新生小鼠视网膜光感受器前体细胞的体外培养及鉴定

目的 对新生小鼠视网膜光感受器前体细胞进行分离和鉴定.方法同窝新生小鼠14只,随机分为7组(P1-P7),每组2只,出生后连续7d每天取1组幼鼠,分离视网膜并进行体外培养,观察其生长状况.传代后培养2周,然后采用细胞荧光免疫组化法检测细胞中的5-溴-2'脱氧尿嘧啶(BrdU)、细胞神经上皮干细胞蛋白(Nestin)、神经视网膜亮氨酸拉链(Nrl)和视蛋白(Opsin)的表达情况,并对视网膜光感受器前体细胞性质进行分析.结果 在特定的培养基中,部分P1-P7各组新生小鼠视网膜细胞贴壁生长,荧光免疫组化标记显示传代后生长细胞大部分为BrdU阳性细胞.P1-P7组Nestin阳性率分别为31.0%、31.6%、32.3%、30.2%、31.2%、30.9%、29.5%.Nrl阳性细胞数出生后3d开始明显增多,随后小幅增加,P1-P7组阳性率分别20.6%、35.2%、65.5%、68.6%、71.6%、73.O%、73.3%.P1-P4组细胞不表达Opsin,P5-P7组少量细胞表达Opsin.结论 新生小鼠视网膜存在视网膜光感受器前体细胞,该细胞具有分裂增殖能力及向视网膜光感受器细胞分化和表达视蛋白的潜能,并于生后3d明显增多,此后逐渐分化为成熟的光感受器细胞.

In vitro cultivation and identification of retinal photoreceptor precursor cells from newborn mice

Objective To isolate and identify the photoreceptor precursor cells isolated from newborn mouse retina. Methods Fourteen newborn littermate mice were randomly divided into seven groups (P1-P7,2 each).The retinal cells were isolated from the newborn mice on postnatal 1-7d respectively, and then cultured in vitro. The cellular growth state was observed under inverted phase contrast microscope. After in vitro expansion,5-bromo-2-deoxy-uridine (BrdU),neuroepithelial stem protein (Nestin),neurd retina leucine zipper (Nrl) and Opsin in cultured cells were detected by immunohistochemistry and immunofluorescence techniques. Results Most of retinal cells isolated from the seven groups (P1-P7) of newborn mice proliferated and adhered to the plate in the particular medium. Most of the growing cells were BrdU-positive cells. A part of the cells could express neural stem cell specific antigen-Nestin,the positive expression rate of Nestin in the seven groups (P1-P7) were 31.0%,31.6%,32.3%,30.2%,31.2%,30.9% and 29.5%, respectively, with no significant difference. Some growing cells expressed Nrl, and the expression of Nrl was increased significantly on the third day after birth, and then increased in small range, the positive expression rate of Nrl in the seven groups (P1-P7) were 20.6%,35.2%,65.5%,68.6%,71.6%,73.0% and 73.3%,respectively.The positive expression of Opsin was found only in a few cells of P5-P7.while the cells in groups P1-P4 did not express Opsin. Conclusions The photoreceptor precursor cells are presented in neonatal mouse retina.They have the ability of proliferation and may differentiate into retinal photoreceptor cells, having a potentiality of expressing Opsin. The photoreceptor precursor cells are increased significantly on the third day after birth, and then differentiate into mature retinal photoreceptor cells gradually.