体外原代培养人视网膜色素上皮细胞

目的：探讨建立体外原代培养人类视网膜色素上皮细胞(RPE)技术。为研究溶血磷脂酸(lysophosphatidic acid，LPA)对培养的人类视网膜色素上皮细胞DNA合成与增殖的作用。方法：取自愿贡献的意外事故成年眼球，用2．5g／L胰酶消化获取人RPE细胞、150mL／L胎牛血清的DMEM培养液培养，细胞接近融合状态时进行传代培养。取自愿贡献的眼球并游离其视网膜色素上皮细胞，用DMEM加100mL／L血清及MEM氨基酸和庆大霉素，进行细胞传代培养。用溶血磷脂酸、转移因子β2(TGF—β2)及LPA TGF—β2进行视网膜色素上皮细胞增殖试验，用细胞染色法进行细胞计数，提取视网膜色素上皮细胞DNA，用Spectrophotometer进行DNA定量测定。结果：RPE原代细胞镜下为圆形，大小不一，内含较多的色素颗粒，胞核无法辨认。原代细胞培养贴壁后3d增殖速度明显加快，至4～5d即可基本融合，细胞浆内色素颗粒则随传代次数增多而逐渐减少。第3代培养的视网膜色素上皮细胞膜表面可见微绒毛，细胞质内细胞器丰富，线粒体量多，体积较小，内外膜分界清晰，嵴较短。色素颗粒散在分布于胞浆内，多数细胞质内数量较少呈高电子密度包含物。LPA对原代培养的视网膜色素上皮细胞增殖有促进作用。含有和／或缺乏10mL／L小牛血清的两种LPA(10μmol／L)均明显刺激视网膜色素上皮细胞增殖，但这种细胞的增殖被TGF-β2所抑制，LPA(10μmol／L)引起视网膜的色素上皮细胞DNA合成增加是正常对照组的两倍。

Primary culture of human retinal pigment epithelium In vitro

AIM: To establish the primary culture of human retinal pigment epithelium(hRPE) cells and study the effects of lysophosphatidic acid(LPA) on cultured hRPE cells proliferation and DNA synthesis in vitro.METHODS: Adult human RPE cells were harvested as intact sheets from volunteers′ eyes, using the enzyme dispase. The primary cultured RPE cells were observed by scanning and transmission electron microscopy and passaged in vitro. Human RPE cells were fed with Dulbecco′s Modified Eagle′s medium supplemented with 10% serum MEM aminoacids and gentamicin. Proliferative responses were assessed with LPA, transforming growth factor β 2 (TGF- β 2), and LPA plus TGF- β 2. Cell counting was performed either by counting stained cells or use of a hemacytometer. DNA synthesis was assessed by extracting the DNA and measuring DNA quantity with a spectrophotometer.RESULTS: The just digested hRPE cells were round,and contained large quantity of pigments, but those pigments in the RPE cells decreased along with the cell division. The third passage of cultured RPE cells maintained a lot of mitochondria and microvilli. Lysophosphatidic acid (LPA10 μ mol/L) significantly enhanced RPE cell proliferation in both the presence and absence of 10mL/L fetal bovine serum. Proliferative effects of LPA were inhibited by TGF- β 2 (2μg/L). LPA (10μmol/L) also significantly increased DNA synthesis in RPE cells.The increase in DNA synthesis was twice that in proliferation, suggesting that LPA also increases the amount of DNA/cell.CONCLUSION: LPA can stimulate the proliferation of hRPE in vitro. The third passage maintains the same biological activities of cells as the primary passage. The proliferative effects of LPA suggest that this serum lipid can play a role in stimulating RPE cell proliferation during normal wound healing and development of proliferative vitreoretinopathy.