维甲酸对人视网膜色素上皮细胞Cx43蛋白和mRNA表达的影响

Retinoic acid on expression of Cx43 protein and mRNA in cultured human retinal pigment epithelial cells

目的观察体外培养的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞中缝隙连接蛋白(connexin43,Cx43)表达特点,以及药物维甲酸(retinoic acid,RA)对RPE细胞Cx43mRNA和蛋白表达的影响。方法取体外传代的第4代人RPE细胞,分为药物组、空白对照组和对照组。药物组分别用终浓度为0.1×10-6mmol·L-1、10-6mmol·L-1、10×10-6mmol·L-1的RA培养,对照组和空白对照组分别加入等体积的无水乙醇、培养液。用免疫组织化学法和原位杂交观察Cx43蛋白和mR-NA的表达情况;用Western blot和RT-PCR半定量法测定RA对体外培养人RPE细胞Cx43蛋白和mRNA表达的灰度值。结果免疫组织化学染色显示体外培养的人RPE细胞表达Cx43,表达的部位主要为细胞浆和细胞膜;Western blot半定量结果显示RA对人RPE细胞Cx43蛋白表达有明显的促进作用,3种浓度药物组灰度值分别是1.1167±0.0031、1.0240±0.0006、0.8560±0.0030;原位杂交定位显示Cx43mRNA主要表达在细胞核;RT-PCR半定量结果显示RA使RPE细胞的Cx43mRNA表达量明显增加,3种浓度药物组灰度值分别是1.3091±0.0053、1.1797±0.0009、0.8390±0.0042。结论体外培养的人RPE细胞表达Cx43;RA可上调Cx43蛋白和mRNA的表达,从而抑制细胞生长。

Objective To investigate the characteristics of expression of connexin43（Cx43） in the cultured human retinal pigment epithelial（RPE） cells and the effects of retinoic acid（RA） on expression of Cx43 protein and mRNA of RPE cells. Methods Cultured human RPE cells in vitro were selected,and were divided into drug group,blank control group and control group.The concentrations of RA were 0.1×10-6 mmol·L-1,10-6 mmol·L-1,10×10-6 mmol·L-1.Control group and blank control group were added with isochoric absolute alcohol and culture fluid,respectively.Immunohistochemistry method and in situ hybridization were performed to investigate the distribution of Cx43 protein and mRNA on cultured human RPE cells;Western blot and RT-PCR semiquantitative method were used to detect the effect of RA on gray values of Cx43 protein and mRNA on cultured human RPE cells in vitro. Results Immunohistochemistry revealed that cultured human RPE cell expressed Cx43,which located on cell plasm and membrane;Western blot showed that RA could obviously promote the expression of Cx43 in human PRE cells.Gray values were 1.116 7±0.003 1,1.024 0±0.000 6,and 0.856 0±0.003 0 for three different concentrations of RA.In situ hybridization showed that Cx43 mRNA mainly expressed in cell nuclear;RT-PCR seminquantitative method showed that RA could obviously promote the expression of mRNA in RPE cells.Gray values were 1.309 1±0.005 3,1.179 7±0.000 9,0.839 0±0.004 2. Conclusions Cx43 can be found in cultured RPE cells in vitro;RA can promote the expression of Cx43 protein and mRNA in human RPE cells.