Impact Of Slit2to The Proliferation、Migration And Angiogenesis Of HCMEC Cells

Objective:The impact of the hypoxia-inducible human retinal pigment epithelialcells(RPE) which transfected by Adenovirus-mediated Slit2andAdenovirus-mediated Slit2ShRNA to the proliferation of human choroidalmicrovascular endothelial cells(HCMEC)and impact of exogenous Slit2onmigration and angiogenesis of HCMEC cells. To explored the possible roleof Slit2/Robo4signaling pathway in choroidal neovascularization,andprovide new direction for the treatment of choroidal neovascularization.Methos:The culture and identification of the RPE and HCMEC invitro;hypoxic conditions were achieved by adding200ｕmol/l CobaltChloride to medium to mimic hypoxia.RPE cells and HCMEC cells werecultured in a contact co-culture system by Transwell chamber;CCK8assaywas carried out to determine the number of cells at the12th,24th and48thhour in each well.The expression of the Slit2、Robo1、Robo4、Rac1、cdc42 were identified by immunohistochemistry and RT-PCR in HCMECcells.Western-blot was used to detect the expression of the Robo4inHCMEC cells dealed with different concentrations exogenous Slit2.theimpact of exogenous150ng/ml Slit2on migration and angiogenesis ofVEGF-induced HCMEC cells.Western-blot was used to detect theexpression of the Rac1and cdc42.Result：There are significant differences statistically in differentgroups(F=98.122,P=0.00000119) and times(F=3388.913,P=0.000),theinteraction of the category and time points(F=82.863,P=0.000),the A valueof Slit2treated group was higher than other groups（with hypoxia groupP=0.001,others P=0.000),the A value of Slit2shRNA treated group waslower than other groups(with hypoxia group P=0.003,with emptyadenovirus group P=0.008at the48th,others P=0.000).The expression ofSlit2、Robo4、Rac1and cdc42is reside in HCMEC cells.Robo4wassignificantly increased in the level of the protein in HCMEC cells dealedwith different concentrations exogenous Slit2.Exogenous150ng/ml Slit2could inhibit the migration and angiogenesis of VEGF-induced HCMECcells.The expression of Rac1had significant difference between Positivecontrol group and group2（p<0.05),The expression of Rac1had nosignificant difference between negative control group and group1 （p>0.05).The expression of cdc42has no obvious statistical significancein the four groups（p>0.05).Conclusion：Under hypoxia the RPE cell can promote the proliferation of HCMECcell,High-expression of Slit2can significantly promote the proliferation ofHCMEC cells,When Slit2shRNA is siliencing the Slit2in RPE cell,it cansignificantly inhibit the proliferation of HCMEC cells.Exogenous Slit2could inhibit the migration and angiogenesis of VEGF-induced HCMECcells,Slit2/Robo4signaling pathways may effect the expression of Rac1.