| Objective To investigate the effects of retinoic acid (RA) on cell growth , function of gap junction and degree of connexin43(Cx43) expression in cultured human retinal pigment epithelial (RPE) cells. Analyze the relationship of the restrain of RA on RPE cell growth to the function gap junction connection and the expresses of Cx43; Obtain a controlled intravitreous delay release system by injection of biodegradable polymers(alginate sodium-retinoic acid microspheres, AGS-RA microspheres) to the rabbit vitreous; Study the potential use of this RA delivery system in a rabbit model of laser injury to inhibition the subretina proliferation after laser coagulation.Methods Immunohistochemistry methods was performed to investigate the distribution of protein Cx43 on cultured human RPE cell; MTT methods was used to observed the effects of retinoic acid(RA) on cell growth; Determined the function of the cell gap junction of human RPE cells by LY methods, We have observed its change induced by varies concentration RA. Immunohistochemistry methods was also performed on cultured human RPE cell to investigate the RA affecting the express of gap junction protein Cx43 on RPE cells, and then we analysised the degree of the gray measured and have a statistic treatment by computer. Western blot experiment was use to fix quantify of the protein expresses; In situ hybridization of Cx43mRNA was performed in RPE cells; RT-PCR was carried out for mRNA half quantitative; Dissolved by organic solvent, RA was mixed with 1.5% alginate sodium and made to microspheres by a kind of special electrostatic generator; The spectrophotometer was use to measured the medicine content of AGS-RA microspheres; Vitro test was use spectrophotometer to analysis the release law of AGS-RA micropheres; We inject the AGS-RA microspheres to thevitreous cavity of rabbit and observed its toxicity effect by electrical physiology, scanning electron microscope and light microscope; At the same time we observed medicine dynamics characteristic of the microspheres by HPLC; We have established laser injury animal model (0.20 s , 200^m, 420m W). AGS-RA tiny ball, blank tiny ball or BBS was injected to the vitreous cavity after laser coagulation immediately; 1, 2, 3, 4, 6 W after laser injury, The eyes were enucleated and does retina pathology respectively by continuous section and 6 laser spots was analysis. We measures area, width and the altitude of laser spot respectively and carries out statistics analysis.Results: Immunohistochemistry reveal that cultured human RPE cell express protein Cx43 local on cell membrane and plasma. RA can restrain the growth of human RPE cell obviously. Its function tie to the dose of RA. LY test indicate that RA can make the intercellular communication increased and tone up the express of protein Cx43 on human RPE cells by dose depend manner. Western blot experiment shows that RA makes protein express increase; In situ hybridization of Cx43mRNA shows that mRNA express major in cell nucleus; RT-PCR test shows that RA make mRNA of RPE cell express obvious decreased; The size of microspheres we manufacture is 95.244 ±8.6265 micron; Medicine contain quantity of RA is 1.7644 ±0.0453 ug/mg; Vitro test show that RA in the microspheres is release evenly; Inject medicine to vitreous cavity show no poisonous side effect; The medicine dynamics research of microspheres showed that medicine is being even in 6 W releasing; The continuously section of retina pathology showed that retina structure change and proliferate occurred after laser injury; The width of laser damage proliferate area does not have obvious difference ( P = 0.1205 ) between groups. Area and the altitude of laser proliferate area of blank and contrast group had no obvious difference, but had notable discrepancy comparing with corresponding experiment group ( p < 0.05 ) ; Within a group have notable discrepancy ( p < 0.001) at the different time point; The altitude of the scar in the blank and contrast... |
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