Experimental Studies On Inhibition Of Human Proliferating Hemangiomas Growth By Recombinant Adenovirus-mediated Antisense MMP-2 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.Degradation of extracellular matrix is key step of angiogenesis. matrix metalloproteinases(MMPs) are important protein kinases which participate in degradation of extracellular matrix(ECM), its degrade all of extracellular matrix. ECM is consanguine connected with angiomatous occurrence and proliferation and fadeaway. MMPs are important protein kinases which advance angiogenesis, it was regarded . MMPs are drone which treat proferating hemangioma. By infecting antisense MMP-2 gene therapy in treating proferating hemangioma reduces activity of MMP-2, it will become important way of treatment proferating hemangioma.This research adopts the strategy that MMP-2 antisense gene could inhibit target gene expression on mRNA level; We use vascular endothelial cells (VEC) and Grafts of human proferating hemangioma as targets, which were cultured from human proferating hemangioma, They are infected with antisense MMP-2, then we study the effect of this method on cell' sbiological behavior and angiogenesis in vitro and in vivo, explore the signification of antisense gene therapy in treating proferating hemangioma, and establish foundations for further clinical applying investigations. Thee main work is introduced as follows:l.The propagation (amplification)> purification, identify, titer determination of Ad-GFP and Ad-aMMP-2mediated by recombinant adenovirus.Objective: To propagate and purify the recombinant adenovirus of Ad-GFP and Ad-aMMP-2 and determine their titer and identify suitable gene . 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 CsCL gradient centrifugation and identified suitable gene by PCR and titrated with fluorescence microscopy or cytopathetic effect (CPE). Results: Suitable gene contains a 500-bp fragment at the 5' end of human MMP-2 cDNA which was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV. Enough fourth adenovirus suspension was harvested. The titers of Ad-GFP and Ad-aMMP-2 were 9.5X10" efu/ml and 1.0X10" pfu/ml respectively. Conclusions: Many Ad-GFP and Ad-aMMP-2 were obtained successfully with high titers . Ad-aMMP-2 contains suitable gene. 2. Effect of Ad-aMMP-2 transfection on human proferating hemangiomas in vitro.Objective: To investigate the effect of antisense Ad-aMMP-2 transfection on VEC, and set foundations for further experiments in vivo. Methods: The different MO I of Ad-GFP was used to determine the transfection efficiencyof VEC(after identification of morphologic and immunohistochemistral staining and electron microscope). Three effects of Ad-aMMP-2 on human VEC were analyzed by MTT assays cell cycles and apoptosis; reverse transcription PCR (RT-PCR) analyses was performed to determine the expression level of endogenous MMP-2mRNA;Immunohistochemistral staining and Western Blotting and Gelatin Zymography analyses were performed to determine the level of endogenous MMP-2 expression. Results:Our results indicated that the exogenous antisense MMP~2cDNA was successfully infected VEC, the ideal infection efficiency of VEC is 84. 3% when MOI is 100. After infecting Ad-aMMP-2, it had no obvious effects on VEC growth, proliferation index and apoptosis. By analyse of immunohistochemistral staining and Western Blotting and Gelatin Zymography, down-regulation of the endogenous MMP-2mRNA and MMP-2 were observed after Ad-aMMP-2 infection. Conclusions: ?After 2 times dealed with G418 , we can gain VEC of purification. VEC originate from human proferating hemangioma by culturing. ? The adenovirus vector could introduce GFP reporter gene into VEC . ? In vitro, Ad-aMMP-2 can not inhibit the growth of VEC, but it can inhibit MMP-2 secretion of VEC. 3. Influence of human proferating hemangiomas growth in nude mice by infecting of Ad-aMMP-2 in vivo.Objective: To investigate the effect of antisense MMP~2cDNA infection on the growth of grafts of human proferating hemangiomas in nude mice. Methods: Grafts of human proferating hemangiomas were transplanted into the two sides of axilla of 18 nude mice in one hour and observed growth, after them were cut into 5mmX4mmX3mm . 45 days later, there are three groups: Group PBS, Group Ad-GFP, and Group Ad-aMMP-2.per Group 5 nude mice , they accepted the first differenttherapy and repeated every other day for four times altogether. Tumor sizes were measured with ruler one time 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 and proliferation index were detected by FCM method. Frozen tumor tissue section of mice injected with Ad-GFP was cut and was performed to ascertain expression of GFP gene. The MMP-2 expression was checked with immunohistochemistry staining by MMP-2 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 grafted sites. Tumor growth speed is more slowly in Group Ad-aMMP-2 than that in other groups. GFP gene can express effectively in tumor mass. Ad-aMMP-2 infection can suppress the growth of tumors derived from grafts of human proferating hemangiomas, and there were no obvious side effects. Comparing with other groups, Ad-aMMP-2 resulted more cell apoptosis. MMP-2 expression was inhibited significantly in Group Ad-aMMP-2 so that the MVD was decreased accordingly. Conclusions: Many results suggest anti-MMP-2 therapy may be a new method against human proferating hemangiomas, whose main mechanism is to induce ischemia by restraining VEC secretion MMP-2.