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Background and Purpose myopia ophthalmology common disease affect the patient's work, learning and life, the incidence increased year by year, has become a global social issues. For myopia research has been 200 years of history, but its pathogenesis is inconclusive. Therefore, to clarify the pathogenesis of myopia is increasingly becoming a major problem in the field of life sciences need to be resolved. Animal studies myopia in 30 years after the 20th century has made great progress, and established two animal models of myopia: form deprivation myopia (form deprivation myopia, FDM), refers to suturing the eyelids or wearing dispersion lens severely damaged caused by the animal's body feel myopia. Away from the focal myopia (defocus myopia), forced animal near vision or wear a negative ball lenses make objects focus behind the retina, causing adjustment and axial extension, resulting in myopia. Since the establishment of since the animals myopia model, people have a more in-depth study of the occurrence, development and prognosis of myopia. The study found the induced myopia substances from retinal neuroepithelial. The next level the retinal neuroepithelial First, the role of a messenger in the retinal pigment epithelium (retinal pigment epithelium, RPE) cells and uveal cells, thus producing the biochemicals (called secondary messenger), acting on the sclera, regulation the scleral growth of sclera remodeling, axial extension. Autocrine or paracrine loops involving cytokines, growth factors and tyrosine kinase receptors may play an important role in the RPE cells of pathological change. All-trans retinoic acid (all-trans retinoic acid, ATRA) is a derivative of vitamin A, can effectively regulate cell differentiation and development. ATRA teratogenicity, and is an important factor of eye development. The study showed that ATRA retina, sclera development and specific cell types (such as photoreceptor cells, amacrine cells) differentiation and maturation has important implications. Amacrine cells known inner retinal layers involved in the metabolism of ATRA, while the output of retinal amacrine cells in the retina image processing, the output of this process with visual control eye growth. In recent years, studies have shown that, ATRA inhibited proliferation of RPE cells, similar myopia pathological changes. Known myopia messenger of bFGF and TGF-β in recent years to study more in-depth, however, can affect eye growth hepatocyte growth factor (hepatocytegrowth factor, HGF), matrix metalloproteinase -2 (matrix metalloproteinase-2, MMP-2) the study less. This study to establish the the RPE cells myopia model, from the the cell microscopic point of qualitative quantitative observation of ATRA induced RPE cells in vitro myopic changes Immunocytochemistry Determination of HGF RPE cells, MMP-2 expression by ELISA method for the determination of HGF secretion in RPE cells, in order to investigate the mechanism of ATRA inhibit RPE cells, and in this role myopia related factor of HGF, MMP-2 changes and relationships. Materials and Methods 1. Selected healthy 1-week-old black rabbit 12, eyes the ball after anesthesia under sterile conditions, from the limbus 2mm ring cut and removal of the anterior segment and vitreous, will be made of the posterior segment of the eye eye cup and fixed with 0.25% Trypsin 0.02? TA about 2ml room temperature digestion 3min digestion down nerve cortex aspiration, then add trypsin at 37 ° C, 5% CO 2 incubator digestion 15min Join FBS-containing DMEM digestion was terminated the straw gently pipetting the RPE cells off, collected cell suspension centrifugal 5min centrifuge tube was added 20? S of DMEM were seeded in culture flasks, in cell incubator of Primary culture. This method can be used to pass on generation to generation 3-5 experimental study. Application vimentin and keratin polyclonal antibodies, the the immunocytochemical staining identification of RPE cells. Growth in RPE cells at different time points (24h, 48h, 72h), added 5nM/ml, 10nM/ml, 20nM/ml ATRA, application Desk trypan blue exclusion RPE cell survival rate in the experimental determination of ATRA after. Growth in RPE cells at different time points, added 5nM/ml, 10nM/ml, 20nM/ml concentration of ATRA Immunocytochemistry staining RPE cells was measured of myopia related factors of HGF, MMP-2 expression. Growth in RPE cells at different time points, added 5nM/ml 10nM/ml, 20nM/ml concentration of ATRA, RPE cells HGF secretion was measured by enzyme-linked immunosorbent assay. 6. Statistical analysis was performed using SPSS 10.0 statistical software, the test indicators data used (?) ± s, x 2 test used to compare the different concentration the ARTA groups in each treatment time point RPE cell viability. Different time and different the ARTA concentration group RPE cells secrete HGF amount compared using two-way ANOVA, LSD-t test was used to compare between the multiple and various concentrations of each time point. Α = 0.05 as the level of inspection. Results 1. Successfully established RPE cell culture models, observe the morphological characteristics of normal RPE cells: RPE cells were oval or irregular polygon, you can often see the white transparent nucleus nucleolus, covered with rich melanin granules in the cytoplasm, distributed concentrically surround the nucleus. After RPE cells 2.ATRA role in morphological characteristics: 5nM/ml 10nM/ml, 20nM/ml the ATRA inhibited proliferation of RPE cells, cells increases flat projections reduce pigment dispersion. Different concentrations of ATRA for 24h, 48h and 72h after RPE cells have increased flat projections reduce partial cleavage vitality weakened performance. Trypan blue exclusion assay suggested: the 5nM/ml 10nM/ml, 20nM/ml ATRA for 24h after cell survival rates were 99.50%, 91.67% and 87.44% (x 2 = 10.80 P = 0.002); 48h after cell viability were 99.02%, 88.17% and 80.44% (x 2 = 18.59, P <0.001); cell viability after 72h were 98.44%, 86.94 % and 71.08% (x 2 = 29.46, P <0.001). The immunocytochemical staining HGF and MMP-2 expression prompted: RPE cells of HGF and positive expression of MMP-2 is located in the cytoplasm of the RPE cells, the positive expression with ATRA concentration increased with ATRA for time extension also increased. Positive expression in the same concentration and duration of action of HGF is more obvious than the expression of MMP-2 positive. 5.ELISA detect HGF secretion Tip: RPE cells secrete the amount of HGF with the the ATRA concentration increased and any two groups were P <0.05. ARTA role duration of RPE cells secrete little effect on the amount of HGF, any two groups were (P> 0.05). The conclusion 1.ATRA concentration ≥ 5 nmol / mL can cause growth inhibition of RPE cells, decreased survival, similar myopia RPE cells change. 2.ATRA role of HGF in RPE cells, increased expression of MMP-2, and the increase of HGF.
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