Studies On The Anti-angiogenesis Activity Of The Polysaccharides From Marine Microalgae

In this thesis,the extraction conditions of the polysaccharides from Karenia Mikimoti were optimized.Different molecular polysaccharides from two microalgae(KPS,CPS)were obtained by fractional precipitation.The effects of inhibiting the activity of tumor angiogenesis and its expression of related proteins at the molecular level were also studied.The effects of soxhlet extraction method,ultrasonic extraction method,flash extraction method and flash extraction combined with protease hydrolysis on the extraction of polysaccharides were studied.The optimum conditions were as follows: the enzymolysis temperature was 40 ?,the pH was 8.5,the dosage of enzyme was 2.5% of the weight of algae powder,the time of enzymolysis was 2 h.The extraction rate of polysaccharides was 9.71% at above conditions.The Karenia polysaccharides KPS1,KPS2,KPS3 and Chlorella polysaccharides CPS1,CPS2 and CPS3 were obtained from 30%,50% and 80% ethanol precipitation.The KPS1,KPS2,CPS1 and CPS2 had the potential on tumor angiogenesis inhibition in chicken chorioallantoic membrane model.The effect of different KPS and CPS on the proliferation without or with HBC medium induced HUVECs was determined by using MTT colorimtric assay.It showed no effect on the growth of normal HUVECs treated with different KPS and CPS.It promoted the proliferation of HUVECs with the addition of 10% HBC medium significantly,while different marine microalgae polysaccharides had some inhibition on the proliferation of induced human umbilical vein endothelial cells at a certain extent.The effect of different KPS and CPS on the migration with HBC-medium induced HUVECs was also investigated by using “scratch wound” assay.It could significantly promote the migration of human umbilical vein endothelial cells with the addition of 10%HBC medium compared with the control group.KPS1,KPS2 could inhibit the migration of induced human umbilical vein endothelial cells at a certain concentration and showed a dose-dependent manner.The inhibitory effect of CPS1 and CPS2 on the migration of human umbilical vein endothelial cells induced by HBC-medium was higher than that of CPS3,and CPS2 showed a good concentration gradient at the concentration range of 5-100?g / mL.The expression of MMP-9 and VEGF protein induced by the HBC-medium in HUVECs had a significant increase compared with the control group by western blot test.The expression of MMP-9 was significantly inhibited by KPS1 at 100?g / mL(p <0.05).It suggested KPS maybe inhibit the tumor angiogenesis by suppressing the angiogenesisfactor MMP-9 related protein expression to achieve its inhibition of tumor angiogenesis activity.KPS2 and KPS3 inhibited the expression of VEGF-related protein in different concentration,and KPS2 had significant inhibitory effect at the concentration of 50?g / mL and 100?g / mL.Chlorella polysaccharide did not significantly inhibit the expression of MMP-9 related proteins in human umbilical vein endothelial cells.It suggested that the inhibition of tumor angiogenesis activity might not achieve by the expression of MMP-9 factor-related proteins.Chlorella polysaccharide had different degrees of reduction of VEGF protein expression in HUVECs,which it had a significant inhibitory effect on CPS1 and CPS3 at a concentration of 100?g / mL.It suggested that Chlorella polysaccharide had a certain inhibition of endothelial cells HUVECs on VEGF Protein expression.A further study showed that CPS1 at a concentration of 100 ?g / mL and CPS2 at a concentration of 50 and 100 ?g / mL had significant inhibitory effect on VEGF-R1,suggesting that Chlorella polysaccharide might surppress the expression of VEGF-R1 protein induced by the HBC-medium in HUVECs.