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Objective@#To determine the changes of protein expressions in human retinal microvascular pericytes (HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy (DR).@*Methods@#HRMPCs were divided into two groups.The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose, while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose.The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin.Peptides of 2 μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA.The results were further analyzed using bioinformatics software.@*Results@#CCK-8 results showed that the absorbance (A450) of HRMPCs in high glucose group was 0.75±0.04, which was significantly lower than 0.91±0.05 in control group (t=5.784, P=0.000 2). In total, 1 972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1.5). Among them, 13 proteins were up-regulated, including CTNNB1 and CTBP2; while 41 proteins were down-regulated, including SQSTM1 and HMGCS1.The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration.@*Conclusions@#The expressions of many proteins in HRMPCs change under the stimulation of high glucose, which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.
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Objective@#To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes (RMPs) from mice.@*Methods@#Retinas were isolated from mice following with mechanical morcel, enzymatic digestion and filtration.The retinal fragments were incubated with low glucose DMEM with 20% fetal bovine serum after 24 hours pre-incubation.Differential digestion was used for purification of primary RMPs.Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry.Functional assay was evaluated by the pericytes-endothelial cells (ECs) co-culture system.The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission.@*Results@#Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually.The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes.No contact inhibition was observed.Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β (PDGFR-β), a few cells expressed the cellular markers glial fibrillary acidic protein (GFAP), but no cell expressed von Willebrand factor (vWF). The purity rate of RMPs was up to 97%.In the co-culture system, RMPs directly contacted with ECs to form the capillary-like cords in vitro.@*Conclusions@#A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.
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Objective To determine the changes of protein expressions in human retinal microvascular pericytes ( HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy ( DR) . Methods HRMPCs were divided into two groups. The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose,while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose. The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin. Peptides of 2μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA. The results were further analyzed using bioinformatics software. Results CCK-8 results showed that the absorbance ( A450 ) of HRMPCs in high glucose group was 0. 75±0. 04,which was significantly lower than 0. 91±0. 05 in control group (t=5. 784,P=0. 0002). In total,1972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1. 5). Among them,13 proteins were up-regulated,including CTNNB1 and CTBP2;while 41 proteins were down-regulated,including SQSTM1 and HMGCS1. The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose,which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.
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Objective To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes ( RMPs) from mice. Methods Retinas were isolated from mice following with mechanical morcel,enzymatic digestion and filtration. The retinal fragments were incubated with low glucose DMEM with 20％ fetal bovine serum after 24 hours pre-incubation. Differential digestion was used for purification of primary RMPs. Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry. Functional assay was evaluated by the pericytes-endothelial cells ( ECs) co-culture system. The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission. Results Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually. The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes. No contact inhibition was observed. Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β( PDGFR-β) ,a few cells expressed the cellular markers glial fibrillary acidic protein ( GFAP) ,but no cell expressed von Willebrand factor ( vWF) . The purity rate of RMPs was up to 97％. In the co-culture system,RMPs directly contacted with ECs to form the capillary-like cords in vitro. Conclusions A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.