| BackgroundMany retinal diseases, such as retinopathy of prematurity, age-related macu lar degeneration,diabetic retinopathy and this will cause retinal cell death, there by lead to irreversible vision loss. At present, for on these diseases, no effecti ve clinical treatment, the method can be employed retinal photocoagulation, co ndensation, surgery, anti-inflammatory treatment, neurotrophic support, but their clinical efficacy is not significant, that is difficult to restore the patient loss o f vision. In recent years, with domestic and foreign researchers deepening stem cells, regeneration and functional reconstruction of retinal cells has become a h ot topic of stem cell transplantation in the treatment of retinal diseases brought new hope, but also cause the majority of clinical ophthalmology workers s co ncern. Stem cells refer to the growth and development of individual organisms primitive cells, both self-replication and differentiation potential of cells curren tly used for scientific research is mainly embryonic stem cells.Embryonic stem cells (ESCs) are early embryos or primitive gonads isolated a class of cells, it has unlimited vitro proliferation, self-renewal and differentiation characteristic s. Both in vitro and in vivo environment, ES cells can be induced to different iate in almost all cell types of the body. Currently used to treat retinaldiseases including eye derived stem cells derived from stem cells and retinal precursor cells, Muller cells, iris pigment epithelial cells (IPE) and retinal pigment epi thelium (RPE).RPE is constituted by a single layer of pigmented epithelium, arranged in a very regular. Cells were polygonal, it was divided into three part s, namely the top of the body portion and a base portion. RPE cells to nutrie nts and maintain the normal function of the photoreceptor cells in the retina pl ay an important role in the metabolism of photoreceptors, such as passing the required substance involved in vitamin A metabolism, constitute the outer retin al barrier, participation in development of the eye, the development of refractiv e errors occur and growth factor production and so on. RPE cells play an imp ortant role in the development of many diseases such as retinopathy of premat ure children retinopathy, age-related macular degeneration, diabetic retinopathy and other diseases.Our mouse RPE cells were isolated and cultured subretinal t ransplantation of retinal pigment epithelial cells in experimental research, and d iscuss the progress of embryonic stem cells and application of several major e ye-derived stem cells in ophthalmology discussion.Objectiveobserve the effects of subretinal transplantation of retinal pigment epithelial cells on the retinal pigment mouse cortex.Methods1. In vitro isolation, culture and cryopreservation of mouse retinal pigment epit helium:Take C57BL/6 mice 10, mice were sacrificed by spinal dislocation, r emove the eye, cleaning and removal of anterior segment.The eye cup soaked in cleaning solution, tweezers and gently stir up the expansion of the retina ne uroepithelium,optic disc surface with micro scissors to remove the retina neur oepithelium, after cleaning using trypsin digestion solution, after digestion is co mpleted using a micro syringe gently pipetting the eye cup surface, were colle cted broth melanin granules, after conventional centrifugal seeded in culture fla sk into incubator. After completing drawn Cell growth was observed under a microscope every day, two to three days to replace a culture medium after ad herent cells was changed the next day, when the cells reached approximately 9 0% confluence, the recording time used, according to 1:4 ratio pass on.After passage, still obverse cells growth under the microscope every day, two to thre e days to replace a culture medium. Take 1,4 by murine RPE cells, cell coun ts after digestion, seeded in 24-well plates, placed in the incubator,3 wells pe r day cells were counted and averaged growth curve. The first generation of mice RPE cells were seeded in 24-well culture plate,3d removed after washin g, ice-propanol fixed, natural drying sealed cells with climbing film after rinsin g to remove endogenous peroxidase too oxide activity, washed again, and then added RPE65 mouse anti-human protein antibody, incubation, and rinsing was added and goat anti-rat goat anti-rabbit secondary antibody, incubation, washin g, chromogenic diamino benzidine, placed under the microscope If the cytoplas m was showing brown positive, otherwise negative. The control group instead of primary antibody as negative control with PBS solution. After four generati ons of mice RPE cells were grown to confluence of about 90%, the cell lines can be frozen in 20% FBS preparation plus 8% DMSO cryopreservation fluid, the cells were digested with trypsin made into single cell suspension, while c ounting, conventional centrifugation, resuspended after precipitation transferred t o vials, into a foam box, directly into -80℃ refrigerator to frozen for subseq uent experiments after recovery using the cell line.2. Mouse subretinal transplantation of retinal pigment epithelial cells:Remove from-80 ℃ refrigerator frozen mouse RPE cells frozen pipe thawing after cells were transferred to a centrifuge