤的抑制作用[J].中华肿瘤杂志,2010,32(10)：725-728.
[55]Chen Yue, Xiong Qing-feng, He Ling, et al.188Re-DTPA-DG preparation and its experimental imaging in MCF-7 tumor-bearing nude mice[J].Chinese Journal of Nuclear Medicine,2007,27(02),23-25.
[56]Jun Wang, Chun Han, Xiao-Ning Li, et al. Short-term efficacy of intensity-modulated radiotherapy on esophageal carcinoma[J] Chinese Journal of Cancer,2009,28(11):35-36.
[57]Cheng Hui-Hua, Fu Zhi-Chao, Lin Gui-Shan,et al. Sensitivity enhancement of sodium glycididazole in re-radiotherapy against local recurrent esophageal carcinoma after radical radiotherapy [J]. Journal of the Fourth MilitaryMedical University,2007,28(11):35-36.
[58]Peng JP, Chang HC, Hwang CF, et al. Over expression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis[J].Oral Oncol,2005,41:903-908.
[59]Daisuke S,Daniel V, Hidekazu K, et al. Increasing cyclooxygenase-2 (cox-2) gene expression in progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2[J]. Int J Cancer,2006,119:765-770.
[1]全国肿瘤防治研究办公室,卫生部卫生统计信息中心编.中国试点市、县恶性肿瘤的发病与死亡.中国医药科技出版社,2002年,第二卷(1993-1997年),p21-34.
[2]Wang GQ, Jiao, Chang FB, et al. Long-term tesults of operation for 420patients with Early squamous cell esophageal carcinoma discovered by screening[J].Ann Thorac Surg,2004,77 (5):1740-1744.
[3]Russell E. Brown, Abbas E. Abbas, Susan Ellis, et al. A Prospective Phase II Evaluation of Esophageal Stenting for Neoadjuvant Therapy for Esophageal Cancer:Optimal Performance and Surgical Safety [J]. Journal of the American College of Surgeons,2011,212(4):582-588.
[4]肖泽芬,食管癌.见殷蔚伯,谷铣之主编.肿瘤放射治疗学.2002年,第三版,中国协和医科大学出版社,p598-622.
[5]Ting Liu, Hongxia Chen, Hongtai Kim, et al. RAD18-BRCTx interaction is required for efficient repair of UV-induced DNA damage [J]. DNA Repair,2012,11 (2):131-138.
[6]Chen YH, Sanchez Y. CHK1 in the DNA damage response:conserved roles from yeasts to mammals. DNA Repair (Amst),2004,3(8-9):1025-1032.
[7]Ahn J, Urist M, Prives C.The CHK2 protein kinase. DNA Repair (Amst),2004,3 (8-9): 1039-1047.
[8]Rahman S.Z.S, Theresa A.L, Jetze J. T. Synthesis and evaluation of debromohymenialdisine-derived Chk2 inhibitors [J]. Bioorganic & Medicinal Chemistry,2012,20(4):1474-1481.
[9]祝淑钗,王玉祥,李任等shRNA转染对食管癌细胞TE13中CHK1和CHK2表达以及照射后G2/M期阻滞的影响[J].癌症,2007,26(2)：127-131.
[10]Gatei M, Sloper K, Sorensen C, et al. Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of CHK1 on Ser-317 in response to ionizing radiation [J]. Biol Chem,2003,278(17):14806-14811.
[11]Xiao Z, Chen Z, Gunasekera AH, et al.CHKl mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents[J].BiolChem,2003,278(24):21767-2173.
[12]Wang X, Khadpe J, Hu B, et al. An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells [J]. Biol Chem,2003,278(33): 30869-30874.
[13]Yamane K, Taylor K and Kinsella TJ. Mismatch repair-mediated G2/Marrest by 6-thioguanine involves the ATR-CHK1 pathway [J]. Biochem Biophys Res Commun,2004, 318(1):297-302.
[14]Chen Z, Xiao Z, Chen J, et al. Human CHK1 expression is dispensable forsomatic cell death and critical for sustaining G2 DNA damage checkpoin[J].Mol Cancer Ther,2003, 2(6):543-548.
[15]Carrassa L, Broggini M, Erba E.et al.CHKl.but not CHK2,is involvedin the cellular response to DNA damaging agents:differential activity incells expressing or not p53[J].Cell Cycle, 2004,3(9):177-181.
[16]Wang Y, Decker SJ and Sebolt-Leopold J. Knockdown of CHK1, Wee land Mytl by RNA interference abrogates G2 checkpoint and induces apoptosis[J].Cancer Biol Ther,2004,3(3): 305-313.
[17]Zhu W, Chen Y, Dutta A. Replication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoin[J]. Mol Cell Biol,2004,24(16):7140-7150.
[18]Linzer DI, Levine AJ. Characterization of a54KDaltonCellular SV40 tumor antigen present in SV402t transformed cells and uninfected embryonal carcinoma cells [J]. Int J Cell,1979,17 (1):43-52.
[19]Concin N, Zeillinger C, Stimpfel M, et al. p53 dependent radioresistance in ovarian carcinomacell lines[J]. Cancer Lett,2000,150 (2):191-199.
[20]Monica Mangoni, Simonetta Bisanzi, Francesca Carozzi, et al. Association Between Genetic Polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT Genes and Radiosensitivity in Breast Cancer Patients [J]. International Journal of Radiation Oncology,2011,81(1):52-58.
[21]Elitsa Encheva, Sofia Deleva, Rositsa Hristova, et al. Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients[J]. Reports of Practical Oncology & Radiotherapy,2012,17(1):24-31.
[22]Hai Liu, XiaoNan Sun, Shuo Zhang, et al. The dominant negative mutant Artemis enhances tumor cell radiosensitivity [J]. Radiotherapy and Oncology,2011,101(1):66-72.
[23]F. Carrier, E. Diss, N. Nalabothula, et al.The Histone Deacetylase Inhibitor Vorinostat Induces Hyper-radiosensitivity (HRS) In P53 Wild Type Glioblastoma CellsThe Histone Deacetylase Inhibitor Vorinostat Induces Hyper-radiosensitivity (HRS) In P53 Wild Type Glioblastoma Cells [J]. International Journal of Radiation Oncology,2011,81(2): S747-S749.
