体外培养人胚胎来源视网膜干细胞的诱导分化

目的 探讨培养的人胚胎来源视网膜干细胞向视网膜终末细胞分化的可能性。方法 来自16～20周人胚胎的视网膜干细胞进行无血清体外培养,并分别进行有血清条件下体外诱导和用含视网膜色素上皮的眼杯模拟体内条件诱导的观察,采用免疫荧光法检测干细胞和视网膜终末细胞表面抗原的表达,采用实时荧光定量PCR法检测诱导前后细胞nestin基因在mRNA水平的表达差异。结果 从人胚胎视网膜神经感觉层分离出的视网膜干细胞,在体外诱导的条件下,可表达视网膜终末细胞标记PKCα、GFAP、Thy1,少数细胞表达nestin和MAP2;在模拟体内环境诱导后,则不仅表达上述细胞标记,而且rhodopsin和syntaxin表达阳性。实时荧光定量PcR法检测显示：诱导后细胞nestin基因表达量较诱导前细胞明显降低。结论 RPE可以促进体外培养的视网膜干细胞向视杆细胞和无长突细胞分化。(中华眼科杂志,2004,40：448-452)

Differentiation of human embryonic retinal cells in vitro

Objective To study the conditions to induce differentiation of human embryonic retinal cells (HRCs) in vitro. Methods HRCs were isolated from neural retinas of 16th-20th week of gestation embryo and cultured in serum-free media. Retinal cell differentiation was induced by serum-containing media and by the mouse retinal pigment epithelium ( RPE) eye cup whick mimic the retinal environment in vivo. The expression of Nestin and several differentiated retinal cell markers was investigated by immunostaining. Real time RT-PCR analysis was used to evaluate the mRNA level of nestin gene in HRCs and differentiated retinal cells. Results HRCs cultured in serum-containing medium can differentiate into glial cell ( GFAP positive) , ganglion cell( Thy 1 positive) and bipolar cell(PKCa positive). HRCs cultured in mouse RPE eye cup can also express photoreceptor marker rhodopsin and amacrine marker syntaxin. Nestin downregulate significantly in differentiated HRCs. Conclusion RPE appeares to play an important role in the differentiation of retinal photoreceptors and amacrine cells in vitro. ( Chin J Ophthalmol, 2004, 40:448-452)