花背蟾蜍视网膜形态发育及视网膜生理活动对角膜诱导影响研究

发布时间：2018-10-22 20:25

【摘要】：花背蟾蜍(Bufo Raddei Strauch)是本实验室研究眼睛发育常用的两栖类模式动物。本文利用常规组织学技术、透射电镜技术、扫描电镜技术和免疫组织化学技术对花背蟾蜍视网膜进行了如下研究：(1)视网膜形态结构及胚后形态发育过程研究,(2)视网膜生理活动与角膜诱导关系研究,(3)不同龄期蟾蜍视网膜增殖细胞分裂能力研究。利用光镜技术和电镜技术对正常成体花背蟾蜍视网膜形态进行了观察,研究结果显示,光镜下花背蟾蜍视网膜具有脊椎动物视网膜典型的十层结构。透射电镜下观察花背蟾蜍视网膜结构特点为：视网膜色素上皮细胞胞质内存在独特的巨大吞噬体；在光感受器细胞内节可观察到椭球体与肌样体结构,视锥细胞内节未观察到起滤色作用脂滴；光感受器细胞外节周围可见辅助外节和花萼状突起断面；光感受器细胞的内突部分与第二级神经元形成突触带。扫描电镜对视网膜色素上皮观察可见排列整齐的色素细胞突起。视网膜断面观察可看到大量的视杆细胞和少数视锥细胞。视杆细胞外、内节表面分布明显的小叶间沟。围绕在光感受器细胞内节周围可见Muller细胞突起形成的外界膜。利用GFAP抗体对视网膜切片进行免疫组化染色发现,Muller细胞是花背蟾蜍视网膜中唯一的胶质细胞,视野中可见它沿着眼球半径呈单层放射状排列的横断面,Muller细胞主干终末膨大形成的圆锥型终足分布于内界膜。利用半薄切片技术及电镜技术对花背蟾蜍26-39期(胚后发育期)蝌蚪视网膜发育过程观察发现,视网膜分层区域逐渐从中央向边缘区扩展。在26、27、28和35期蝌蚪视网膜的边缘存在一逐渐闭合的裂孔结构。透射电镜观察可见到26至39期色素细胞色素颗粒逐渐增加,突起逐渐伸长。同时可观察到光感受器细胞外节内的膜盘增多,视锥细胞内节特有的含脂质色素脂滴逐渐形成过程。用移植皮肤将蝌蚪角膜替换,移植皮肤在一定因素诱导下透明成为角膜即角膜诱导。利用眶内注射河豚毒素阻断蝌蚪视神经生理活动,观察不同时间点移植角膜中黑素色颗粒数量变化发现,实验组角膜与对照组角膜黑素色颗粒都随着时间延续逐渐减少,但实验组角膜透明过程显著慢于对照组,两者差异显著(p0.05)。此结果说明视网膜生理活动是诱导角膜透明的直接因素之一。为了观察花背蟾蜍视网膜内神经细胞增殖能力与蟾蜍年龄关系,利用Skeletochronology技术对花背蟾蜍年龄进行了鉴定,并对其群体年龄结构和生长模式进行了分析,分析结果显示实验中所用的成体花背蟾蜍年龄可达到6龄。利用BrdU标记分裂细胞的方法,对花背蟾蜍30、35、39期蝌蚪、变态后幼体,以及1龄、2龄、3龄成体视网膜增殖细胞进行显示表明,随着花背蟾蜍生长时间延续,其视网膜中具有分裂能力的神经细胞逐渐减少,2龄期视网膜中几乎没有增殖细胞存在。由此可以推论无尾两栖动物可能是视网膜神经细胞由终生保持分裂能力到短期保持分裂能力的过渡动物。本研究从以上3方面对花背蟾蜍视网膜进行观察研究,在视网膜形态结构方面观察到色素上皮细胞中存在着巨大吞噬体、在胚后发育中观察到的视网膜裂孔、以及神经细胞分裂能力与年龄的关系、视网膜生理活动与角膜诱导关系等,为今后进一步进行相关研究奠定了研究基础。
[Abstract]:Bufo Raddei Strauch (Bufo Raddei Strauch) is one of the most commonly used animal models for the development of eye development in our laboratory. In this paper, the retinal morphology and morphology development were studied by using conventional histological techniques, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunohistochemistry. (2) Study on the relationship between retinal physiology activity and cornea induction, and (3) study on cell division ability of toad retina at different age. The morphology of toad retina was observed by light microscopy and electron microscopy. The results showed that the retinas of toad retina was the typical ten in the retina under light microscope. The structure of toad retina was observed under transmission electron microscope (TEM). An auxiliary outer section and a flower-shaped protrusion section are visible around the outer section of the photoreceptor cell; the inner and second stage neurons of the photoreceptor cell are formed Synaptic band. Scanning electron microscope showed the visible arrangement of pigment fine in retinal pigment epithelium. The observation of retinal cross-section can see a large number of rod cells and a few eyes. In addition to the cells of the cone, the surface distribution of the inner section was significantly smaller than that of the rod cells. An inter-leaf groove formed by a Muller cell protrusion that is visible around the inside of a photoreceptor cell. It was found by immunohistochemical staining of retinal sections with anti-inflammatory antibodies, which was the only glial cell in the retina of Bufo gartoads, and it was seen in the field of view that it was arranged radially along the radius of the eyeball. In cross-section, the cone-type final foot formed by the terminal enlargement of the main stem of Muller cells was distributed in Using semi-thin slice technique and electron microscope technique to observe the development of tadpole's retina during the 26-39 period (embryonic development stage), the retinal delamination area was gradually observed. In the 26, 27, 28, and 35 tadpoles, there was a gradual increase in the edge of the tadpoles retina. According to transmission electron microscope, the pigment particles of 26 to 39 pigment cells were gradually increased. At the same time, it can be observed that the film disc in the outer section of photoreceptor cells is increased, and the specific lipid-containing pigment grease is peculiar to the inside of the cone cell. The process is gradually formed. The tadpole cornea is replaced by transplanting the skin, and the transplanted skin is induced by a certain factor. Using intraorbital injection of dodotoxin to block tadpole's optic nerve physiological activity, the changes of the number of black pigment particles in the cornea were observed in different time points, and the corneal and black pigment particles in the experimental group and the control group were observed. As time continues to decrease, the corneal transparency in the experimental group is significantly slower than that in the control group, both The difference was significant (p0.05). This result shows that the retinal physiology activity is induced One of the direct factors of corneal transparency was to observe the relationship between the proliferation of nerve cells and the age of toad in toad retina. The age structure and growth model of the body were analyzed, and the results showed that By using BrdU, the cells were displayed in 30, 35, 39 tadpoles, perverted larvae, and 1, 2, and 3-year-old retinal proliferative cells. It is shown that with the duration of the growth time of the toad toad, the nerve cells with splitting ability in the retina are gradually decreased, and the age of 2 years There is almost no proliferating cell in the retina of the retina. Thus, it is possible to deduce that the non-proliferative cells may be retinal nerve cells that remain divided by life for life. From the above three aspects, we observed the retinal morphology, observed the presence of giant phagocytes in the epithelial cells of the pigment epithelium, and observed the retina after the embryo development. The relationship between the splitting ability and age of nerve cells, the relationship between retinal physiology activity and cornea induction, etc.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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