[24]Tomasz Targowski, Karina Jahnz-Rozyk, Witold Owczarek, et al. Telomerase activity and serum levels of p53 protein as prognostic factors of survival in patients with advanced non-small cell lung cancer [J]. Respiratory Medicine,2010,104(9):1356-1361.
[25]Chikara K, Toshio Ⅰ, Roppei Y, et al. Prognostic factors after chemoradiotherapy for patients with inoperable esophageal squamous cell carcinoma[J]. Int J Hepato Gastroenterology,2006, 53 (69):366-371.
[26]Oohira G, Yamada S, Ochiai T, et al. Growth suppression ofesophageal squamous cell carcinoma induced by heavy carbon-ionbeams combined with p53 gene transfer [J]. Int J Oncol,2004,25 (3):563.
[27]高献书,卢付河,周志国等.p53基因对食管癌细胞的作用[J].中华放射肿瘤学杂志.2002,11(1)：34-37.
[28]Moon C, Oh Y, Roth J A. Current Status of Gene Therapy for Lung Cancer and Head and Neck Cancer [J]. Clin Cancer Res,2003,9(14):5055-5067.
[29]Semenza GL, Wang GL, A nuclear factor induced by hypoxia via denovo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation [J].Mol Cell Biochem,1992,12(12):5447-5454.
[30]Jiang BH, Rue E, Wang GL, et al. Dimerization,DNA binding, and transactivation properties of hypoxia-inducible factor[J]. Biol Chem,1996,271(30):17771-17778.
[31]Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor levels vary exponentially over a physiologically relevant range of 02 tension [J].Am J Physiol,1996,271(4): 1172-1180.
[32]Matsuyama T, Nakanishi K, Hayashi T, et al. Expression of hypoxia-inducible factor 1 alpha in esophageal squamous cell carcinoma [J]. Int J Cancer Sci,2005,96(3):176-182.
[33]Kimura S, Kitadai Y, Tanaka S, et al. Expression of hypoxia-inducible factor(HIF)-1 alpha is associated with vascular endothelial growth factor expression and tumor angiogenesis in human oesophageal squamous cell carcinoma[J].Eur J Cancer,2004,40(12):1904-1912.
[34]Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[J]. Proc Natl Acad Sci USA, 1995,92(12):5510-5514.
[35]Semenza GL. Regulation of mammalian homeostasis by hypoxia-in-ducible factor 1[J]. Annu Rea Cell Dev Biol,1999,15(8):551-578.
[36]Brown JM. The hypoxic cell:a targer for selective cancer therapy-eighteenth Bruce F. Cain Memorial Award lecture [J]. Cancer Res,1999,59(23):5863-5870.
[37]Vaupel P. Tumor microenvironmental physiology and its implication for radiation oncology[J].Semin Radiat Oncol,2004,14(3):198-206.
[38]Semenza GL, Targeting HIF-1 for cancer therapy [J].Nat Rev Cancer,2003,3(10):721-732.
[39]Garcia-Barros M, Paris F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis[J]. Science,2003,300(5622):1155-1159.
[40]Moeller BJ, Dewhirst MW. Raisingthebar:how HIF-1 helps determine tumor radiosensitivity [J]. Int J Cell Cycle,2004,3(9):1107-1110.
[41]Sohda M, Ishikawa H, Masuda N, et al. Pretreatment evaluation of combined HIF-1 a, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer[J]. Int J Cancer,2004,110(6):838-844.
[42]Garcia-Rarroos M, Pairs F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis [J]. Int J Science,2003,300(5622):1155-1159.
[43]金海林,施瑞华,朱宏等.RNA干扰HIF-1α对食管癌细胞株TE13的生物学特性的影响[J].世界华人消化杂志,2009,17(27)：2762-2766.
[44]张洪珍,祝淑钗,段昕波等.食管鳞癌组织中HIF-1α和survivin的表达及其对放射治疗预后的影响[J].肿瘤,2007,27(2)：139-141.
[45]Sanger DR, Vandewater L, Brown LF, et al. Vascular permeability factor(VPF, VEGF) in tumor biology[J]. Int J Cancer Metastasis Rew,1993,12(3-4):303-324.
[46]smith BD, Smith GL, Car D, et al. prognostic significance of vascular epithelial growth factor, protein levels in oral and orpharungeal squamous cell carcinoma[J].J Clin,2000.18 (10):2046-2052.
[47]Gorski DH, Beckett MA, Jaskowiak NT. et al. Bloekage of the vascular endothelial growth factor stress response increases the antitumor effeets of ionizing radiation[J].Cancer Res.1999,59 (14):3374-3378.
[48]Ohnuma Y, Toda M, Fujita M, et al. Blockade of an angiotensin type I receptor enhance effects of radiation on tumor growth and tumor associated angiogenesis by reducing vascular endothelial growth factor expression[J]. Int J Biomedicine & Pharmacotherapy,2009,63 (2): 136-145.
[49]Maria Grazia Fabrini, Franco Perrone, Mario De Liguoro, et al.A single-institutional brachytherapy experience in the management of esophageal cancer [J]. Brachytherapy,2012, 9 (2):185-191.
[50]封巍,祝淑钗,王玉祥反义血管内皮生长因子对食管癌细胞体外放射增敏作用[J].肿瘤学杂志,2007,13(3)：182-185.
[51]汪洋,何少琴,施学辉等.血管内皮生长因子和增殖细胞核抗原在食管癌放射治疗中的临床意义[J].中华放射肿瘤学杂志,2002,11(4)：248-251.
[52]Shen MR, Jones IM, Mohrenweiseer H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair gene in healthy humans [J]. Int J Cancer Res, 1998,58(4):604-608.
[53]Xing D, Qi J, Miao X, et al. Polymorphisms of DNA repair genes XRCC1 and XPD and their associations with risk of esophageal squamous cell carcinoma in a Chinese population[J] Int J Cancer,2002(5),100:600-605.
