肝细胞生长因子对人视网膜色素上皮细胞表型的影响

The effects of hepatocyte growth factor on phenotype of human retinal pigment epithelium cells

目的探讨肝细胞生长因子(HGF)对人视网膜色素上皮(RPE)细胞形态及细胞表面抗原的影响。方法体外培养人RPE细胞,分别采用0.4%小牛血清和重组人HGF因子刺激人RPE细胞,应用相差显微镜观察细胞形态变化;免疫组织化学方法检测细胞表面抗原细胞角蛋白、波形蛋白、成纤维细胞特异性蛋白-1(Fsp-1)的表达变化;采集经HGF(20ng/mL)刺激后0、12、24、48h的RPE细胞应用RT-PCR方法观察Fsp-1 mRNA的表达变化。结果体外培养的人RPE细胞,在含0.4%小牛血清培养液中呈多角形,贴壁生长融合后呈铺路石样;细胞角蛋白的表达阳性率为89.7%,波形蛋白阳性率为100%,Fsp-1阳性率为45.3%。HGF(20ng/mL)刺激24h后,细胞呈长梭形,具有成纤维细胞形态特征;细胞角蛋白阳性率为50.1%(χ2=38.1,P<0.01),Fsp-1阳性率为91.2%(χ2=48.62,P<0.01),而波形蛋白表达无明显变化。HGF作用后,Fsp-1 mRNA表达升高(F=13.761,P=0.002)。结论HGF促进人RPE细胞向成纤维细胞的表型转化,提示HGF在RPE细胞上皮-间质转化过程中起重要作用。

Objective Studies showed that hepatocyte growth factor（HGF）is an critical regulator for barrier function of retinal pigment epithelium（RPE） ceils and plays important roles during the transformation of epithelium-to-mesenchyme of RPE cells. This study was to investigate the effect of HGF on cell morphology and surface antigen of human RPE cells. Methods Human RPE cells from normal donor were isolated and cultured in vitro and divided into normal culture group（0. 4% calf serum medium）and HGF treated group. 20ng/mL recombined human HGF was co-incubated with RPE cells in 0.4% calf serum medium in the latter group. The morphological change of cultured RPE cells was observed under the phase-contrast microscope. The expressions of cytokeratin（ AE1/AE3 ） , Vimentin and fibroblast specific protein（ Fsp-1 ） in the RPE ceils were detected by immunohistochemistry,and Fsp-1 mRNA level in the RPE cells were evaluated in 0,12,24 and 48 hours after HGF treatment by reverse transcriptase polymerase chain reaction （RT-PCR）. Results After HGF stimulation, the morphology of RPE cells changed from polygon into elongated spindle shape, presenting the characteristics of fibroblasts. The positive cell rate for AE1/ AE3 in HGF group （50. 1% ）was significantly lower than normal group （89.7%） （X2 = 38. 1, P 〈 0. 01 ） , and that of Fsp-1 was obviously increased in HGF group（ 91.2% ）compared with normal group（ 45.3 % ） （X2 = 48.62, P 〈 0.01 ）. However, the positive cell rate for Vimentin had no obvious change between these two groups（P 〉0. 05）. Fsp-1mRNA level increased significantly 12 hours later in HGF group （ F = 13. 761, P = 0. 002）. Conclusion These findings suggest that HGF may play a role in the epithelial-to-mesenchymal transformation of RPE cells.