tube containing RPE cell culture medium in mixing conventional centrifugal, after withdrawing the supernatant resuspended cell pellet transferred to suspension culture flasks, supplemented with mouse RPE cell culture medium to 6 ml. After the recovery of retinal pigment epithelial cells in mice unstable, need passaged twice before making subretinal transplantation, when the cells reached approximately 90% confluence the next passage, washed, digested microscope to cell rounding, there are cells begin when out of the bottle wall, stop digestion. Centrifuged, resuspended precipitate 1:8 ratio passaged to a new culture flask after shaking gently moved into an incubator. Mouse RPE cells were passaged 2 times under the microscope when the cell fusion of more than 90%, digested with trypsin, resuspended after centrifugation the cell pellet mix added to the culture to prepare approximately 1 × 106 cells/ml concentration RPE cell suspension was transplanted mice. The 7-day-old C57BL/6J mice were divided into normal group, OIR model group and OIR model cell transplantation group,10 in each group. OIR OIR model group and model and cell transplantation groups established OIR model:Mice were placed together with the oxygen concentration in female rats (75 ± 2)% oxygen chamber, the inside temperature (23 ±2) ℃,12 h of sunlight per day, feeding 5 d after feeding into the normal environment 5 d. Normal mice and female mice together in an airtight chamber, temperature (23 ±2) ℃, sunlight every day 12 h, keeping 5 d. OIR model of cell transplantation group mice were way outside subretinal transplantation. Mice were anesthetized by intraperitoneal, cut above the eye conjunctiva a little exposed sclera, anterior chamber by micro shovel needled release a little aqueous humor after limbus 2 mm at the rear by a micro syringe slightly toward the eye sclera parallel oblique piercing ball wall, into the cell sap, the same operation procedures OIR model mice, mice injected RPE cell culture medium, surgery coated erythromycin ointment to prevent infection. Fluorescence microscope in each group of mice RPE layer. Each group of mice retinal frozen sections, fixed, dehydrated, embedded, sliced in a humid chamber,1:50 dilution was added fluorescently labeledRPE65, after washing, horseradish peroxidase secondary antibody incubation, washing, staining nuclei transferred onto a glass slide after rinsing, dry naturallydark coverslip observed and photographed under a fluorescence microscope. RPE cells of mice in each group RPE65 using Western blot (Western blot), Bestrophin,ZO-1 protein relative expression. By RT-PCR to detect the mouse RPE cells RPE65, Bestrophin, ZO-1 mRNA relative expression.Results1.The primary mouse RPE cells cell body present with round, fusiform, irregular shape, contains a wealth of melanin granules, no nucleus,12h after the majority of adherent cells, adherent cells were visible nuclei after cell fusion 4d monolayer growth. After passage of the first generation of cell viability is strong, cell fusion after 2～3d, mostly fusiform shape and irregular shape, more melanin granules, but the contrast of primary cells becomes faint,4th generation cell, less melanin granules cells gradually transparent. The results showed that the growth curve first 1,4-generation mouse retinal pigment epithelial cells are the first to enter the cell 3d logarithmic growth phase,4～5d grow significantly, and then into the stable, where the first generation of mouse retinal pigment epithelial cell growth rate slightly faster than the 4th generation. The first generation of mouse retinal pigment epithelial cells by immunocytochemistry RPE65 protein showed mice retinal pigment epithelial cells were positive, the cytoplasm showed brown, blank control group were negative.2. Fluorescence microscopy revealed normal mice RPE layer structure were neatly arranged in a single layer, OIR model mice RPE layer discontinuity, disorganized, and the position of the missing part of RPE cells, OIR model mice RPE layer cell transplantation significantly increased thick, was a multi-layer arrangement, neatstructure, cell survival is good. Western blot analysis showed that the normal group, OIR model group, OIR model cell transplantation group RPE65 protein relative expression levels were 123.750±6.752,100.000±15.492,124.000±5.292; Bestrophin relative protein expression were 95.250±15.414, 52.750±15.392,109.000±20.833; ZO-1 protein relative expression levels were 93.750±6.946,93.500±6.807,83.500±8.583. Statistical analysis Results are shown among groups were compared using ANOVA, homogeneity of variance test p> 0.05, F= 7.272,2.438,11.358, differences among groups RPE65 protein, Bestrophin protein statistically significance, p<0.05, and Zo-1 protein was no significant difference. Multiple comparisons between groups using LSD-t test, which RPE65 protein relative expression between normal group and the OIR model group difference was statistically significant p<0.05, between OIR OIR model cell transplantation group and model group, the difference was statistically significant p< 0.05, while the OIR model between cell transplantation group and normal group showed no significant difference, p> 0.05. Bestrophin protein relative expression between normal