【作者】 贾宗平；

【导师】 俞诗源；

【作者基本信息】 西北师范大学 ， 细胞生物学， 2007， 硕士

【摘要】 两栖类动物鳃的发育是一个复杂的过程,包括外鳃期、内鳃期、内鳃退化等阶段。本研究以花背蟾蜍发育过程中的鳃为研究对象,运用石蜡切片、半薄切片、透射电镜、免疫组化等相关技术,研究了花背蟾蜍鳃的发生,发育,退化过程中的形态结构及细胞凋亡的变化规律,探论了细胞凋亡与鳃发育之间的关系。结果表明花背蟾蜍鳃的发生是从胚胎发育第15期鳃原基开始出现,随着发育,鳃原基分化出鳃板,鳃板继续分化出鳃弓,接着由鳃弓分化出外鳃,到胚胎发育20期形成以外鳃为呼吸器官的呼吸系统。外鳃进一步发育,形成了三排指状外鳃,到22-23期,达到发育高峰。到胚胎发育25期,外鳃被鳃盖包在鳃盖腔里面,内鳃开始形成,同时外鳃开始退化消失。内鳃的发育是首先由鳃弓上的突起形成鳃叶,其次鳃叶上出现初级突起,随着进一步发育出现次级突起和三级突起,直到内鳃发育最高峰(胚胎发育的36期)。随后进入变态期,内鳃开始退化,首先是三级突起开始萎缩,其次是次级突起和初级突起,最后是鳃弓,直至鳃弓完全消失,变态完成。电镜结果表明花背蟾蜍外鳃与内鳃具有相同的呼吸上皮,为扁平状,上皮细胞质中富含粗面内质网及空泡和线粒体;气血屏障由由单层上皮细胞,内皮细胞以及二者之间的基底膜构成,随着个体的发育,气血屏障逐渐变薄。在电镜下可见外鳃(25期)与内鳃退化时期(37-39期)鳃上的凋亡细胞,鳃细胞凋亡的形态学改变表现为:细胞体积明显缩小,形状改变:细胞胞质空泡化十分显著,胞浆浓缩,胞质减少,细胞质电子密度大,呈深色:胞质内细胞器结构模糊不清,数目明显减少或消失;细胞核凝集,染色质固缩,积聚成致密块,并伴有“凋亡小体”出现。但凋亡细胞出现机率最多的是外鳃与内鳃退化时期,即发育过程中的25期与37、38、39期。TUNEL免疫组化结果表明花背蟾蜍胚胎发育以下阶段都存在细胞阳性反应,但反应程度不同,23、24、27、31、35、36期有少量的细胞呈阳性,而在25、37、38期有大量的细胞呈阳性。表明在花背蟾蜍胚胎鳃的整个发育过程中都存在着细胞凋亡,但只有在外鳃和内鳃退化阶段出现大量的细胞凋亡。说明细胞凋亡是生物体维持机体发育平衡的一种最基本的生物现象。更多还原

【Abstract】 The gill development of the amphibians is a complicated process, it is comprised the outside gill period、inside gill period、inside gill degenerated period etc. The research studied the gill in the Bufo raddei Strauch with the research object, used the methods of the paraffin section、semithin section、transmission electron microscope、Immuno-histochemistry, studied the change rule of the morphological structure and apoptosis in the gill development, discussed the relation of the apoptosis and the gill development.The result showed the gill happened in the 15 stage when the swimbladder primordium appeared, the swimbladder primordium differentiated the gill plate, the gill plate differentiated gill arches, following it formed the outside gill, in the 20 stage, the outside gill is the respiration organ. the outside gill formed the three fingerlike, in the 22、23 stage, it developed at the peak, in the 25 stage, the operculum folded the outside gill in the operculum antrum, the inside gill formed, at the same time the outside gill degenerated. the outside gill disappeared, the gill arches developed to from the branchia, later the branchia appeared primary protuberance, along with developing, it appeared the secondary protuberance and the third protuberance, until the 36 stage, the stage is the inside gill developing at the peak, later it came into the period of the metamorphosis, the inside gill degenerated, the third protuberance began shrank, next the secondary protuberance and the primary protuberance shrank, finally the gill arches shrank and disappeared, the metamorphosis completed.The results of the TEM showed the same respiration epithelium in the inside gill and the gill filament, it is flat, the cytoplast of the epithelium contained of the rough endoplasmic reticulum, vacuole and mitochondrion in rich; the blood-gas barrier is formed of the monolayer epithelium, the endothelial cell and the basilar membrane between the both, following the development the blood-gas barrier attenuate gradually.The apoptosis can be observed in the outside gill period(25 stage) and inside gill period(37-39 stage), the morphological of the apoptosis cell in the gill showed: the volume of the cell reduced, the shape changed; the cytoplast vacuoled, the kytoplasm contracted, the cytoplast reduced,the electron density increased in the cytoplast, the frame of the organelle illegible and the amount of the the organelle decreased evidenctly, the karyon agglutinated, the chromatin pycnosised and accumulated, the apoptpticboclies appeared,but the apoptosis happened in the outside gill and the inside gill degenerated period, it is the 25、37、38、39stage.The result of the TUNEL Immuno-histochemistry showed the positive reaction existed in the development of the Bufo raddei strauch, In the 23、24、27、31、35、36 stage there is a small quantity of the cell, but in 25、37、38 there are abundant cell, it is indicated the apoptosis of the gill in the development of the Bufo raddei strauch, but in the outside gill and the inside gill degenerated period there are abundant apoptosis cell, the apoptosis is a basal biology process in maintaining the balance of the organism.更多还原