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Objective To compare the protective effects of pharmacological batch RC28-E1 and pilot batch RC28-E2 on retinal vascular endothelial cells (RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).Methods RF/6A cells were divided into normal control group,VEGF + FGF group and RC28-E1 groups with different concentrations.The optimal concentration of RC28-E1 was determined by cell counting kit-8 (CCK-8) method.Cells were divided into normal control group,VEGF+FGF group,RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group,and cultured with serum-free culture medium,serum-free culture medium containing VEGF+FGF,serum-free culture medium containing VEGF+FGF+RC28-E1,serum-free culture medium containing VEGF+FGF+RC28-E2,and serum-free culture medium containing VEGF+FGF+ conbercept,serum-free medium containing VEGF+FGF+FGF trap,respectively.Cell proliferation rate was measured by CCK-8 method,cell migration ability was detected by Transwell test,and tube formation ability was detected by Matrigel assay.Results The cell proliferation rate of 0.080 mg/ml RC28-E1 group was significantly lower than that of VEGF+FGF group (P＜0.05).The cell proliferation rate of RC28-E1 group,RC28-E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P＜0.05).The number of migrated cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0.000).The numbers of meshes formed by retinal vascular endothelial cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P =0.003,0.001,0.009,0.018).The number of tube formation in FGF trap group was significantly higher than those in RC28-E1 group,RC28-E2 group,conbercept group and normal control group (P =0.014,0.000,0.008,0.014).Conclusions Under the stimulation of VEGF + FGF,the inhibitory effect of RC28-E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap.There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.
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Objective To compare the pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E1 and RC28-E2 in retinal angiogenesis and neovascularization,and analyze its mechanism.Methods Sixty postnatal Day 4 (P4) C57BL/6J mice were randomly divided into normal control group,vascular endothelial growth factor (VEGF)+fibroblast growth factor 2 (FGF2) group,VEGF+FGF2+RC28-E1 group,VEGF+FGF2+RC28-E2 group,VEGF+FGF2+conbercept group and VEGF+FGF2+FGF trap group by using a random number table,with 10 mice in each group.The mouse retinal explant culture system was established,and stimulated with the corresponding factors and drugs prepared in the starving culture media.The normal controls were treated with the starving media.Then the retinal explants were stained with Isolectin B4 and imaged.The number of filopodia per vascular length was quantified.In addition,ninety-six P7 C57BL/6J mice were randomly divided into normal control group,oxygen-induced retinopathy (OIR) model control group,OIR + RC28-E1 group,OIR + RC28-E2 group,OIR+conbercept group and OIR+FGF trap group by using a random number table,with 16 mice in each group.The normal controls were raised under normoxia for 10 days,and the rest of the groups were raised under hyperoxia for 5 days,then returned to normoxia for another 5 days.On P17,the retinas were isolated and stained with Isolectin B4.The stained retinas were mountedon the slides and photographed.The relative vessel obliteration and neovascularization in retina were analyzed with computer software.Then the protein levels of VEGF and FGF2 were examined by Western blot in the retinas of each group in the OIR experiment.Finally,in the RF/6A cells stimulated with VEGF and FGF2,the activities of the signaling pathways,including MEK-extracellular regulated protein kinases (Erk),protein kinase C (PKC) and protein kinase B (Akt) pathways,were examined by Western blot.All experimental procedures were evaluated and approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (SYXK 2009-0001),and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Results The results of retinal explant cultures showed that the numbers of filopodia per vascular length in VEGF + FGF2 + RC28-E1,VEGF + FGF2 + RC28-E2,VEGF + FGF2 + conbcrcept,and VEGF+FGF2+FGF trap groups were all significantly less than that in the VEGF+FGF2 group (all at P ＜ 0.001).The filopodia number in retinal vascular front in RC28-E1 group was similar to that in the RC28-E2 group (P =0.15),whereas the filopodia numbers in both groups were significantly decreased as compared to those in VEGF+ FGF2+conbercept group and VEGF+FGF2+FGF trap group (all at P＜0.001).The results from the OIR mouse model showed that the relative vessel obliteration area in OIR model control group was dramatically higher than those in the drug intervention groups (all at P＜0.05).There was no statistical significance in the relative vessel obliteration area between OIR+RC28-E1 group and OIR+RC28-E2 group (P =0.17),while the obliteration areas in both RC28-E-intervened groups were significantly lower than those in the OIR+conbercept group and OIR+FGF trap group (all at P＜0.05).The relative neovascular pixels in the intervention groups were significantly lower than those in the OIR model control group (all at P＜0.001).The neovascular pixels in OIR+RC28-E1 group were significantly lower than those in VEGF+FGF2+conbercept group and VEGF + FGF2 + FGF trap group (both at P ＜ 0.05),but comparable to those in OIR+RC28-E2 group (P =0.39).Western blot result showed that,the protein expression of VEGF and FGF2 in the OIR mouse retinas were significantly upregulated compared to those in the normal ones (both at P＜0.001).The upregulation of both genes were normalized by both RC28-E1 and RC28-E2.In addition,the stimulation of VEGF and FGF2 induced an enhanced activity in MEK-Erk pathway in RF/6A cells,whereas RC28-E1 inhibited the overactivation.Conclusions RC28-E1 and RC28-E2 both can inhibit angiogenesis in the retinal explants isolated from neonatal mice;they also reduce vessel obliteration and mitigate neovascularization in the OIR mouse model.Therefore,the pharmacology batch and pilot test batch of RC28-E have similar efficacies and reliable stability,and are superior in the anti-angiogenic and anti-neovascular efficacy to the currently clinically available drugs conbercept and FGF trap.RC28-E1 may suppress pathological neovascularization through inhibiting the overactivation of MEK-Erk pathway in retinal vascular endothelial cells.
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Objective To compare the protective effects of pharmacological batch RC28.E1 and pilot batch RC28.E2 on retinal vascular endothelial cells ( RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Methods RF/6A cells were divided into normal control group, VEGF + FGF group and RC28.E1 groups with different concentrations. The optimal concentration of RC28.E1 was determined by cell counting kit.8 (CCK.8) method. Cells were divided into normal control group,VEGF+FGF group, RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group,and cultured with serum.free culture medium, serum.free culture medium containing VEGF+FGF,serum.free culture medium containing VEGF+FGF+RC28.E1, serum.free culture medium containing VEGF+FGF+RC28.E2,and serum.free culture medium containing VEGF+FGF+conbercept,serum.free medium containing VEGF+FGF+FGF trap,respectively. Cell proliferation rate was measured by CCK.8 method, cell migration ability was detected by Transwell test, and tube formation ability was detected by Matrigel assay. Results The cell proliferation rate of 0. 080 mg/ml RC28.E1 group was significantly lower than that of VEGF+FGF group (P<0. 05). The cell proliferation rate of RC28.E1 group,RC28.E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P<0. 05). The number of migrated cells in RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0. 000). The numbers of meshes formed by retinal vascular endothelial cells in RC28.E1 group,RC28.E2 group, conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group ( P=0. 003,0. 001, 0. 009,0. 018). The number of tube formation in FGF trap group was significantly higher than those in RC28.E1 group,RC28.E2 group, conbercept group and normal control group ( P = 0. 014, 0. 000, 0. 008, 0. 014 ). Conclusions Under the stimulation of VEGF+FGF,the inhibitory effect of RC28.E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap. There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.
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This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.