[54]RobertsonKA, Bullock HA, Xu Y, et al. Altered expression of Apel/ref-1 in germ cell tumors and over expression in NT2 cells confers resistance to bleomycin and radiation[J]. Int J Cancer Res,2001,61(5):2220-2225.
[55]Zdzienicka MZ, Van Schans GP, Natarajan AT, et al. A Chinese hamster ovary cellmutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatin exchanges[J]. Int J Mutagenesis,1992,7(4):265-269.
[56]Fernet M, HallJ. Genetic biomarkers of the rapeutic radiation sensitivity [J]. Int J DNA Repair(Amst),2004,3(8-9):1237-1243.
[57]Madeleen J. U, Lilly-Ann V, Maarten F, et al.Recordings of consultations are beneficial in the transition from curative to palliative cancer care:A pilot-study in patients with oesophageal or head and neck cancer [J]. European Journal of Oncology Nursing,2012, 16(2):109-114.
[58]Guo W, Zhao YP, Jiang YG, et al. ZNRD1 might mediate UV irradiation related DNA damage and repair in human esophageal cancer cells by regulation of ERCC1[J]. Int J Diseases of the Esophagus,2008,21(8):730-736.
[59]王永坤,吴克瑾,林清等.乏氧与放射敏感性的研究进展[J].外科理论与实践, 2011,16(1)：89-91.
[60]党亚正,费晋秀.肿瘤乏氧细胞与放射治疗[J].现代肿瘤医学,2008,16(3)：492-496.
[61]邱娇娇,郑文斐,佐满珍等.甘氨双唑钠和塞来昔布与肿瘤的研究进展[J].肿瘤学杂志,2011,17(3)：225-227.
[62]陈昱明,张纯,成奇峰.甘氨双唑钠对中晚期宫颈癌放射治疗增敏作用的临床研究[J].南方医科大学学报,2007,27(11)：1789-1794.
[63]孙菁,王凤英.甘氨双哇钠对食管癌放射增敏作用的生存分析[J].中华放射肿瘤学杂志,2006,15(4)：297-300.
[64]万志龙,蔡晶,杨燕光等.甘氨双唑钠(CMNa)对食管癌放射治疗增敏作用的临床研究[J].肿瘤基础与临床,2007,20(2)：64-65.
[65]顾红芳,万志龙,刘向阳,等.甘氨双唑钠对食管癌放射治疗增敏作用的研究[J].中国肿瘤,2005,14(12)：825-829.
[66]陆嘉德,Lee Khai Mun局部中晚期食管癌的多学科整合治疗[J].中国癌症杂志,2008,18(5)：333-338.
[67]苗劲柏,申文江,张仁尧,等.局部晚期非小细胞肺癌联合放化疗及放射增敏研究[J].国外医学·临床放射学分册,2005,28(1)：63-65.
[68]卢金利,杨芳.宫颈癌中顺铂放疗增敏的临床药代动力学机制[J].南方医科大学学报,2006,26(8)：1170-1172.
[69]吴剑,戴慧,翁欣然等.小剂量顺铂对中晚期食管癌放疗增敏作用的近期疗效观察[J].中国肿瘤临床,2006,33(13)：768-770.
[70]刘辉,梅步铎,何俊洪.放疗加卡铂增敏治疗食管癌108例的前瞻性随机研究[J].临床肿瘤学杂志,1997,2(3)：25-26.
[71]Sai H, Mitsumori M,Yamauchi C, et al. Concurrent chemoradiotherapy for esophageal cancer:comparison between in termittent standa rd dose cisplatin wit h 5-fluorouracil and daily low-dose cisplatin with continuous in fusion of 5-fluorouracil [J]. In t J C lin Oncol,2004,9(3):149-153.
[72]Kim ES, Khuri FR. Docetaxel and radiation as combined-modality therapy [J]. Oncology (Williston Park),2002, (6Suppl6):97-105.
[73]樊锐太,陈淅娟,孙浩等.多西紫杉醇对中晚期食管癌放疗增敏作用的临床观察[J].郑州大学学报(医学版),2009,44(3)：481-485.
[74]Pasini F, deManzoni G, Pedrazzani C, et al. High pathological response rate in locally advanced esophageal cancer after neoadjuvant combined modality therapy:dose finding of a weekly chemotherapy schedule with protracted venous infusion of 5-fluorouracil and dose escalation of cisplation, docetaxel and concurrent radiotherapy[J]. Ann Onco,2005,16 (7): 1133.
[75]蒋晓东,于金明,吴瑾等.多西他赛乏氧增敏放射治疗食管癌的临床研究[J].实用肿瘤杂志,2010,25(4)：436-440.
[76]Simoens C, Lardon F. Comparative study of the radiosensitizing and cell cycle effects of vinflunine and vinorelbine, in vitro[J]. BMC Cancer,2008,29(8):5.
[77]Bidoli P, Slani S, Candis D, et al. Single-agent chemotherapy with vinorelbine for pretreated or metastatic squamous cell carcinoma of the esophagus[J]. Tumori.2001,87(5):299-302.
[78]Conroy T, Etienne PL. Adenis A, et al. Vinorelbine and cisplatin in metastatic squamous cell carcinoma of the esophagus:response, toxility.quality of life and survival[J]. Ann Oncol, 2002,13(5):721-729.
[79]孙燕.内科肿瘤学[M].北京：人民卫生出版社,2001,426-427.
[80]Ralph Winterhalder, Simone Deschler-Marini, Christine Landmann, et al.Vinorelbine plus low-dose cisplatinum with concomitant radiotherapy for the treatment of locally advanced or inoperable non-metastasized non-small-cell lung cancer (stage Ⅰ-ⅢB):a phase Ⅱ study [J]. Radiotherapy and Oncology,2004,73(3):321-324.
[81]李志琳,马家宝,吴双等.长春瑞滨对中晚期食管癌放疗增敏作用的疗效观察[J].四川医学,2008,29(11)：1485-1487.
