Experimental Study On The Anti-angiogenesis Effect Of Gecko Poly-eptides Mixture A

OBJECTIVE: To observe the effect of gecko peptide mixtures A (GPMA) onangiogenesis in vitro or in vivo, and to explore its mechanism.METHODS:（1） A vascular model， origined from chicken embryochorioallantoic membrane (CAM model), was built, and50eggs were incubated inwarm-box to nine-day-age eggs, and50nine-day-age eggs were divided randomly into5groups which each group were included10eggs. Five groups were high-dose geckopolypeptide mixture group, middle-dose gecko polypeptide mixture group, low-dosegecko polypeptide mixture group, negative control group and positive control group. Awindow was made on the shell to expose of chick chorioallantoic membrane, andmethyl cellulose sheet served as drug carrier was placed on neoangiogenic area ofchorioallantoic membrane. Then different concentrations of gecko polypeptide mixturewere given to the experimental group and inhibitory effect of gecko polypeptide onangiogenesis of chicken embryo chorioallantoic membrane were observed. EquivalentNormal Saline was injected to negative control group and paclitaxel solution wereinjected to positive control group.After48hours, inhibitory effect on angiogenesis wereobserved in methyl cellulose sheet covering area and the surrounding area.(2) Human umbilical vein endothelial cells (HUVEC) observed by invertedmicroscope were separated and cultured to survey morphological changes ofHUVEC cultured in vitro and the effects of different concentration (0.4mg/mL,0.2mg/mL,0.1mg/mL,0.05mg/mL,0.025mg/mL) of the gecko peptidemixture A onthe proliferation of HUVEC were measured by MTT assay. (3) EC109cells were treated separately by0.2mg/mL and0.1mg/mL geckopeptide mixtures A for24hours, and expression of VEGF protein in treated EC109cells were examined by immunohistochemical method.(4) mice in model of mouse H22transplanted tumor were established and treatedseparately by80mg/kg，40mg/kg and20mg/kg gecko peptide mixtures A, andexpression of VEGF protein in treated mice were examined by immunohistochemicalmethod and Western-blot methodRESULTS:（1）In negative control group, the number of large vascular, thevessel density, and vascular branches were significantly larger than these in the geckopeptide group. Diameter of vascular in control group was larger than those in geckopolypeptide group. In CAM, very few of large blood vessels were observed in high-gecko-polypeptide-dose group, and diameter of vessels were very fine, and vascularbranches were significantly reduced. And small blood vessels were hardly seen, andeven the emergence of hemolysis were appeared. Some chicken embryo diedindividually. By counting the vascular under dissect microscope, inhibitory rate was90%in40μ g/egg polypeptide group, which was significantly different from the controlgroup.(2) Under inverted microscope, human umbilical vein endothelial cells wereclear boundary, abundant cytoplasm and flat polygonal cells, and seen to grow inmonolayer, arrange in cobblestone monolayer. t After treated24hours, each group cellswere arranged disorderly, especially5-FU positive drug and high-dosed groups, thenumber of cells were decreased obviously. MTT results showed that the inhibition ratewas increased with time-long and the increasing drug concentration.(3) In vitro EC109tumor cell culture experiments, expression of VEGF in EC109tumor cells were reducedin0.2mg/mL or0.1mg/mL gecko peptide mixtures A group treated separately.(4) Inmice transplanted with H22tumor model, injecting (80mg/kg),(40mg/kg), low(20mg/kg) dose gecko peptide mixtures of A, inhibition the production of VEGF proteinin tumor tissues were observed in immunohistochemistry and Western-blot experiment. CONCLUSION: Gecko peptide mixtures A can inhibit the normal and tumorangiogenesis, mechanism of inhibition of angiogenesis may be induced by the inhibitionof vascular endothelial or production of VEGF in tumor cells.