骨桥蛋白,视网膜老化模型,动物造模 z-bio 2022-01-27 698

　　Introduction: Osteopontin (OPN) is a secreted glycosylated phosphoprotein encoded by the SPP1 gene. OPN is a cytokine that binds to cell surface integrins and CD44 mutants. Integrins perform multiple functions in cells, affecting cell survival and apoptosis, inflammation, micromineralization, cell attachment and migration, intracellular signal transduction, chemotaxis, and maintenance of homeostasis after injury. Regulation of neuronal regeneration after injury. OPN is present in the nervous system, the developing adult rodent brain and the adult rodent brain, and neurons in the olfactory bulb, retina, striatum, and brain stem produce OPN. OPN may be weakly expressed under physiological conditions, but its expression may increase in inflammation and neurodegenerative diseases. In the retina, OPN is expressed by retinal ganglion cells (RGC), which transmit visual signals to the brain. GC is the most sensitive to ischemia and excitotoxicity, and may help to up-regulate OPN expression and prevent experimental glaucoma. Retinal glial cells, astrocytes, microglia and Muller cells maintain retinal homeostasis, provide structural support, and affect metabolism, neuronal phagocytosis, immune response and other activities. Reactive astrocytes express OPN after various types of brain injury, and are related to microglia, which act as macrophages. Astrocytes suggest that microglia activated after central nervous system injury can up-regulate OPN and may play an important role in the pathogenesis of neuroinflammation. OPN binds to the integrin αVβ3 receptor. It may be a chemokine that mobilizes astrocytes and microglia in the process of glial scar formation after ischemic injury. In fact, OPN may be involved in the activation of glial cells, cell repair, migration of glial and macrophages, and matrix remodeling of reactive astrocytes. Based on this, the effect of OPN on retinal astrocytes and microglia is worth studying. In addition, because OPN was found in the secretions of Mueller cells, we studied their morphology to understand how OPN affects these retinal glial cells. Aging is the main risk factor for neurodegenerative diseases, and OPN has age-dependent neuroprotective effects. Some of the changes in normal retinal aging include loss of RGC, astrocytes expressing GFAP (glial fibrillary acidic protein), increased cytoplasmic organelles, and the acquisition of an activated microglia phenotype. Therefore, it is important to compare the effects of OPN loss on the retina of young (3 months old) and old (20 months) mice. The purpose of this study is to study the effects of OPN deletion in OPN knockout mice on RGCS, astrocytes, microglia and Muller cells. In addition, the effect of aging on OPN function is unknown, so the effect of this defect has been studied in animals of various ages.

　　Methods: Animals: Female knockout animals (B6129S6(CG)-SPP1TM1BLH/J) and C57BL/6J mice, 3 months old (n = 3 wild type and n = 3 knockout) and 20 months old (n = 4 wild type) In these experiments, n = 4 knockouts were used. Animals can eat and drink freely, the light-dark cycle is 12/12, and the temperature is 21°C. Tissue collection: After cervical dislocation, the animals were sacrificed and the eyes were removed. Remove the cornea, lens and vitreous, and carefully extract the retina. Immediately fix 4% paraformaldehyde (PFA) prepared in 0.1 M phosphate buffer (pH 7.4) on the retina for 5 hours, and then spread it on filter paper. The eyes were removed and immediately fixed with 4% PFA overnight to obtain sections. It was then frozen and stored in 30% sucrose in 0.1M phosphate buffer at 4°C for 24 hours and embedded in OCT. Frozen sections (14μm thick) are stored at -20°C. Immunochemistry: As mentioned above, the entire retina is immunostained. The fixed retina was washed with phosphate buffered saline (PBS, pH 7.4) and shaken with PBS-TX-100-BSA (0.25% TrITO-X100 and 1% bovine serum albumin) solution at 4°C. Closed overnight. .. Then incubate the retina with the same antibody (diluted with PBS-TX-100-BSA) at 4°C for 1 day. The anti-RBPMS (RNA binding protein with multiple splicing) guinea pig antibody (1:4000) detects RGC, and the anti-GFAP mouse antibody (1:1000) detects astrocytes. Then wash the retina 3 times with PBS, 15 minutes each time, and then combine it with the secondary antibody diluted 1:1000 with PBS-BSA (1%) (Alexa Fluor 555 conjugated with goat anti-guinea pig antibody and Alexa Fluor 488 conjugated with goat anti Mouse antibody)... Rinse the retina 3 times with PBS for 10 minutes each time, place the retina on the section, and then fix the slide with PBS:glycerol (1:1). As mentioned earlier, microglia and Muller cells were immunostained on the sections. The sections were washed twice with PBS-TX-100 for 10 minutes each, and then incubated with rabbit anti-IBA1 primary antibody (1:2000) overnight to detect microglia. Use rabbit anti-glutamine synthetase antibody (1:10000) for detection. Multicellular. GC is also labeled with anti-RBPMS guinea pig antibody. After washing twice with PBS, mix with secondary antibody (Alexa Fluor 555 or 488 goat anti-rabbit or anti-guinea pig) for 1 hour. The sections were washed twice with PBS for 10 minutes each, and the coverslips were fixed with PBS:glycerol (1:1).