[82]Janmaat ML, Gallegos-ruiz MI, Rodriguez JA, et al. Predictive factors for outcome in a phase II study of gefitinib in second-line treatment of advanced esophageal cancer patients[J]. J Clin Oncol,2006,24(10):1612-1619.
[83]Rodriguez CP, Adelstein DJ, Rice TW, et al. A Phase II study of perioperative concurrent chemotherapy, gefitinib, and hyperfractionated radiation followed by maintenance gefitinib in locoregionally advanced esophagus and gastroesophageal junction cancer[J]. J Thorac Oncol,2010,5(2):229-235.
[84]Safran H, Suntharalingam M, Dipetrillo T, et al. Cetuximab with concurrent chemoradiation for esophagogastric cancer:Assessment of toxicity[J]. Int J Radiat Oncol Biol Phys,2008, 70(2):391-395.
[85]Lorenzen S, Schuster T, Porschen R, et al. Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus:a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie[J]. Ann Oncol,2009,20(10):1667-1673.
[2]邹小农.食管癌流行病学[J].中华肿瘤防治杂志,2006,13(18)：18-21.
[3]韩企夏,施学辉.食管癌[A].见汤钊猷.现代肿瘤学[M].第二版.上海：上海医科大学出版社,2000：691.
[4]蔡凯,胡联民,陈刚,等.后程加速超分割三维适形放疗食管癌的临床研究[J].实用肿瘤杂志,2011,26(6)：631-634.
[5]殷慰伯.食管癌的放射治疗进展[J].实用肿瘤杂志,2006,21(2)：99-108.
[6]肖泽芬,食管癌[A].见殷蔚伯,谷铣之.肿瘤放射治疗学[M].第三版.北京：中国协和医科大学出版社,2002：610.
[7]KARIM A, MCCARTHY K, JAWAHAR A, et al. Differential cycloooxygenase-2 enzyme expression in radiosensitive versus radioresistant glioblastoma multiforme cell line[J]. AnticancerRes,2005,25:675-679.
[8]NIX P, LIND M, GREENMAN J, et al. Expression of cox-2 protein in radioresistant laryngeal cancer[J].Ann Oncol,2004,15:797-801.
[9]TERAKADO N, SHINTANI S, YANO J, et al. Overexpression of cyclooxygenasse-2 is associated with radioresistance in oral squamous cell carcinoma [J].Oral Oncol,2004,40:383-389.
[10]Liu Y, Zhao S, Liu G, et al. The value of cyclooxygenasse-2 to predict the effect of radiotherapy in nasopharyngeal carcinoma [J]. Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2007,21 (5):199-202.
[11]谢传高,王兴鹏.环氧合酶—2与消化系统肿瘤[J].国外医学.消化系疾病分册,2002,22(1)：20-23.
[12]Karim A, McCarthy K, Jawahar, et al. Differential eyelooxygenas-2 enzyme expression in radiosensitive Versus radioresistant glioblastoma multiform cell lines[J].Antieaneer Res,2005,25 (1B):675-679.
[13]Nix P, Lind M, Greenman J, et al. Expression of COX-2 protein in radiosensistant laryngeal cancer [J].Ann Oncol,2004,15(5):797-801.
[14]Terakado N, Shintani S, Yano J, et al. Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma [J]. Oral Oncol,2004,40(4):383-389.
[15]Huang WZ, Fu JH, Wang DK, et al. Overexpress of cyclooxygenase-2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients [J]. Dis Esophagus 2008,21(8):679-684.
[16]殷蔚伯,谷铣之.肿瘤放射治疗学.[M].北京：中国协和医科大学出版社,2002 610-611.
[17]刘彤华,夏求洁,刘复生等.消化道疾病.白希清.病理学(第二版)[M]北京：科学出版社，1992：215-217.
[18]Williams CS, Dubois RN. Prostaglandin endoperoxide synthase:why two isoform? [J]Am J physiol,1996,270(3):393-400.
[19]Williams CS, Tsujii M, Reese J, et al. Host cyclooxygenase-2 modulates carcinoma growth [J] J ClinInvest,2000,105(11):1589-1594.
[20]PENG JP, CHANG HC, HWANG CF, et al. Overepression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis[J].Oral Oncol,2005,41:903-908.
[21]KARIM A, MCCARTHY K, JAWAHAR A, et al. Differential cycloooxygenase-2 enzyme expression in radiosensitive versus radioresistant glioblastoma multiforme cell line[J]. Anticancer Res,2005,25:675-679.
[22]Kuipers GK, Radiosensitization of human glioma cells by cyelooxygenase-2 inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration [J].Int J Radiat Bio,2007,183 (10):677-685.
[23]朱辉,王慧娟,贺春雨,等.食管鳞状细胞癌组织中COX-2和MGMT蛋白的表达.[J].郑州大学学报：医学版,2011,46(1)：125-128.
[24]Lagorec C, Paraf F, Vidaud D, et al. cyelooxygenase-2 is expressed frequently and early in Barrett's esophagus and assoiated adenocarcinomar [J]. His topathology,2003,42 (5): 457-460.
[25]燕春艳,涂玉林,钟苗,等Bcl-2siRNA的设计合成及其对HL-60 bcl-2基因表达的干扰作用[J].白血病·淋巴瘤,2006,15(5)：323-326.
[26]Hao JH, Gu QL, Liu BY, et al. Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA [J]. Chin Med J(Engl),2007,120(23): 2105-2111.
[27]袁月秀,王言奎,罗兵等.化学合成siRNA对宫颈癌细胞SiHa中E6基因表达的抑制作用[J].中国癌症杂志,2007,12(17)：914-919.
[28]Tchurikov NA, Chistyakova LG, Zavilgelsky GB, et al. Gene-specific silencing by expression of parallel complementary RNA in Escherichia coli. J.Biol.Chem,2000,275 (34):26523-26529.
[29]Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature,2001,409(6818):363-366.
[30]Kihara A, Kabeya Y, Ohsumi Y, et al. Beclinphosphatidy linositool 3-kinase complex functions at the trans-Golgi network. EMBO Rep,2001,2:330-335.
