花背蟾蜍（Bufo raddei Strauch）眼早期发育过程中细胞凋亡和分子分化的研究

发布时间：2018-05-13 08:33

本文选题：预定视网膜色素上皮 + 预定神经视网膜　；参考：《兰州大学》2012年博士论文

【摘要】：脊椎动物视网膜的发育是一个复杂的形态学和分子生物学过程。在由视杯向成熟的视网膜的发育过程中,存在着细胞分化和转分化现象,但是在视泡发育成预定视网膜色素上皮(PRPE)和预定神经视网膜(PNR)的过程中,相关的研究比较少,并且其在形态变化中所涉及的生物学机制还不清楚。本实验就是通过形态学和免疫组织化学实验,研究视泡发育过程中细胞凋亡和分子分化的机制。本实验以花背蟾蜍为材料,通过苏木精-伊红(HE)染色观察视泡发育过程的组织形态学变化；然后通过TUNEL染色法观察视泡发育过程中预定视网膜色素上皮和预定神经视网膜中的细胞凋亡(apoptosis)。最后通过星形胶质细胞胶质原纤维酸性蛋白(GFAP)、神经丝蛋白(RT97)和色素合成过程的催化酶—酪氨酸酶(Tyrosinase, Tyr)的免疫组织化学染色,研究视泡发育过程中的分子分化。形态学染色结果发现,在整个视泡发育时期,预定神经视网膜和预定视网膜色素上皮组织结构变化明显。间脑两侧向外突出,形成视泡。然后视泡向上突起,最终与表皮外胚层接触。预定视网膜色素上皮的厚度逐渐变薄,而预定神经视网膜的厚度逐渐增加。同时,发现细胞凋亡存在于整个视泡发育过程,但是凋亡细胞的密度随着视泡发育进程而逐渐降低。在同一发育时期内,腹部和前部预定神经视网膜中凋亡细胞的密度在不同的发育模式中相对较高(P0.01)；背腹轴发育模式的视网膜凋亡细胞密度大于前后轴发育模式,而且预定视网膜色素上皮的凋亡细胞密度大于预定神经视网膜中的凋亡细胞密度(P0.01)。胚胎期的视网膜免疫组织化学染色发现,GFAP和RT97表达水平的变化趋势接近一致。视泡发育初期,GFAP和RT97在预定视网膜色素上皮中的表达水平很高,相反在预定神经视网膜表达非常少。视泡发育末期,GFAP和RT97在预定神经视网膜表达水平提高,而在预定视网膜色素上皮中降至最低值。在绝大多数发育时期,GFAP和RT97在腹部和前部PNR中的表达水平在不同的发育模式中相对比较高(P0.01)。GFAP和RT97抗体在视网膜前后轴发育模式中的表达水平要高于背腹轴发育模式中的表达水平(P0.01)。在视泡发育初期,Tyr在预定视网膜色素上皮的表达水平很低,而在预定神经视网膜的表达水平却很高。在随后的发育时期内,Tyr在预定视网膜色素上皮的表达水平逐渐提高,而在预定神经视网膜的表达水平降至最低值。在背腹轴发育模式中,Tyr在腹部预定视网膜色素上皮的表达水平相对较高(P0.01)。在视泡发育前期,Tyr在前部预定视网膜色素上皮的表达水平相对较高(P0.01);但是到了发育后期,Tyr在后部预定视网膜色素上皮的表达水平相对较高(P0.01)。与视泡形成初期相比,GFAP、RT97和Tyr在视网膜中的表达水平变化明显(P0.01)。本实验结果说明细胞凋亡是两栖动物视泡发育过程中的一种普遍生物学现象,它存在于各个发育阶段,并形成了空间和时间的变化模式。细胞凋亡现象在背腹轴发育模式中比较明显,因此细胞凋亡的作用可能在视泡的背腹轴发育模式中更为重要。在同一发育时期,预定视网膜色素上皮中的凋亡细胞的密度要高于预定神经视网膜中的凋亡的细胞密度,而且预定视网膜色素上皮的厚度变的比预定神经视网膜薄,因此细胞凋亡可能是引起眼发育早期形态分化的原因之一。 GFAP、RT97和Tyr表达的差异性,说明在视泡发育过程中,发生了分子分化。预定神经视网膜和预定视网膜色素上皮来源于相同的组织,因此在眼发育早期,GFAP、RT97和Tyr同时表达,但是它们的表达水平不同。数据显示这三种蛋白不同的表达水平可能和预定神经视网膜和预定视网膜色素上皮在视泡中不同的位置有关。这三种抗体的表达在视泡发育5-6小时后有个交叉点,此时视泡正好与外胚层接触。从分子分化的观点上认为,特定的分子分化起始于视泡和外胚层的接触后,这三种抗体的表达显示出视网膜发育过程中的不同细胞类型的分化。在这个交叉点后,神经元、神经胶质细胞和色素细胞根据它们将来的功能而不是在视网膜中的位置,开始表达一些特异性的蛋白质。
[Abstract]:The development of vertebrate retina is a complex morphological and molecular biological process . In the course of development of visual cup to mature retina , there is a phenomenon of cell differentiation and differentiation , but in the course of development of retinal pigment epithelium ( PRPE ) and pre - defined nerve retina ( pnr ) , the related research is less , and its biological mechanism involved in morphological change is not clear . This experiment is to study the mechanism of cell apoptosis and molecular differentiation in the process of cell development through morphological and immunohistochemical experiments .

In this experiment , the morphological changes of the visual cell were observed by HE staining .
The apoptosis of the retinal pigment epithelium and the pre - determined retinal pigment epithelium was observed by TUNEL staining . Finally , the molecular differentiation in the process of cell development was studied by immunohistochemical staining of GFAP , RT97 and catalytic enzyme - tyrosinase ( Tyr ) in the process of pigment synthesis .

At the same time , the density of apoptotic cells was relatively high in different developmental patterns ( P0.01 ) .
The apoptotic cell density of the retinal pigment epithelium was greater than that in the pre - and posterior - axis development model , and the density of apoptotic cells was greater than that in the pre - determined retinal pigment epithelium ( P0.01 ) .

The expression levels of GFAP and RT97 in the pre - determined retinal pigment epithelium were higher than those in the development model of the dorsal axises ( P0.01 ) .

The results indicate that apoptosis is a common biological phenomenon in the development of cell apoptosis , which exists in various developmental stages and forms a pattern of spatial and temporal changes . Apoptosis is more important in the development of dorsal axises .

The difference between GFAP , RT97 and Tyr indicates that molecular differentiation occurs during the development of the cell . The expression levels of these three antibodies may be related to the different positions of the predetermined nerve retina and the predetermined retinal pigment epithelium in the development of the eye . The data show that the expression levels of these three proteins may be related to the different positions of the predetermined nerve retina and the predetermined retinal pigment epithelium in the cell . The expression of these three antibodies is shown to be the differentiation of different cell types in the development of the retina . After this crossing point , the neurons , glia and pigment cells begin to express some specific proteins based on their future function , rather than in the retina .

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

【参考文献】