group and OIR model group difference was statistically significant p<0.05, between OIR OIR model cell transplantation group and model group, the difference was statistically significant p<0.05, and the OIR model cell transplantation group and normal group no significant difference between, p> 0.05. ZO-1 protein relative expression between normal group and the OIR model group was statistically significant difference between the p<0.05, OIR OIR model cell transplantation group and model group, the difference was statistically significant p<0.05, and the OIR model and cell transplantation group between the normal group no significant difference, p> 0.05.RT-PCR test results showed that the normal group, OIR model group, OIR model cell transplantation group RPE65 mRNA relative expression levels were 0.895±0.118,0.393±0.123,0.928±0.169; relative Bestrophin mRNA expression amount were 0.818±0.161,0.317±0.120,0.735±0.278; ZO-I mRNA relative expression levels were 0.841±0.151,0.391±0.103,0.883±0.176.Using spss 19.0 statistical software for analysis, among groups were compared using ANOVA homogeneity of variance test was p> 0.05, F= 28.155,20.769,11.066, more statistically significant difference between groups, p<0.05, multiple comparisons between groups using LSD-t test, in which the relative expression of RPE65 protein, there was statistically significant between the p<0.05, OIR OIR model cell transplantation group and model group, the difference between the normal group and model group, the difference was OIR statistical significance p<0.05, while among OIR model cell transplantation group and normal group had no significant difference, p> 0.05. Bestrophin protein relative expression between normal group and OIR model group difference was statistically significant p<0.05, between OIR OIR model cell transplantation group and model group, the difference was statistically significant p <0.05, and the OIR model cell transplantation group and normal group no significant difference between, p> 0.05. ZO-1 protein relative expression, there was no statistically significant difference between groups, p> 0.05. The results showed that RPE65, Bestrophin protein relative expression of the normal group, OIR OIR model cell transplantation group than in the model group increased, while no significant difference in cell transplantation group OIR model group and the normal group; ZO-1 protein expression between the relative amount of each group no significant difference. RT-PCR test results showed that the normal group, OIR model group, OIR model cell transplantation group RPE65 mRNA relative expression levels were 0.895±0.118,0.393±0.123,0.928±0.169; relative Bestrophin mRNA expression amount were 0.818±0.161,0.317±0.120,0.735±0.278; Zo-1 mRNA relative expression levels were 0.841±0.151,0.391±0.103,0.883±0.176. Using spss 19.0 statistical software for analysis, among groups were compared using ANOVA (one-way ANOVA), homogeneity of variance test was p> 0.05, F= 28.155,20.769,11.066, more statistically significant difference between groups, p <0.05, multiple comparisons between groups using LSD-t test, which RPE65 mRNA relative expression, there was statistically significant between the p<0.05, OIR OIR model cell transplantation group and model group, the difference between the normal group and model group, the difference was OIR statistical significance p<0.05, while among OIR model cell transplantation group and normal group had no significant difference, p> 0.05. Bestrophin mRNA relative expression between normal group and OIR model group was statistically significant difference between the p<0.05, OIR OIR model cell transplantation group and model group, the difference was statistically significant p<0.05, and the OIR model cell transplantation group and normal group no significant difference between, p> 0.05. ZO-1 mRNA relative expression between normal group and OIR model group had a significant difference p <0.05, between OIR OIR model cell transplantation group and model group, the difference was statistically significant p<0.05, and the OIR model and cell transplantation group between the normal group no significant difference, p> 0.05.Conclusions1.In vitro modified enzyme digestion can be successfully isolated and cultured and cryopreserved mouse RPE cells, high purity, adherent rate, cell viability is strong, providing a source of cells for subsequent experiments.2.subretinal injection of RPE cells can promote the proliferation of retinal pigment mouse cortex, RPE layer thickened, it is multilayered arrangement, neat structure, cell survival is good. RPE65, Bestrophin protein relative expression of the normal group, OIR OIR model cell transplantation group than in the model group increased, while no difference OIR model group, cell transplantation group and normal group, ZO-1 protein relative expression OIR model cell transplantation group, normal group There were no differences between the OIR model group; RPE65, Bestrophin, ZO-1 mRNA relative expression of the normal group, OIR OIR model cell transplantation group than in the model group increased, while no significant difference in cell transplantation group OIR model group and the normal group. |
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