[31]Siddhesh D, David G, Diane J. DNA-based therapeutics and DNA delivery systems:A comprehensive review [J].The AAPS J,2005,7(1):E61-E77.
[32]Castanotto D, Li H, Rossi JJ. Function siRNA expression from transfected PCR products. RNA,2002,8(11):1454-1460.
[33]PENG JP, CHANG HC, HWANG CF, et al. Over expression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis [J]. Oral Oncol,2005, 41(9):903-908.
[34]DAISUKE S.DANIEL V,HIDEKAZU K, et al. Increasing cyclooxygenase-2 (cox-2) gene expression in progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2[J]. Int J Cancer,2006,119(4):765-770.
[35]ChantrainCF, Henriet P, Jodele S, et al..Mechanisms of Pericyte recruitment in tumor angiogenesis:a new rolefor metalloproteinases[J].Eur J Cancer,2006,42(3):310-318.
[36]Son KN> Hwang J, Kwon BS, et al. Human CC chemokine CCL23 enhances expression of matrixmetalloproteinase-2 and invasion of vascular endothelial cells [J]. Biochem Biophys Res Cornrnun,2006,340(2):498-504.
[37]Edwards JG, Mclaren J, Jonea JL,et al. Matrix metalloproteinases-2and9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura[J].Br J Caneer,2003,88(10):1553-1559.
[38]Mook OR,Freder Iksw M,Van Noorden CJ. The role of gelatinases in colorectal cancer progression and metastasis[J].Biochim BiophysActa,2004,1705(2):69-89.
[39]Patel BR,Shan PM, Rawal UM,et al. Activation of MMP2 and MMP9 in patients with oral squamous cell carcinoma[J].Surg Oncol,2005,90(3):81-88.
[40]John A, Tuszynski GThe role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis[J].Pathol Oncol Res,2001,7(1):14-23.
[41]Yu XF, Han ZC. Matrix metalloproteinases in bone mmarrow:roles of gelatinases in physiological hematopoiesis and hematopoietic malignancies [J]. Histopathol,2006,21(5): 519-531.
[42]Adams JM,Cory S.The bcl-2 protein family:arbiters of cell survival. Science,1998,281 (5381):1322-1326.
[43]O'Neill J, Manion M, Schwartz P, et al. Promises and challenges of targeting Bcl-2 anti-apoptotic proteins for cancer therapy. Biochim Biophys Acta,2004,1705(l):43-51.
[44]Leung RK, Whittaker PA. RNA interference:from gene silencing to gene-specific therapeutics. Pharmacol Ther,2005,107(2)222-239.
[45]Kirkin V, Joos S, Zornig M. The role of Bcl-2 falllily members in tumor genesis. Biochim Biophys Acta,2004,1644(2-3):229-249.
[46]McCabe ML, Dlamini Z. The molecular mechanisms of oesophageal cancer. Int Immunopharmacol,2005,5(7-8):1113-1130.
[47]刘军叶,郭鹞,郭国祯.Bcl-2基因特异性小干涉RNA增强食管癌细胞辐射敏感性的实验研究[J].国际放射医学核医学杂志,2006,30(2)：110-113.
[48]Hao JH, Gu QL, Liu BY, et al. Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA. Chin Med J,2007,120(23):2105-2111.
[49]Hara T, Omura-Minamisawa M, Chao C, et al. Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 over expressing radioresistant tumor cell. Int J Radiat Oncol Phy,2005,61:517-528.
[50]Scopa CD, Vagianos C, Kardamakis D, et al.Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with rectal cancer. Appl Immunohistochem Mol Morphol 2001，9(4):329-334.
[51]艾义国，饶旭光.马立民，等.食管癌组织环氧化酶-2和Bcl-2蛋白的表达与临床病理的关系研究[J].汕头大学医学院学报,2009.1(22)：21-24.
[52]Kim D, Kim S, KoH, et al.Akt/PKB promotes cancer cell invasion via increased motility and metalbproteinase production [J]. FASEB J 2001,15(11):1953-1956.
[53]Zhong H, Chiles K, Feldser D,et al. Modulation of hypoxia inducble factor 1 alpha expression by the epidermal growth factor/phosphatidy linositol3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells implications for tumor angiogenesis and therapeutics[J].cance Res,2000,60(6):1541-1544.
[54]冯学泉,王金环,徐新女,等.碱性成纤维细胞生长因子小干扰RNA与人类免疫缺陷病毒1型病毒蛋白R基因对裸鼠移胶质瘤的抑制作用[J].中华肿瘤杂志,2010,32(10)：725-728.
[55]Chen Yue, Xiong Qing-feng, He Ling, et al.188Re-DTPA-DG preparation and its experimental imaging in MCF-7 tumor-bearing nude mice[J].Chinese Journal of Nuclear Medicine,2007,27(02),23-25.
[56]Jun Wang, Chun Han, Xiao-Ning Li, et al. Short-term efficacy of intensity-modulated radiotherapy on esophageal carcinoma[J] Chinese Journal of Cancer,2009,28(11):35-36.
[57]Cheng Hui-Hua, Fu Zhi-Chao, Lin Gui-Shan,et al. Sensitivity enhancement of sodium glycididazole in re-radiotherapy against local recurrent esophageal carcinoma after radical radiotherapy [J]. Journal of the Fourth MilitaryMedical University,2007,28(11):35-36.
[58]Peng JP, Chang HC, Hwang CF, et al. Over expression of cyclooxygenase-2 in nasopharyngeal carcinoma and association with lymph node metastasis[J].Oral Oncol,2005,41:903-908.
[59]Daisuke S,Daniel V, Hidekazu K, et al. Increasing cyclooxygenase-2 (cox-2) gene expression in progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2[J]. Int J Cancer,2006,119:765-770.
[1]全国肿瘤防治研究办公室,卫生部卫生统计信息中心编.中国试点市、县恶性肿瘤的发病与死亡.中国医药科技出版社,2002年,第二卷(1993-1997年),p21-34.
