| Background and ObjectiveMyopia is a most commonly disease in ophthalmology, which greatly affects people's life. The incidence of myopia is increasing year by year. After studied of more than 200 years, there is still no satisfactory explanation to myopia pathogenesis. There are a lot of inconvenience and complications. It remains urgent task. Therefore we need to study the outbreak mechanism of myopia. In the last 30 years of 20 century, scientists built up tow animal experiment of myopia which made tremendous progression. The one is form deprivation myopia (FDM). The rate of eye will accelerate its growth and become highly myopia if deprived of form vision. The other is defocus myopia, which forced animal look at near or wear negatively spherical lens in order to modulation and accelerate its growth. After establish animal experimental model, people have made a more in-depth research to the occurrence, development and turnover of myopia. Study shows that neuroepithelium of retinal produce various neurotransmitters and growth factors induce to myopia. The neuroepithelium of retinal generated first messenger, which action to RPE cell and uvea cell. Subsequently, which generated second messenger and regulate sclera. These factors work by means of cooperate parallel and across lead to sclera remodeling and axial extension. Some cytokine, growth factor and tyrosine kinase receptors may play important role on RPE cell through autocrine and paracrine secretion. The Vitamin A derivative, all-trans retinoic acid (ATRA), affects the proliferation and differentiation of some types of cells, including photoreceptors and amacrine cell. ATRA is known to be teratogenic and is thought to be an important factor in ocular development. In both mice and chicks, dorsal-ventral patterning of retina is determined by gradients of ATRA. Recently, evidence was provided that amacrine cells in the inner retina are involved in retinoid metabolism. It is also assumed that retinal amacrine cell are at the output level of the retinal image processing that is involved in the visual control of eye growth. It is known that the retina by itself can determine whether the focused image is in front or behind its surface. The retina releases yet unknown messengers to control the growth rates of the underlying tissues so that, during development, the image remains at the photoreceptor plane. Recent studies show that ATRA effect inhibitory action to the hyperplasia of RPE cell, which similar to pathology of myopia. However, the relationship between the change of HGF and MMP-2 in RPE cell after ATRA action in vitro has been rarely reported.This study was to establish myopia model of RPE cell in vitro. Form microcosmic aspect, such as qualitatively and quantitatively, observe the change of HGF and MMP-2 in RPE cell with the intervention of ATRA. The Trypan blue reject test was used to analyze the cell vitality. Immunocytochemistry were used to analyze the expression of HGF and MMP-2. Enzyme linked immunosorbent assay were used to analyze the secretory volume of HGF. To explore the mechanism of ATRA in inhibiting RPE cell, as well as the change and relation of HGF and MMP-2 in inhibiting RPE cell.Materials and Methods1. Whole eye were enucleated from 7 day old black rabbit, and the anterior section, vitreous were discard. The posterior segment were made to cupula optica and fixed. Firstly, neuroepithelium of retina was digestived by 0.25%Trypsin+0.02%EDTA and drawn off. Secondly, RPE cell was digestived by 0.25%Trypsin+0.02%EDTA in incubator and stopped with FBS. Cell suspensions were collected by centrifuge tubes, which are centrifugated and rinsed. The RPE cell of third or fifth generation was used in the study.2. The identification of RPE cell was performed. The antibodies in immunocytochemistry studies were anti-cytokeratin and anti-vimentin antibodies.3. Different concentration of ATRA was added during the different period of cells growth, and then the cell vitality was analyzed using the Trypan blue reject test. The expression of HGF and MMP-2 was tested using an immunocytochemistry method. The secretion of HGF was tested using ELISA.4. These datas were analyzed by statistical software SPSS 10.0, (x|-)±s was demonstrated experimental data,χ~2 test were used to analyze vitality of RPE cell in different concentration and action time of ARTA. Tow factor analysis of variance were used to analyze the secret of HGF in ELISA. LSD-t was used to analyze the multiple comparisons in different concentration and action time of ARTA. Setα=0.05 as the criteria of test.Results1. Cultured RPE cell was established. To observe normal RPE cellular morphology characteristics: RPE cell was oval or irregular polygon and white transparent nucleus nucleolus. Cytoplasm covered with rich melanin granules, which concentric circles liken circled cell nucleus.2. To observe RPE cellular morphology characteristics after ATRA action: 5nM/ml,10nM/ml,20nM/ml of ATRA inhibited RPE cell. The cell became enlargement and applanation, dispersion of pigment. Different concentration of ATRA action 241k 48h and 72h, cell become enlargement and applanation, dispersion of pigment, partly disintegrate, vitality decrease.3. The Trypan blue reject test show: 5nM/ml,10nM/ml,20nM/ml of ATRA acted to RPE cell. After 24h, the cell vitality respective is 99.50%,91.67% and 87.44%(χ~2=10.80, P=0.002). After 48h, the cell vitality respective is 99.02%,88.17% and 80.44%0cM8.59, P<0.001). After 72h, the cell vitality respective is 98.44%,86.94% and 71.08%(χ~2=29.46, P<0.001).4. The immunocytochemistry method show: HGF and MMP-2 positive expression display as brown, which were mainly distributed in cytolymph of RPE cell, which increased along with an increase in ATRA concentration and action time.5. The enzyme linked immunosorbent assay show: The secretion of HGF raise along with an increase in ATRA concentration. There was significant difference between negative group and experiments group in secretion. (P <0.05).Conclusions1. Greater than or Equal to 5nM/ml ATRA could inhibit the growth of RPE cell, and lead to the change of morphology and cell viability. This is similar to the myopia pathological change.2. After ATRA action, the expression of myopia correlator HGF and MMP-2 were up-regulated in RPE cell. And the increase of HGF is more obviously. |
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