　　Image capture: Use ZEN software to capture images through a digital camera and a fluorescence microscope. The mosaic area is defined by overlapping photomicrographs of the defined area of the retina. Once the mosaic is defined, the outline of the retina can be measured and the surface area of the retina can be calculated. GC quantification: The ZESS software was used to semi-automatically count the number of RGCS, and the t test was used to compare the RGC density of wild-type and OPN-excluded retinas. In addition, the Mann-Whitney U test was used to test the difference between the two groups. Use IBM SPSS statistical software V.21 for statistical analysis. Astrocyte morphology: Calculate the proportion of the retinal surface occupied by astrocytes and compare it with OPN knockout mice of the optic nerve in the center of the retina (1 mm in diameter). The morphological branches of the above astrocytes are very complicated, which hinders quantification. Results: The RGCs and astrocytes in the retinas of wild-type and OPN knockout mice were labeled with RBPMS or GFAP antibodies, respectively. The retina of OPN knockout mice has several areas with very low cell density. In particular, it is located in the peripheral area 2.5 mm from the optic nerve. In these areas, the low density of RGCS and astrocytes is further exaggerated with age. Evaluate the number of RBP-labeled RGCs in the entire retina and each retinal area, and calculate its density (RGCS/MM2). The average RGC density of 3-month-old wild-type mice (2607.15±38.36gcs/mm2) is higher than the average RGC density of OPN knockout mice (1953.37±29.75gcs/mm2), and the average RGC density is 20-one month old Mice are animals (wild), the type is 2101.86±84.73gcs)/MM2). The average RGC density of 20-month-old OPN knockout mice is 832.77±114.28gcs/mm2. Therefore, OPN deficiency can reduce the RGC density by an average of 25.09% at 3 months, and further reduce to 60.7%. In the retina of wild-type mice, astrocytes cover the entire retina, which is a network of retinal blood vessels connected in stars. In contrast, OPN knockout mice seem to have fewer astrocytes. The number of astrocytes is small, the branches are short, and the process is low, suggesting atrophy of astrocytes. This effect is more severe in 20-month-old mice than in 3-month-old mice. Astrocytes are obviously insufficient, and some cells lose the morphology of astrocytes. It is worth noting that there are many areas without astrocytes in the retina of OPN knockout mice, and the density of RGCS is low. The area of astrocytes (33.05%±0.95) of the 3-month-old wild-type mice was larger than that of the OPN knockout mice (16.19%±3.47). Without OPN, the density of astrocytes is reduced by 51.01%. In 20-month-old mice, the proportion of wild-type retinal astrocytes (21.67%±1.02) is still higher than that of OPN knockout mice (9.14%±1.95), and the loss of OPN may be reduced Retina density. comfirmed. Astrocytes. In addition, in 3-month-old OPN knockout mice, the number of RGCS and astrocyte coverage between the two were very similar to those of 20-month-old wild-type mice. the difference. Age difference. At 3 or 20 months, no signs of microglia activation were detected in the OPN knockout retina. In the presence and absence of OPN, the morphology of microglia is similar. In addition, in all cases, microglia are located inside the retina. This means that microglia are not active, because migration to the subretinal space is a sign of activation. Finally, in Muller cells between wild-type and OPN knockout mice, no differences were found between the ages of 3 to 20 months. In both cases, the bone marrow cells labeled with glutamine synthetase antibody pass through the retina, and the process surrounds the cell bodies of retinal neurons. Although the morphology of the retina of 20-month-old mice can be changed, Muller cells continue to surround the RGCS cell body and photoreceptor cells.

　　Conclusion: Lack of OPN may cause premature aging. However, microglia and Muller cells do not seem to be affected by OPN deficiency. Therefore, OPN may be a candidate molecule for the development of treatments for neurodegenerative diseases, in which case astrocytes may be a specific target.