[2]Wang GQ, Jiao, Chang FB, et al. Long-term tesults of operation for 420patients with Early squamous cell esophageal carcinoma discovered by screening[J].Ann Thorac Surg,2004,77 (5):1740-1744.
[3]Russell E. Brown, Abbas E. Abbas, Susan Ellis, et al. A Prospective Phase II Evaluation of Esophageal Stenting for Neoadjuvant Therapy for Esophageal Cancer:Optimal Performance and Surgical Safety [J]. Journal of the American College of Surgeons,2011,212(4):582-588.
[4]肖泽芬,食管癌.见殷蔚伯,谷铣之主编.肿瘤放射治疗学.2002年,第三版,中国协和医科大学出版社,p598-622.
[5]Ting Liu, Hongxia Chen, Hongtai Kim, et al. RAD18-BRCTx interaction is required for efficient repair of UV-induced DNA damage [J]. DNA Repair,2012,11 (2):131-138.
[6]Chen YH, Sanchez Y. CHK1 in the DNA damage response:conserved roles from yeasts to mammals. DNA Repair (Amst),2004,3(8-9):1025-1032.
[7]Ahn J, Urist M, Prives C.The CHK2 protein kinase. DNA Repair (Amst),2004,3 (8-9): 1039-1047.
[8]Rahman S.Z.S, Theresa A.L, Jetze J. T. Synthesis and evaluation of debromohymenialdisine-derived Chk2 inhibitors [J]. Bioorganic & Medicinal Chemistry,2012,20(4):1474-1481.
[9]祝淑钗,王玉祥,李任等shRNA转染对食管癌细胞TE13中CHK1和CHK2表达以及照射后G2/M期阻滞的影响[J].癌症,2007,26(2)：127-131.
[10]Gatei M, Sloper K, Sorensen C, et al. Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of CHK1 on Ser-317 in response to ionizing radiation [J]. Biol Chem,2003,278(17):14806-14811.
[11]Xiao Z, Chen Z, Gunasekera AH, et al.CHKl mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents[J].BiolChem,2003,278(24):21767-2173.
[12]Wang X, Khadpe J, Hu B, et al. An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells [J]. Biol Chem,2003,278(33): 30869-30874.
[13]Yamane K, Taylor K and Kinsella TJ. Mismatch repair-mediated G2/Marrest by 6-thioguanine involves the ATR-CHK1 pathway [J]. Biochem Biophys Res Commun,2004, 318(1):297-302.
[14]Chen Z, Xiao Z, Chen J, et al. Human CHK1 expression is dispensable forsomatic cell death and critical for sustaining G2 DNA damage checkpoin[J].Mol Cancer Ther,2003, 2(6):543-548.
[15]Carrassa L, Broggini M, Erba E.et al.CHKl.but not CHK2,is involvedin the cellular response to DNA damaging agents:differential activity incells expressing or not p53[J].Cell Cycle, 2004,3(9):177-181.
[16]Wang Y, Decker SJ and Sebolt-Leopold J. Knockdown of CHK1, Wee land Mytl by RNA interference abrogates G2 checkpoint and induces apoptosis[J].Cancer Biol Ther,2004,3(3): 305-313.
[17]Zhu W, Chen Y, Dutta A. Replication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoin[J]. Mol Cell Biol,2004,24(16):7140-7150.
[18]Linzer DI, Levine AJ. Characterization of a54KDaltonCellular SV40 tumor antigen present in SV402t transformed cells and uninfected embryonal carcinoma cells [J]. Int J Cell,1979,17 (1):43-52.
[19]Concin N, Zeillinger C, Stimpfel M, et al. p53 dependent radioresistance in ovarian carcinomacell lines[J]. Cancer Lett,2000,150 (2):191-199.
[20]Monica Mangoni, Simonetta Bisanzi, Francesca Carozzi, et al. Association Between Genetic Polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT Genes and Radiosensitivity in Breast Cancer Patients [J]. International Journal of Radiation Oncology,2011,81(1):52-58.
[21]Elitsa Encheva, Sofia Deleva, Rositsa Hristova, et al. Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients[J]. Reports of Practical Oncology & Radiotherapy,2012,17(1):24-31.
[22]Hai Liu, XiaoNan Sun, Shuo Zhang, et al. The dominant negative mutant Artemis enhances tumor cell radiosensitivity [J]. Radiotherapy and Oncology,2011,101(1):66-72.
[23]F. Carrier, E. Diss, N. Nalabothula, et al.The Histone Deacetylase Inhibitor Vorinostat Induces Hyper-radiosensitivity (HRS) In P53 Wild Type Glioblastoma CellsThe Histone Deacetylase Inhibitor Vorinostat Induces Hyper-radiosensitivity (HRS) In P53 Wild Type Glioblastoma Cells [J]. International Journal of Radiation Oncology,2011,81(2): S747-S749.
[24]Tomasz Targowski, Karina Jahnz-Rozyk, Witold Owczarek, et al. Telomerase activity and serum levels of p53 protein as prognostic factors of survival in patients with advanced non-small cell lung cancer [J]. Respiratory Medicine,2010,104(9):1356-1361.
[25]Chikara K, Toshio Ⅰ, Roppei Y, et al. Prognostic factors after chemoradiotherapy for patients with inoperable esophageal squamous cell carcinoma[J]. Int J Hepato Gastroenterology,2006, 53 (69):366-371.
[26]Oohira G, Yamada S, Ochiai T, et al. Growth suppression ofesophageal squamous cell carcinoma induced by heavy carbon-ionbeams combined with p53 gene transfer [J]. Int J Oncol,2004,25 (3):563.
[27]高献书,卢付河,周志国等.p53基因对食管癌细胞的作用[J].中华放射肿瘤学杂志.2002,11(1)：34-37.
[28]Moon C, Oh Y, Roth J A. Current Status of Gene Therapy for Lung Cancer and Head and Neck Cancer [J]. Clin Cancer Res,2003,9(14):5055-5067.
[29]Semenza GL, Wang GL, A nuclear factor induced by hypoxia via denovo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation [J].Mol Cell Biochem,1992,12(12):5447-5454.
