Experimental Studies On The Growth Inhibition Of Human Malignant Melanoma By Adenovirus Mediated Antisense VEGF₁₆₅ Gene

Judah Folkman first brought up the theory of tumor' s angiogenesis in 1970' s. Many researches showed that angiogenesis is the premise of solid tumor growth and metastasis, capturing nutrition and evacuating waste. Micro vessel density (MVD) of tumor is closely related to the growth and prognosis of almost all the solid tumor. Vascular endothelial growth factor (VEGF) is a kind of important cytokine stimulating vascularization by promoting mitosis and permeability of vascular endothelial cell. The treatment of anti-tumor angiogenesis targeting VEGF is the hot research point at present.This research adopts the strategy that VEGF₁65 antisense gene could inhibit target gene expression on mRNA level; We use ECV₃04 cells and A₃75 cells as targets, the former can express VEGFR, the letter can express VEGF. They are transfected with antisense VEGF₁65, then we study the effect of this method on cell' s biological behavior and angiogenesis in vitro and in vivo, explore the signification of antisense gene therapy in treating malignant melanoma, and establish foundations for further clinical applying investigations. Thee main work is introduced as follows: 1.The propagation (amplification),purification,titer determination of Ad-GFP and Ad-aVEGF₁65 mediated by recombinant adenovirus. Objective: To propagate and purify the recombinant adenovirus of Ad-GFPand Ad-aVEGFies and determine their titer. Methods: All recombinant adenovirus were reproduced and propagated in 293 cells. Then we extract adenovirus suspension by freezing and thawing 293 cells repeatedly. The adenovirus suspension was purified by CsC12 gradient centrifugation and titrated with fluorescence microscopy or cytopathetic effect (CPE). Results: Enough fourth adenovirus suspension was harvested. The titers of Ad-GFP and Ad-aVEGF were 9. 5X109 efu/ml and 1.2X1010 pfu/ml respectively. Conclusions: Many Ad-GFP and Ad-aVEGFi65 were obtained successfully with high titers.2. Effect of Ad-aVEGF166 transfection on human malignant melanoma in vitro. Objective: To investigate the effect of antisense Ad-aVEGF,65 transfection on ECVsm and A375 cells, and set foundations for further experiments in vivo.Methods: The different MOI of Ad-GFP was used to determine the transfection efficiency of ECVsiwand A375 cells. Three effects of Ad-aVEGFms on human ECV304 and A375 cells were analyzed by MTT assay> cell cycle^ and apoptosis; In situ hybridization (ISH) analyses was performed to determine the expression level of endogenous VEGF-mRNA;Immunohistochemistral staining and ELISA analyses were performed to determine the level of endogenous VEGF expression. Results:Our results indicated that the exogenous antisense VEGFcDNA was successfully infected ECV304 cells and A375 cells, the ideal infection efficiency of ECV304 cell is 80.2% and that of A375 cell is 84. 3% when MOI is 100. After transfecting Ad-aVEGF165, A375 cell culture supernatant fluid could inhibit ECV304 cell growth markedly and decrease its proliferation index comparing with other groups. VEGF immunohistochemistral staining showed that ECV304cell did not express VEGF. Ad-aVEGF had no obvious effects on A376cells' growth, proliferation index and apoptosis. Down-regulation of the endogenous VEGFmRNA and VEGF wereobserved after Ad-aVEGF infection. Conclusions: (J)The adenovirus vector could introduce GFP reporter gene into ECV304 and A375 cells. (2) ECV304 cell does not express VEGF. (D In vitro, Ad-aVEGFi6S can not inhibit the growth of A375 cells, but it can inhibit VEGF secretion of A375 cells. 3. Influence of human malignant melanoma growth in nude mice by infecting of recombinant adenovirus.Objective: To investigate the effect of antisense VEGF165cDNA infection on the growth of A375 cells in nude mice. Methods:A375 cells were injected s. c into the axilla of the nude mouse. After the tumor formed, we cut it into 16 pieces equally, then transplanted into another 15 nude mice. There are three groups: Group PBS, Group Ad-GFP, and Group Ad-aVEGF. One week later, they accepted the first different therapy and repeated every other day for four times altogether. Tumor sizes were measured with ruler two times a week. Four weeks after treatment, the mice were sacrificed and their tumors were excised for naked eye observation, HE staining and electron microscopy. Tumor cell apoptosis was detected by TUNEL-AP method. Frozen tumor tissue section of mice injected with Ad-GFP was cut and was performed to ascertain expression of GFP gene. The VEGF expression was checked with ISH and immunohistochemistral staining by VEGFmRNA and VEGF protein. At last, the micro-vessel density (MVD) in tumor mass was counted by VIII factor immunohistochemistry staining. Results: The visible and palpable nodules had developed at all the injected sites. Tumor growth speed is more slowly in Group Ad-aVEGF than that in other groups. GFP gene can express effectively in tumor mass. Ad-aVEGF infection can suppress the growth of tumors derived from A375 cells, and there were no obvious side effects. Comparing with other groups, Ad-aVEGF resulted more tissue necrosis, but it had no obvious effect on cell apoptosis. VEGF expressionwas inhibited significantly in Group Ad-aVEGF so that the MVD was decreasedaccordingly. Conclusions: Many results suggest anti-VEGF therapy may bea new method against human malignant melanoma, whose main mechanism isto induce ischemia, but is not induce apoptosis.