早期人胚胎视网膜色素上皮细胞的体外培养及生物学特性

目的探索早期人胚视网膜色素上皮(RPE)细胞体外培养以获得高纯度的RPE细胞的方法,并进一步研究其生物学特性。方法取意外流产4h以内人胚(6～10周)眼球,通过混合酶消化法获取RPE细胞。用F12培养液体外培养并作细胞形态学、S-100和Keratin免疫组织化学鉴定;通过Trizol一步法提取总RNA,逆转录聚合酶反应(RT-PCR)分析体外培养RPE细胞酪氨酸酶基因的表达。结果用F12培养液可获得纯度高、活性好的人胚RPE细胞;体外培养的RPE细胞不表达酪氨酸酶基因。结论早期人胚RPE细胞具有在体RPE细胞的部分特征;体外培养的RPE细胞未检测到酪氨酸酶RNA表达,故随着细胞分裂增殖,色素颗粒逐渐减少,提示体内外RPE细胞在功能上存在某些差异。

The in vitro culture of human embryonic retinal pigment epithelium cells and its biological characteristics

Objective To explore the method to obtain high purity of human retinal pigment epithelium (hRPE) cells in the culture of them, and to study their biological characteristics further. Methods The eyes of 6-10 weeks old human embryos after aborting were used within 4 hours. RPE cells were obtained after the isolation by combined digestion of mixed enzyme, and then were cultured in F12 medium. The RPE cells were identified by their morphological features and were further confirmed by immunohistochemical method through the RPE cells markers: S-100 and keratin. The total RNA of the RPE cells were extracted by Trizol method, and semi-quantitative RT-PCR method was used to analyze the mRNA expression of tyrosinase. Results REP cells with high purity and activity were obtained through the culture with F12 media. The RPE cells cultured in vitro didn't express their functional marker: tyrosinase. Conclusion Cultured early human embryonic RPE cells have some biological characteristics of that in vivo. There is no expression of tyrosinase in RPE cells cultured in vitro indicates that their function, to some extent, is different with those in vivo.