[30]Jiang BH, Rue E, Wang GL, et al. Dimerization,DNA binding, and transactivation properties of hypoxia-inducible factor[J]. Biol Chem,1996,271(30):17771-17778.
[31]Jiang BH, Semenza GL, Bauer C, et al. Hypoxia-inducible factor levels vary exponentially over a physiologically relevant range of 02 tension [J].Am J Physiol,1996,271(4): 1172-1180.
[32]Matsuyama T, Nakanishi K, Hayashi T, et al. Expression of hypoxia-inducible factor 1 alpha in esophageal squamous cell carcinoma [J]. Int J Cancer Sci,2005,96(3):176-182.
[33]Kimura S, Kitadai Y, Tanaka S, et al. Expression of hypoxia-inducible factor(HIF)-1 alpha is associated with vascular endothelial growth factor expression and tumor angiogenesis in human oesophageal squamous cell carcinoma[J].Eur J Cancer,2004,40(12):1904-1912.
[34]Wang GL, Jiang BH, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[J]. Proc Natl Acad Sci USA, 1995,92(12):5510-5514.
[35]Semenza GL. Regulation of mammalian homeostasis by hypoxia-in-ducible factor 1[J]. Annu Rea Cell Dev Biol,1999,15(8):551-578.
[36]Brown JM. The hypoxic cell:a targer for selective cancer therapy-eighteenth Bruce F. Cain Memorial Award lecture [J]. Cancer Res,1999,59(23):5863-5870.
[37]Vaupel P. Tumor microenvironmental physiology and its implication for radiation oncology[J].Semin Radiat Oncol,2004,14(3):198-206.
[38]Semenza GL, Targeting HIF-1 for cancer therapy [J].Nat Rev Cancer,2003,3(10):721-732.
[39]Garcia-Barros M, Paris F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis[J]. Science,2003,300(5622):1155-1159.
[40]Moeller BJ, Dewhirst MW. Raisingthebar:how HIF-1 helps determine tumor radiosensitivity [J]. Int J Cell Cycle,2004,3(9):1107-1110.
[41]Sohda M, Ishikawa H, Masuda N, et al. Pretreatment evaluation of combined HIF-1 a, p53 and p21 expression is a useful and sensitive indicator of response to radiation and chemotherapy in esophageal cancer[J]. Int J Cancer,2004,110(6):838-844.
[42]Garcia-Rarroos M, Pairs F, Cordon-Cardo C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis [J]. Int J Science,2003,300(5622):1155-1159.
[43]金海林,施瑞华,朱宏等.RNA干扰HIF-1α对食管癌细胞株TE13的生物学特性的影响[J].世界华人消化杂志,2009,17(27)：2762-2766.
[44]张洪珍,祝淑钗,段昕波等.食管鳞癌组织中HIF-1α和survivin的表达及其对放射治疗预后的影响[J].肿瘤,2007,27(2)：139-141.
[45]Sanger DR, Vandewater L, Brown LF, et al. Vascular permeability factor(VPF, VEGF) in tumor biology[J]. Int J Cancer Metastasis Rew,1993,12(3-4):303-324.
[46]smith BD, Smith GL, Car D, et al. prognostic significance of vascular epithelial growth factor, protein levels in oral and orpharungeal squamous cell carcinoma[J].J Clin,2000.18 (10):2046-2052.
[47]Gorski DH, Beckett MA, Jaskowiak NT. et al. Bloekage of the vascular endothelial growth factor stress response increases the antitumor effeets of ionizing radiation[J].Cancer Res.1999,59 (14):3374-3378.
[48]Ohnuma Y, Toda M, Fujita M, et al. Blockade of an angiotensin type I receptor enhance effects of radiation on tumor growth and tumor associated angiogenesis by reducing vascular endothelial growth factor expression[J]. Int J Biomedicine & Pharmacotherapy,2009,63 (2): 136-145.
[49]Maria Grazia Fabrini, Franco Perrone, Mario De Liguoro, et al.A single-institutional brachytherapy experience in the management of esophageal cancer [J]. Brachytherapy,2012, 9 (2):185-191.
[50]封巍,祝淑钗,王玉祥反义血管内皮生长因子对食管癌细胞体外放射增敏作用[J].肿瘤学杂志,2007,13(3)：182-185.
[51]汪洋,何少琴,施学辉等.血管内皮生长因子和增殖细胞核抗原在食管癌放射治疗中的临床意义[J].中华放射肿瘤学杂志,2002,11(4)：248-251.
[52]Shen MR, Jones IM, Mohrenweiseer H. Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair gene in healthy humans [J]. Int J Cancer Res, 1998,58(4):604-608.
[53]Xing D, Qi J, Miao X, et al. Polymorphisms of DNA repair genes XRCC1 and XPD and their associations with risk of esophageal squamous cell carcinoma in a Chinese population[J] Int J Cancer,2002(5),100:600-605.
[54]RobertsonKA, Bullock HA, Xu Y, et al. Altered expression of Apel/ref-1 in germ cell tumors and over expression in NT2 cells confers resistance to bleomycin and radiation[J]. Int J Cancer Res,2001,61(5):2220-2225.
[55]Zdzienicka MZ, Van Schans GP, Natarajan AT, et al. A Chinese hamster ovary cellmutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatin exchanges[J]. Int J Mutagenesis,1992,7(4):265-269.
[56]Fernet M, HallJ. Genetic biomarkers of the rapeutic radiation sensitivity [J]. Int J DNA Repair(Amst),2004,3(8-9):1237-1243.
[57]Madeleen J. U, Lilly-Ann V, Maarten F, et al.Recordings of consultations are beneficial in the transition from curative to palliative cancer care:A pilot-study in patients with oesophageal or head and neck cancer [J]. European Journal of Oncology Nursing,2012, 16(2):109-114.
[58]Guo W, Zhao YP, Jiang YG, et al. ZNRD1 might mediate UV irradiation related DNA damage and repair in human esophageal cancer cells by regulation of ERCC1[J]. Int J Diseases of the Esophagus,2008,21(8):730-736.
