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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 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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In view of the deficiency of class hour and the limitations of classroom teaching in the course of ocular fundus disease, a network teaching platform, based on Browser/Server structure, was explored and constructed to assist classroom teaching. The network platform was constituted with teaching demonstration system, communication-test system, search system, and help system, mainly including 18 functional modules of learning-world et al. The students can be guided with the modules such as navigation, acting as a self-regulated learner through the modules such as learning-world, searching and downloading the related learning materials through the modules such as searching and discussing different learning topics with other students and their teachers through the modules such as forum. The network platform was used for classroom teaching of ocular fundus disease in auxiliary , and the result showed it was helpful to breaking the time and space constraints in conventional teach-ing, expanding the teaching content, solving the difficulties in teaching, improving the students' learning initiative, and realizing the interactive teaching.