全反视黄酸对培养的人视网膜色素上皮细胞的影响

The effect of all-trans retinoic acid on human RPE cells in cell culture

目的观察全反视黄酸(all-trans retinoic acid,ATRA)对培养的视网膜色素上皮细胞(retinal pigment epitheli-um,RPE)的形态、增殖以及对RPE细胞中转化生长因子-β2(transforming growth factor-β2,TGF-β2)表达的影响,并探讨ATRA与近视的关系。方法RPE传代至第3代后以1×105/孔的密度接种于96孔板或以1×106的浓度接种于250ml培养瓶,以10%FBS的F12培养液培养48h后转为0.5%FBS的F12培养液,12h后培养液转换为F12和1nM/ml、5nM/ml、10nM/ml、20nM/ml、30nM/ml的ATRA。以未加ATRA组作对照。48h后观察RPE细胞形态,并以MTT法测定其增殖情况,以流式细胞技术检测其细胞凋亡情况,以免疫组化方法检测RPE细胞TGF-β2表达,并以酶联免疫吸附实验(enzyme-linked immunosorbnent assay,ELISA)方法测定其上清液的TGF-β2浓度。结果不同浓度的ATRA对RPE细胞的形态、增殖有不同的影响。<10nM/ml的ATRA对RPE细胞的增殖无影响,细胞形态正常,MTT法显示在这些浓度下ATRA对RPE的增殖影响差异无显著性(P>0.05);10nM/ml、20nM/ml、30nM/ml的ATRA抑制RPE的增殖,MTT法显示ATRA对RPE有明显的抑制作用(P值分别为P<0.05,P<0.01,P<0.001),该效应呈剂量依赖性反应。RPE细胞增大变平,突起减少,部分裂解,活力减弱。流式细胞技术检测提示,10nM/ml、20nM/ml、30nM/ml浓度下细胞凋亡率分别为14.58%、19.95%和21.69%。免疫组化方法检测提示,当RPE的ATRA浓度大于10nM/ml时,TGF-β2的表达随浓度增高而增加。ELISA结果显示,ATRA浓度>10nM/ml时,RPE分泌TGF-β2的浓度增加。结论1nM/ml、5nM/ml的ATRA可维持培养的人RPE的生长,≥10nM/ml引起RPE的增殖抑制,并可诱导RPE的凋亡。10nM/ml及以上的ATRA引起的RPE病理性改变为理解近视眼的RPE改变提供了一条思路,≥10nM/ml的ATRA诱导RPE的TGF-β2表达增加可能导致TGF-β2拮抗碱性成纤维细胞生长因子的作用增强,使后者抑制近视发生的作用减弱而促进近视的发展。

Objective To evaluate the role of all-trans retinoic acid （ATRA） on the morphology, proliferation, apeptosis and function in the secretion of TGF-β2 in cultured human retinal pigment epithelial cells （RPE）. Methods RPE was plated into 96-well dishes at a density of 1 × 10^5/well or plated into 250 ml bottles at a density of 1 × 10^6. Forty-eight hours after seeding, the medium was changed to an F-12 medium centring 0.5 % FBS. Twelve hours after the first medium change, the medium was changed to an F-12 medium containing the above concentration of FBS plus 1 nM/ml, 5 nM/ml, 10 nM/ml, 20 nM/ml, and 30 nM/ml of ATRA （Sigma）. The cells cultured without ATRA were taken as the control. After another 48 hours, the morphology of RPE was studied, and cell proliferation and apoptosis were analyzed using the MTT assay and flow cytometer, respectively. The TGF-β2 expressed by RPE was tested using an immunohistechemistry method. The study was repeated three times. Results Different concentrations of ATRA have different effects on morphology, proliferation and opoptosis of the RPE ceils. 1 nM/ml and 5 nM/ml of ATRA did not affect the proliferation of RPE and its morphology remained normal; but 10 nM/ml, 20 nM/ml, or 30 nM/ml of ATRA inhibited the proliferation of RYE and the cells increased in size and became flattened. The process reduced the quantity of cells and some cell splitting, and the effect was concentration-dependent. The immunohistochemistry method showed that the expression of TGF-β2 increased along with an incaease in ATRA concentration. Flow cytometry showed that the rate of apoptosis was 14.58%, 19.95% and 21.69%, respectively. Conclusion Lower concentrations of ATRA do not affect the proliferation of RPE and cell morphology remain normal. However, concentrations of 10 nM/ml more and of ATRA can induce a change in pathology that is shnilar to the change in RPE cells in myopia with an increase in the secretion of TGF-β2, which can promote myopia. ATRA may relate to the onset or development of myopia.