| ObjectiveThe purpopse was to observe the effect of all-trans retinoic acid (ATRA) on the proliferation, secretion of TGF-β2 and the second messenger in cultured guinea pig's retinal pigment epithelial(RPE) cells. And to investigate the effect of phospholipase C inhibitor U73122 and adenylate cyclase inhibitor SQ22536 on the proliferation and function of secreting TGF-β2 in the RPE cells.Then analyze from which signal pathway RPE cells regulate the secretion of TGF-β2 and to further identify the role of RPE in the myopia development.MethodsRPE cells were from the 3-week old guinea pig eyes, the 2-3 passage cells in logarithmic growth phase were used for the following experiment. Cells were verified by keratin immunohistochemistry. When cells nearly grow confluence, the medium was changed to DMEM-F12 medium without FBS for 24 hours before used.1. ATRA group:The effect on the cells proliferation after added different concentrations ATRA was analyzed using the MTT assay. The TGF-β2 secreted by RPE cells was tested using an ELISA kit at the times of 2,4,6,8,16 hours after The medium was changed to DMEM-F12 containing 10×10-6M ATRA. Intracellular 1,4,5-trisphosphate(IP3) and cyclic adenosine monophosphate (cAMP) were measured at the time of 0min,5min,30min,2h and 6h by the ELISA method and radioimmunoassay.2. U73122 and SQ22536 group:The effect on the cells proliferation after added different concentrations U73122 and SQ22536 was analyzed using the MTT assay. The TGF-β2 secreted by RPE was tested using an ELISA method at the times of 2,4, 6,8,16 hours after the medium was changed to 1×10-5M U73122 or SQ22536.Results1. The primary cultured guinea pig's retinal pigment epithelial cells came to grow after 48 hours. The cells were big and flat, and full of pigment. There was karyokinesis in some cells. When the cells grew confluens, they were almost hexagonal. The cells'keratin immunohistochemistry result showed the positive reaction.2. The cell proliferation was inhibited by 40×10-6M ATRA. After added 10×10-6M ATRA, the secretion of TGF-β2 was increased within 6h, but decreased at 16h. The intracellular IP3 was inhibited at each time point. But the amount of cAMP was increasd at 30min and 2h.3. The MTT result showed that the RPE cells proliferation was inhibited by U73122 compared with the control groups (p<0.05). And the effect was concentration dependent. The result of the ELISA test showed that the secretion of TGF-β2 was increased. The SQ22536 had not significant effect on the RPE cells proliferation. And the ELISA test showed that the secretion of TGF-β2 was no significant difference.Conclusions1. The cell proliferation can be inhibited by high concentration ATRA and U73122. The SQ22536 had not significant effect on the RPE cells proliferation.2.10×10-6M ATRA added to the RPE cells, the secretion of TGF-β2 was increased first(within 6h), and then time related decreased.3. The intracellular IP3 was inhibited at each time point. But the amount of cAMP was increasd at 30min and 2h after added 10×10-6M ATRA.4. After added 10×10-6M U73122 to the RPE cells, the secretion of TGF-β2 was increased. But the SQ22536 has no significant influence on the secretion of TGF-β2 in most of the time points.The ATRA can increase the secretion of TGF-β2 in short term in guinea pig's retinal pigment epithelial cell. And this result may has relation with the decreased IP3 and increased cAMP. When used the phospholipase C inhibitor U73122 which can decreas the IP3, the TGF-β2 increased. This reslut further verified the relationship. |
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