[59]王永坤,吴克瑾,林清等.乏氧与放射敏感性的研究进展[J].外科理论与实践, 2011,16(1)：89-91.
[60]党亚正,费晋秀.肿瘤乏氧细胞与放射治疗[J].现代肿瘤医学,2008,16(3)：492-496.
[61]邱娇娇,郑文斐,佐满珍等.甘氨双唑钠和塞来昔布与肿瘤的研究进展[J].肿瘤学杂志,2011,17(3)：225-227.
[62]陈昱明,张纯,成奇峰.甘氨双唑钠对中晚期宫颈癌放射治疗增敏作用的临床研究[J].南方医科大学学报,2007,27(11)：1789-1794.
[63]孙菁,王凤英.甘氨双哇钠对食管癌放射增敏作用的生存分析[J].中华放射肿瘤学杂志,2006,15(4)：297-300.
[64]万志龙,蔡晶,杨燕光等.甘氨双唑钠(CMNa)对食管癌放射治疗增敏作用的临床研究[J].肿瘤基础与临床,2007,20(2)：64-65.
[65]顾红芳,万志龙,刘向阳,等.甘氨双唑钠对食管癌放射治疗增敏作用的研究[J].中国肿瘤,2005,14(12)：825-829.
[66]陆嘉德,Lee Khai Mun局部中晚期食管癌的多学科整合治疗[J].中国癌症杂志,2008,18(5)：333-338.
[67]苗劲柏,申文江,张仁尧,等.局部晚期非小细胞肺癌联合放化疗及放射增敏研究[J].国外医学·临床放射学分册,2005,28(1)：63-65.
[68]卢金利,杨芳.宫颈癌中顺铂放疗增敏的临床药代动力学机制[J].南方医科大学学报,2006,26(8)：1170-1172.
[69]吴剑,戴慧,翁欣然等.小剂量顺铂对中晚期食管癌放疗增敏作用的近期疗效观察[J].中国肿瘤临床,2006,33(13)：768-770.
[70]刘辉,梅步铎,何俊洪.放疗加卡铂增敏治疗食管癌108例的前瞻性随机研究[J].临床肿瘤学杂志,1997,2(3)：25-26.
[71]Sai H, Mitsumori M,Yamauchi C, et al. Concurrent chemoradiotherapy for esophageal cancer:comparison between in termittent standa rd dose cisplatin wit h 5-fluorouracil and daily low-dose cisplatin with continuous in fusion of 5-fluorouracil [J]. In t J C lin Oncol,2004,9(3):149-153.
[72]Kim ES, Khuri FR. Docetaxel and radiation as combined-modality therapy [J]. Oncology (Williston Park),2002, (6Suppl6):97-105.
[73]樊锐太,陈淅娟,孙浩等.多西紫杉醇对中晚期食管癌放疗增敏作用的临床观察[J].郑州大学学报(医学版),2009,44(3)：481-485.
[74]Pasini F, deManzoni G, Pedrazzani C, et al. High pathological response rate in locally advanced esophageal cancer after neoadjuvant combined modality therapy:dose finding of a weekly chemotherapy schedule with protracted venous infusion of 5-fluorouracil and dose escalation of cisplation, docetaxel and concurrent radiotherapy[J]. Ann Onco,2005,16 (7): 1133.
[75]蒋晓东,于金明,吴瑾等.多西他赛乏氧增敏放射治疗食管癌的临床研究[J].实用肿瘤杂志,2010,25(4)：436-440.
[76]Simoens C, Lardon F. Comparative study of the radiosensitizing and cell cycle effects of vinflunine and vinorelbine, in vitro[J]. BMC Cancer,2008,29(8):5.
[77]Bidoli P, Slani S, Candis D, et al. Single-agent chemotherapy with vinorelbine for pretreated or metastatic squamous cell carcinoma of the esophagus[J]. Tumori.2001,87(5):299-302.
[78]Conroy T, Etienne PL. Adenis A, et al. Vinorelbine and cisplatin in metastatic squamous cell carcinoma of the esophagus:response, toxility.quality of life and survival[J]. Ann Oncol, 2002,13(5):721-729.
[79]孙燕.内科肿瘤学[M].北京：人民卫生出版社,2001,426-427.
[80]Ralph Winterhalder, Simone Deschler-Marini, Christine Landmann, et al.Vinorelbine plus low-dose cisplatinum with concomitant radiotherapy for the treatment of locally advanced or inoperable non-metastasized non-small-cell lung cancer (stage Ⅰ-ⅢB):a phase Ⅱ study [J]. Radiotherapy and Oncology,2004,73(3):321-324.
[81]李志琳,马家宝,吴双等.长春瑞滨对中晚期食管癌放疗增敏作用的疗效观察[J].四川医学,2008,29(11)：1485-1487.
[82]Janmaat ML, Gallegos-ruiz MI, Rodriguez JA, et al. Predictive factors for outcome in a phase II study of gefitinib in second-line treatment of advanced esophageal cancer patients[J]. J Clin Oncol,2006,24(10):1612-1619.
[83]Rodriguez CP, Adelstein DJ, Rice TW, et al. A Phase II study of perioperative concurrent chemotherapy, gefitinib, and hyperfractionated radiation followed by maintenance gefitinib in locoregionally advanced esophagus and gastroesophageal junction cancer[J]. J Thorac Oncol,2010,5(2):229-235.
[84]Safran H, Suntharalingam M, Dipetrillo T, et al. Cetuximab with concurrent chemoradiation for esophagogastric cancer:Assessment of toxicity[J]. Int J Radiat Oncol Biol Phys,2008, 70(2):391-395.
[85]Lorenzen S, Schuster T, Porschen R, et al. Cetuximab plus cisplatin-5-fluorouracil versus cisplatin-5-fluorouracil alone in first-line metastatic squamous cell carcinoma of the esophagus:a randomized phase II study of the Arbeitsgemeinschaft Internistische Onkologie[J]. Ann Oncol,2009,20(10):1667-1673.