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Background The proliferation and migration of vascular endothelial cells is a primary link during angiogenesis.Studies showed that heat shock protein B6 (HspB6) promotes the secretion of multiple angiogenesis-related factors and therefore leads to neovascularization.Understanding the effects of neutralizing HspB6 antibody on the biological behavior of human choroidal vascular endothelial cells has an important significance in the target treatment of choroidal neovacularization diseases.Objective This study was to address the role and mechanism of neutralizing HspB6 antibody in tube formation of human choroidal vascular endothelial cells.Methods Human choroidal vascular endothelial cell line was normally cultured and harvested for total RNA extraction.Expressions of HspB6 mRNA and protein in human choroidal vascular endothelial cells were detected by reverse transcription PCR (RT-PCR) and flow cytometry (FCM).The cells were seeded on 96-well plate covered with matrigel at the density of 2×104/hole.Then the neutralizing HspB6 antibody at the concentration of 100 μg/Land 500 μg/L was added into the medium respectively,and the control cells were set without the addition of HspB6 antibody.The number of capillary tubes was calculated 12 hours after culture by three-dimensional matrigel assay.In addition,0,50,100,500 μg/L of neutralizing HspB6 antibody were added into the cell medium separately for 24hours,cell counting kit-8 (CCK-8) method was employed to assay the inhibitory rate(IR) of the cells.Transwell test was used to count the cell number across chamber membrane for the evaluation of migration ability of the cells.The apoptosis of the cells was assayed by FCM.Results Both HspB6 mRNA and protein were expressed on human choroidal vascular endothelial cells.The number of capillary tube formation of human choroidal vascular endothelial cells was (67.25±5.75),(60.39±6.41) and (39.76±10.73) /field in the 0,100 and 500 μg/L neutralizing HspB6 antibody groups,with significant difference among them (F =10.210,P =0.012),and the tube number was significantly less in the 500 μg/L neutralizing HspB6 antibody group compared with 0 μg/L neutralizing HspB6 group (P =0.005).The IR of neutralizing HspB6 antibody to the cellular proliferation and migration was enhanced with the increases of concentration and time lapse(Fconcentration =7.485,P =0.002 ; Ftime =16.684,P =0.001).The number of the cells through Transwell chamber membrane was 14.0 ± 2.5,11.1 ± 0.8,6.6 ± 0.1,6.7 ± 0.2 in the 0,50,100,500 μg/L neutralizing HspB6 antibody group respectively,and that in the 100 μg/L and 500 μg/L neutralizing HspB6 antibody group was lessened in comparison with the 0 μg/L neutralizing HspB6 antibody group(both at P=0.000).The apoptosis rate of the cells was (22.73 ± 2.53)％ in the neutralizing HspB6 antibody group,which was significantly lower than (13.33±2.08) ％ of the control group (t=4.967,P=0.008).Conclusions Neutralizing HspB6 antibody inhibits capillary tube formation of human choroidal endothelial cells in vitro in dose-and timedependent manner,probably through suppressing the proliferation and migration and promoting the apoptosis of choroidal endothelial cells.
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BackgroundIt has been proved that as a chemokine,interferon-inducible protein-10(IP-10)can regulate the immuno-inflammatory reaction.Some new researches showed that IP-10 also played role in regulating the neovascular vessel formation.Corneal neovascularization (CNV) is associated with multiple cellular factors,but its mechanism is below clear.Objective The present study was to address the roles of exogenous mouse IP-10 in alkali burn-induced CNV.Methods Eighty-two SPF BALB/c mice were used in this experiment and grouped according to random number table.The corneal alkali burn models were established by putting the filter paper with 1 mol/L NaOH at the central corneas of the left eyes for 40 seconds.10 mg/L IP-10 was topically administered from the first day or 14 days after modeling in the early intervene group( 10 eyes)or middle-late intervene group(5 eyes).CNV area was measured as a percentage of whole cornea.0.2％ sodium hyaluronate(HA) as vehicle was utilized in the model control group.Angiogenic factor expression in corneal tissue in the early intervene group was quantified by reverse transcriptase polymerase chain reaction (RT-PCR)and compared with model control group.All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and complied with the standards of Guidelines for the Care and Use of Laboratory Animals of Soochow University. ResultsThe CNV percentage was(88.67±10.22) ％ in the model control mice,showing a significant increase in comparison with that of IP-10 early intervene group (70.06±12.21)％ (t=3.77,P=0.00).In 21 days after corneal alkali burn,the CNV percentage was(87.33±13.47)％ in the model control mice,and that of the IP-10 middle-late intervene group was ( 86.56± 12.47 ) ％ without significant difference between them ( t =1.26,P＞0.05 ).Two days or four days after IP-10 early intervene,the expressions of chemokine receptor type 3 ( CXCR3 ) in corneal tissue were significant higher than model control group( t =3.13,3.07,P＜0.05 ),but the expressions of vascular endothelial growth factor (VEGF) in cornea were lowed ( t =5.99,6.27,P＜0.01),and so were transforming growth factor-β1 (TGF-β1) (t =8.50,P＜0.01;t =4.53,P＜0.05).Conclusions The early topical administration of the exogenous mouse IP-10 can inhibit CNV by up-regulating CXCR3 expression and down-regulating VEGF and TGF-β1 expression in cornea.However,middle-later usage of the IP-10 is uneffective.