Study On The Expression Of Vascular Endothelial Growth Factor (VEGF) In Esophageal Carcinoma And The Effect Of Antisene VEGF On The Radiation Sensitivity Of Esophageal Cancer Cell In Vivo & In Vitro

Esophageal cancer (EC) is one of the most common malignant diseases in china. The disease are charactered with poor prognosis and higher metastasis and high mortality. Although the different available multi-modalities of therapies have currently been used in the treatment for this disease, the prognosis of esophageal cancer has not been markedly improved. Radiotherapy is an effective modality for middle-advanted esophageal cancer, with over half of all esophageal cancer patients receiving radiation therapy during their course of treatment. Although widely used, necessarily to improve the cure rate by radiation therapy alone. With development of the basic research on oncological fields, it was found the progression of tumors depends on angiogenesis. The vascular endothelial growth factor (VEGF) is an important factor which elicits the angiogensis in tumors. It takes an important role in the growth,differentiation,metastasis,proliferation of tumors. Some recent preclinical studies suggest that the combination of radiotherapy and angiogenic blockade enhance the therapeutic ratio of ionizing radiation by targeting both tumor cells and tumor vessels. At present, there is great interest in combining antiangiogenic strategy with conventional cytotoxic therapies such as radiotherapy to improve therapeutic gain. The mechanisms by which tumor response to radiation is enhanced by these new agents, however, are not currently understood. By now, combining antisense VEGF and radiotherapy in esophageal cancer treating have not been reported. This study contains three parts of work:The first,the relationships between the expression of VEGF in esophageal cancer The second, The effect of antisense VEGF on the radiation sensitivity of esophageal cancer cells in vitro.The third, The effect of antisense VEGF on radiation sensitivity esophageal cancer in vivo. Part Ⅰ: Study on the expression of VEGF in esophageal cancer Objective: To study expression of VEGF in esophageal cancer. Methods:Detecting the expression of VEGF in esophageal cancer cell lines (TE-1,TE-13,ECA109)with immunohistochemistry staining, by reverse transcriptase-polymerase chain reaction(RT-PCR) and western blotting; Expression of VEGF protein were determined in 36 esophageal cancer tissues by western blotting .Expression of VEGF protein in 22 esophageal cancer patients'plasma were determined by Enzyme-linked immunosorbent assay. Results: ⑴There are the expression of VEGF in esophageal cancer cell lines (TE-1 , TE-13 , ECA109) and tissue of esophageal cancer with immunohistochemistry staining, by reverse transcriptase-polymerase chain reaction(RT-PCR) and western blotting.⑵The level of VEGF protein was also significantly correlated to lymph node metastasis, invading depth of the tumors and patients'prognosis. The level expression of VEGF protein in esophageal cancer patients'plasma during radiotherapy are changing. Conclusions: Esophageal cancer cells and tissues have the higher expression protein of VEGF. There were relationship between overexpression of VEGF and significantly metastasis and prognosis of esophageal cancer patients. Part ⅡThe effect of antisense VEGF on the radiation sensitivity of esophageal cancer cells in vitro Objective: To investigate the effect of antisense VEGF on the radiation sensitivity of esophageal cancer cells in vitro. Methods: ⑴Vector plasmid were enlarge and adopt throught DH5αand identify by limited enzyme.⑵The fragments of VEGF antisense cDNA,empty vector plasmid DNA and antisense oligodeoxynucleotide were transfected into TE-1 cells mediated with lipofectamine respectively. We confirmed the expression of VEGF in TE-1 cells by RT-PCR analysis,insitu hybridization and western blotting.The growth rate among these groups were detected by MTT assay. Apoptotic cells were detected in transfected cells by FCM assay.The radiation sensitivity of transfected esophageal cancer cells were detected by clonogenic survival curves . After irradiation , We confirmed the expression of VEGF in transfected cells by RT-PCR , western blotting.The growth rate among different groups were detected by MTT assay. Apoptotic cells were detected by FCM assay. Results: ⑴The antisense VEGFcDNA and oligodeoxynucleotide could be transfected into TE-1 cells mediated with lipofectamine.⑵The growth rate of these groups had not detected differences by MTT assay.⑶The decreased expression level of VEGF protein were found in antisene group by RT-PCR analysis,western blotting and insitu hybridization .Apoptotic cells were not found in transfected cells by FCM assay.Distribution of cell cycle also were similar among the four groups.⑷The increased radiation sensitivity of transfected esophageal cancer cells were detected by clonogenic survival curves.⑸After irradiation, the four groups'growth rate had not markedly increased differences detected by MTT assay, Apoptotic cells were also found no differences in four groups cells by FCM. Conclusions: The results indicated that VEGFmRNA and VEGF protein expression were dramatically blocked by transfection with antisense VEGF . The increased radiation sensitivity of transfected esophageal cancer cells were detected by clonogenic survival curves. Part ⅢThe effect of antisense VEGF on radiation sensitivity of esophageal cancer in vivo Objective: To investigate the effect of antisense VEGF on the radiation sensitivity of esophageal cancer cells in vivo. Methods: 32 male BLBA/C/nu nude mice were randomly divided into eight groups and eight kinds of prepared cells were subcutaneously injected on the paw pat of mice(2×10 6/100μl/mice): ⑴The antisense VEGFcDNA group: This group was divided into four groups A:TE-1 control group (TE-11group)B: TE-1 irradiation group (TE-1-R1group)C: TE-1 antisense VEGFcDNA group(TE-1-A group) D: TE-1 antisense VEGFcDNA and irradiation group(TE-1-AR group) ⑵antisense oligodeoxynucleotide group:...