全反式视黄酸对兔视网膜色素上皮细胞的影响

The effect of retinoic acid on rabbit RPE cells in vitro

目的观察全反式视黄酸(ATRA)对培养的兔视网膜色素上皮(RPE)细胞的作用,研究肝细胞生长因子(HGF)、基质金属蛋白酶-2(MMP-2)在此作用下的变化情况及关系。方法取原代兔RPE细胞,传代培养至第5代,加入ATRA后观察RPE细胞的形态,用台盼蓝排斥实验测定细胞活力,用免疫组织化学法检测RPE细胞HGF、MMP-2的表达,并用ELISA法测定RPE细胞培养液中HGF的浓度。结果不同浓度、不同作用时间的ATRA对RPE细胞的形态、活力影响不同。随ATRA浓度增加、作用时间延长RPE细胞存活率下降,HGF、MMP-2表达增加,HGF分泌量随ATRA的浓度增高而增加(P<0.05)。结论ATRA的浓度≥5nmol/mL时可引起RPE细胞的生长抑制,存活率降低,类似于近视眼RPE细胞改变。ATRA作用下RPE细胞中HGF、MMP-2表达增加,且HGF的增加更明显。

Objective Researches showed that all-trans retinoie acid（ATRA） has an inhibitory effect on the proliferation of retinal pigment epithelium （RPE） cells, and this event is similar to the pathogenesis of myopia. Other studies also demonstrated that hepatocyte growth factor （HGF） plays an action to generation of myopia. Present study was to evaluate the role of ATRA on the morphology,vitality, hypertrophy and function in the secretion of HGF and matrix metalloprateinase-2 （MMP-2） in cultured rabbit RPE ceils. Methods The RPE cells were isolated from black rabbits. The ceils were digested in 0.25% typsin ＋ 0.02% EDTA and cultured in DMEM containing 10% fetal bovene serum and then identified with CK8 and Vimentin. The fifth generation of ceils were collected and treated with 5,10,20 nmol/mL of ATRA respectively. After 24,48 and 72 hours, the morphology of RPE cells was studied using immunohistochemistry, and cell vitality was analyzed using the trypan blue reject test. The expression of HGF and MMP-2 in the RPE cells was detected using immunohistochemistry. The secretion of HGF by RPE was evaluated using ELISA. Results The flattening and splitting of cells were found from 24 through 72 hours after various concentrations of ATRA treatment. The cell vitality was gradually decreased following the increase of the time and concentration of ATRA treatment （P 〈 0. 01 ）. The expression intensity of HGF and MMP-2 in the cells was gradually enhanced with the increase of time and concentration of ATRA, and secretion of HGF by RPE cells followed the same pattern （P 〈 0. 05 ）. Conclusion ARTA at the concentration of ≥5 nmol/mL can induce the morphological change of cultured RPE cells. The expression of HGF and MMP-2 is upregulated in RPE cells stimulated by ARTA.