【作者】 吴慧；

【导师】 樊青霞；

【作者基本信息】 郑州大学 ， 肿瘤学， 2012， 博士

【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。更多还原

【Abstract】 IntroductionEsophageal 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.更多还原