| 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.
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