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Hepatocellular carcinoma of various treatments for the cancer cell itself, genetic performance is unstable due to liver cancer cells, the early detection difficult, surgical resection is low and easy to chemotherapy and radiotherapy treatment was well tolerated, and thus find effective The therapeutic approach is the key to improve the survival rate of liver cancer. Neovascularization target tumor biological treatment has become in recent years a new hotspot, compared with conventional treatment with broad-spectrum, the amplified anti-tumor effect, and the toxic side effects, resistance is not easy. In addition, with the development of modern molecular biology techniques, antisense technology more and more people's attention, especially antisense oligodeoxynucleotide (ASODN) is expected to become the first clinical application of gene therapy drugs, indicating that a tumor Treatment of a new era. Tumor angiogenesis by a variety of regulatory factor adjustment, wherein the vascular endothelial growth factor (vascular endothelial growth factor, VEGF) on the proliferation of vascular endothelial cells, hydrolysis of the base film, the strongest role of migration and blood vessel, and the high specificity . In a variety of tumors over-expression promotes tumor angiogenesis, in order to maintain the strong metabolism of tumor cells and the tumor cells can be further proliferation and metastasis. Therefore, VEGF and its receptor is an ideal target for the anti-angiogenic treatment of tumors. It was believed that VEGF is a paracrine manner to regulate tumor angiogenesis, VEGF secreted by tumor cells selective effect on tumor vascular endothelial cells on specific VEGF receptor (Flt-1 Fourth Military Medical University doctoral thesis Chinese Yang and KDR), and by the tyrosine kinase mediated signal transduction, regulation of endothelial cell differentiation and angiogenesis. But in recent years, studies have found that a variety of tumor cell expression of VEGF receptor and VEGF and its receptors interventions can change these tumor cells in vitro proliferative activity and other biological characteristics. This shows that the tumor is likely to exist VEGF autocrine mechanism. Studies have shown that human liver carcinoma (HCC) in the expression of VEGF and VEGF expression and liver cancer growth and metastasis. Autocrine mechanism of VEGF has not been reported. In this study, a variety of hepatocellular carcinoma cell lines in vitro model, people, VEGF 11mNA sequence 5 'translation initiation first 5Al74-bit base antisense target, synthetic ZI poly ASODN, and its sense strand companion ODM as controls. By laser confocal microscopy ASODN distribution in liver cancer cells' inert conditions, quantitative analysis of the concentration and time characteristics of its role, and in InRNA, protein and protein secretion levels detected ASODN hepatoma cell VEGF expression inhibition. From explore hepatoma cells VEGF autocrine mechanism, using RTFCR immunocytochemical staining was used to detect a variety of VEGF receptor expression in hepatocellular carcinoma cell lines. MTH method, morphological observation and flow cytometry analysis means the the ASODN proliferation of liver cancer cells in vitro activity, morphology, cell cycle and the impact of membrane surface expression of VEGF receptor. The main results are as follows: l. VEGF ASODN in S deleted the CJ72 cell in the concentration with the role of extension of time, and gradually increased, 12h can reach a higher concentration after 48 h reached a peak and, thereafter, gradually decreased, but the effect is sustainable for more than 72 h; intracellular after the ASODN content with the role of concentration increases, but when the reach a certain concentration of action, the increase is not obvious, 5 u mol / L ASODN ideal role of concentration. VEGF ASO Chau deleted in S CJ cell distribution site are mainly located in the cytoplasm, the nucleus of a small amount of the distribution. Mitotic cells to the ASODN intake capacity is much larger than the non-dividing cells and ASODN mainly distributed in the nucleus in dividing cells. 2. RT-PCR and immunocytochemical staining SMMC-772 cells the VEGF InRNA and protein expression: VE * F * S * DN inhibit the expression of S mmmm * -7721 cells *** F. 3. The complete bovine aortic endothelial cells Tian AEC) in vitro culture and identification, and to observe its biological characteristics. - Fourth Military Medical University doctoral thesis the Chinese arrest to 4. SMMC7721 cell culture supernatant can stimulate the proliferation of BAEC and increases with the increase in the number of SMMC-772 inoculated, but inhibit the proliferation of BAEC highest biological activity, excessive number of cells when the cell number of knife XIO '. VEGF ASODN can inhibit SMMC-772 cell culture supernatant of BAEC proliferation stimulation and inhibition rate and dose linear relationship, the greater the dose, the stronger the inhibition: SO * N is essentially no inhibitory effect, and * SO * N no direct role in the SAEC proliferation. 5. Body eve of cultured SMMC-7721 cells a a * -1 weak expression: HHCC and HCpGZ cells express KDR. 6. VEGF ASODN of different concentrations inhibit the proliferation of HHCC cells, and inhibition with ASODN concentration increased and enhanced SODN no effect on proliferation of HHCC cells: VEGFASODN SMMCJ72 and L929 cells no effect on proliferation. 7. HHCC cells after VEGF ASODN role of morphological changes. Performance of cells arranged in a more sparse, mitotic figures reduced, the reduction of cell atypia, cell surface microvilli, mitochondrial swelling, vacuolization, chromatin margination. HHCC cell morphology of the SODN and the control group no significant change and no significant change: the treatment groups S MMMMO7 knife cell morphology. 8. Flow cytometry analysis of the VEGF ASODN role in cell cycle and HHCC cells in S phase decreased, the Q of the proportion of the increase, and a small pre-apoptotic peak: SMMC-772 cells of each treatment cycle no significant change . 9. Flow cytometry showed that SMMC-7721 cell surface expression of N-l of ASODN, SODN and percentage of positive control group were 20%, 21.7% and 19 a%, no significant difference among the three groups. HHCC cell surface expression of KDR, ASODN, SODN and the percentage of positive control group were 19%, 29.8% and 31%, wherein ASODN group the HHCC membrane sheet
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