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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.
ABSTRACT
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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To investigate the clinicopathologic features of granular cell tumor(GCT) of the breast in order to make a diagnosis and give treatment strategy of this disease.Two cases of granular cell tumor (GCT) of the breast were reported,and the clinical presentation,pathological examination and surgical procedure were analyzed.Clinical presentations were atypical,and the tumor was characterized by clear or ill-defined boundary,abundant cytoplasm,full of eosinophilic granular,and infiltrative growth.Immunohistochemical study revealed that tumor cells were positive with S-100,CD68 and vimentin but negative with cytokeratin (CK) and epithelial membrane antigen(EMA).GCT of the breast is rare,most of which is benign tumor that may be misdiagnosed as malignancy.Wide resection is the main curative treatment.The prognosis is good.The understanding of this disease should be strengthened to avoid being operated excessively.Long-term follow-up should be performed.
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Malignant neoplasm recurrence is the most important prognostic factor for patients.Looking for tar-get gene is an important part of cancer research.Annexin Ⅰ (ANXA1 )is to be discovered in the seventies century, composition by 13 calcium -phospholipid binding proteins annexin superfamily members of the first molecules. ANXA1 expressed in a variety of malignant tumors,ANXA1 expression levels were significantly reduced or absent in most tumor tissues.The study found that changed ANXA1 expression in tumor cells may have a causal relationship with the malignant phenotype of tumor cells.This paper aims to investigate the relationship between annexin Ⅰ and malignant neoplasm metastasis.
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Background Retinal microvascular pericytes (RMPs) have been played increasing attention as an emerging key in pathogenesis of various retinal angiogenic diseases including diabetic retinopathy,and RMPs are thought to be a potential target for treatment.Yet the study has been hindered by the difficulty of obtaining source of tissue and isolating pure population.Objective This study was to establish a simple method of isolation,purification and cultivation of primary RMPs for rat.Methods Eyeballs were extracted from clean male Sprague Dawley rats and immersed by 75％ alcohol for 1 minute.The retinas were isolated and mechanical morcel.Trypsin (2.5 g/L) was firstly used and followed by type Ⅰ collagenase (2 g/L) for the digestion of the retina for 15 minutes,respectively.Retinal microvascular fragments were screened by 100 μm and 55 μm filter screen.DMEM containing 20％ fetal bovine serum was added for the cultivation and passaged of the cells.The cells were purified by exchanging medium and partial enzymatic digestion.The morphology and growth status were monitored under the phase contrast microscope,and α-smooth muscle actin (α-SMA),platelet-derived growth factor receptor-β (PDGFR-β),yon Willebrand factor (vWF),glial fibrillary acidic protein (GFAP) antibodies were used for the identification of RMPs.Results RMPs migrated out of fragments after 24-48 hours of plating.On day 7,RMPs appeared in primary cultures as loose colonies.The cells reached confluence to about 80％-90％ on day 14-16.The subcultures grew faster than the primary and reached confluence on day 12-14.The culture showed typical morphology of pericyte with large irregular triangular cell body and multiple long processes,and they could be repeatedly passaged 9 times without obvious loss of characteristic phenotype.Fluorescence assay exhibited that 96％ of the cells showed positive immunofluorescence for α-SMA and PDGFR-β,confirming the purity of RMPs in culture.However,only a few of them were positive for GFAP and the cells response for vWF was absent.Conclusions High purity of rat RMPs can be obtained easily by our method without high cost-consuming.Hcrc wc cstablished a simple mcthod for the primary culture of rat RMPs.
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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.
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Peritoneal carcinomatosis after operation is a major cause of treatment failure in gastric cancer with few options for treatment.Preoperative intraperitoneal chemotherapy seems to be an effective therapy for maximal containment of the malignant process of gastric cancer.Thus we reviewed the background,clinical efficacy a,drug selection,other issues of the infusion chemo theropy.