巢蛋白和神经胶质纤维酸性蛋白在大鼠视网膜发育中的动态表达

The dynamic expression of nestin and glial fibrilary acidic protein in the development of retina in rats

目的 观察巢蛋白（nestin）和神经胶质纤维酸性蛋白（GFAP）在大鼠视网膜发育中的动态变化.方法 48只Wistar大鼠,其中24只大鼠分为出生后1 d,1、2、3、4、7、12、20周8组,每组3只.制作眼球矢状位冰冻切片,采用共聚焦激光显微镜观察nestin和谷氨酰胺合成酶（GS）以及GFAP和GS免疫荧光染色情况.18只大鼠分为出生后id,1、2、3、4、12周,每组3只.提取大鼠视网膜总RNA,实时定量逆转录聚合酶链反应（RT-PCR）检测nestin、GFAP和GS mRNA表达.选择6只出生后7～12 d新生鼠,取眼球体外培养Müller细胞,行GS和（或）nestin免疫荧光染色,共聚焦激光显微镜观察荧光染色情况.结果 出生后1 d,nestin免疫阳性细胞贯穿神经视网膜全层,主要定位于视网膜前体细胞放射状排列的细长纤维中,在视网膜内侧出现GFAP阳性的星形胶质细胞.出生后1周出现表达GS的Müller细胞,同时表达nestin,但不表达GFAP;GFAP阳性细胞仍然位于视网膜内侧.出生后2～12周,nestin在Mfiller细胞上的表达逐渐减少直至消失,GFAP在星形胶质细胞中的表达强度投有显著变化.体外培养的Müller细胞表达nestin,不表达GFAP.Nestin和GFAP mRNA在视网膜中的表达与免疫荧光染色结果相一致.结论 随大鼠视网膜不断发育,Müller细胞上nestin表达逐渐减少,成年大鼠视网膜Müller细胞不再表达nestin;新生鼠和成年鼠Müller细胞均不表达GFAP.

Objective To observe the dynamic expression of nestin and glial fibrilary acidic protein（GFAP） in the development of retina in rats. Methods In 48 Wistar rats, 24 were divided into 8 groups with 3 rats in each according to their age （1 day, 1 week, and 2, 3, 4, 7, 12, and 20 weeks old）. The sagittal freezing sections of the eye were made; nestin/glutamine synthetase （GS） and GFAP/GS were stained by immunofluorescence and were observed under the confocal microscope. Total RNA was extracted from 18 rats which were divided into 6 groups according to the age （1 day, 1 week, and 2, 3, 4, and 12 weeks old） with 3 rats in each. The expression of nestin, GAFA and GS mRNA were detected by real-time postnatal day 7-12 rats; the expression of nestin and GFAP was detected by immunostaining study. Double immunofluorescence was carried out between nestin/GS and GFAP/GS. Results One day after the birth,nestin positive ceils were found in the whole retinal neuroblast layers with elongated retinal progenitor cells;the GFAP positive astrocytes were observed in the inner retina. One week after the birth, Mailer glial cells expressed GS and nestin but not GFAP; GFAP positive cells localized in the inner retina. Two to 12 weeks GFAP in astrocytes didn＇t change much. The Mailer cells expressed nestin but no GFAP in vitro. The expression of nestin and GFAP mRNA in retina was accordant with the results of immunofluorescence ceils in neonatal and adult rats.