倒置相差显微镜观察完整晶状体上皮及纤维细胞的形态变化

发布时间：2018-07-02 13:42

本文选题：晶状体 + 形态变化　；参考：《复旦大学》2012年硕士论文

【摘要】：前言晶状体是由晶状体上皮细胞(lens epithelial cells, LECs)和晶状体纤维细胞构成的透明器官,其独特的细胞形态和规律的排列结构对维持晶状体的透明性具有重要作用[1,3]。白内障的发生正是由于两种细胞发生了形态结构的改变,增加了对光的散射和吸收,导致晶状体的透明度下降[1,2,4]。晶状体细胞形态学的研究,对了解晶状体的生理功能和白内障的病理过程具有重要意义。传统的晶状体细胞的形态学研究大多是将晶状体固定切片后进行光镜、电镜观察,然而在样本固定切片的过程中,化学及物理因素会引起晶状体细胞形态结构的改变破坏,并且该方法不能对同一样本进行连续观察[5-7]。离体培养的晶状体上皮细胞脱离了原来的解剖位置,细胞的形态发生了变化,并不适合于形态学的观察研究[8-10]。由于观察手段的缺乏,目前尚无研究对完整晶状体上皮细胞及纤维细胞的形态变化特征进行观察和描述。本研究通过对倒置相差显微镜环形光阑的调整,得到了一种观察完整晶状体上细胞形态变化的方法,并用该方法观察记录了晶状体上皮细胞的增殖、凋亡及纤维细胞受损后的形态变化过程,为晶状体细胞的生理病理研究提供了形态学资料。第一部分倒置相差显微镜观察完整晶状体上皮细胞的形态变化目的倒置相差显微镜观察完整晶状体上皮细胞分裂增殖及凋亡活动的形态变化特征。方法体外培养Wistar大鼠(200-250g)晶状体。将完整晶状体置于倒置相差显微镜(Leica DMI3000)下,调整环状光阑以获得最佳的观察效果。培养的晶状体分为三组：第一组为IGF-I组,大鼠晶状体在20ng/ml的IGF-I中孵育15小时,诱导晶状体上皮细胞(LECs)分裂增殖；第二组为紫外照射组,利用相差显微镜本身的340-380nm紫外激发光(物镜40×)照射晶状体前表面10分钟,诱导上皮细胞发生凋亡；第三组为空白对照组,大鼠晶状体体外培养7天。应用倒置相差显微镜观察记录上皮细胞分裂增殖和凋亡的形态变化过程。应用凋亡试剂盒(Hoechst33342/YO-PRO(?)-1/PI)对晶状体上皮细胞(LECs)进行染色,观察测定细胞凋亡结果采用环状光阑PHO搭配高倍物镜(20×,40×)观察完整晶状体的上皮细胞,能获得最佳的观察效果；空白对照组晶状体前表面存在均匀散在小隆起,晶状体上皮细胞(LECs)光镜下无法分辨,培养7天后未见明显的形态改变；在IGF-I中孵育15小时后,晶状体前表面出现大量细胞分裂相,染色体及细胞轮廓清晰可见,分裂细胞的数量、位置及分裂时相可以直接观察得到；晶状体上皮细胞受到紫外线照射后,细胞核移向一边,原位留下圆形“空穴”结构。凋亡试剂盒(Hoechst33342/YO-PRO(?)-1/PI)检测发现,紫外照射区的上皮细胞全部凋亡,照射区以外的上皮细胞未受影响。结论倒置相差显微镜下,完整晶状体上皮细胞在分裂增殖和凋亡过程中表现出了独特的形态学变化。第二部分倒置相差显微镜观察完整晶状体纤维细胞的形态变化目的倒置相差显微镜观察完整晶状体纤维细胞损伤后的形态变化特征。方法体外培养Wistar大鼠(200-250g)晶状体,分为四组：第一组为紫外照射组,利用相差显微镜本身的340-380nm紫外激发光(物镜40×)照射晶状体后表面10分钟；第二组为H202组,大鼠晶状体在100μM的H202中孵育48小时；第三组为高浓度半乳糖组,大鼠晶状体在150μM的半乳糖中孵育72小时；第四组为空白对照组,大鼠晶状体体外培养7天。应用倒置相差显微镜观察记录晶状体纤维细胞损伤后的形态特征。结果空白对照组晶状体后表面浅层纤维形态及排列结构清晰可见,培养7天后未见明显的形态改变；纤维细胞受到紫外线照射后,出现两种形态的损伤,受照部位纤维变形凝聚成小球,附近的纤维虽然本身未出现改变,但其间距变宽；晶状体在100μM的H202中孵育48小时后,赤道部出现环形损伤,并逐渐向中央推进；晶状体在150μM的半乳糖中孵育72小时后,表层纤维的出现放射样损伤。结论倒置相差显微镜下,不同因素诱导的完整晶状体纤维损伤的形态学表现不同。
[Abstract]:Preface
Lens is a transparent organ composed of lens epithelial cells (lens epithelial cells, LECs) and crystalline fibrous cells. The unique morphology and regularity of the cells play an important role in maintaining the transparency of the lens. The occurrence of cataracts is due to the morphological changes of the two kinds of cells. The scattering and absorption of light causes the transparency of the lens to decrease the morphology of [1,2,4]. lens cells, which is of great significance to understanding the physiological function of the lens and the pathological process of cataract.
Most of the morphological studies of the traditional lens cells are fixed and sectioned by light microscopy and electron microscopy. However, the chemical and physical factors may cause changes in the morphological structure of the lens cells during the fixed section of the sample, and the method can not continuously observe the crystalline form of the [5-7]. in vitro culture. The body epithelial cells are separated from the original anatomical position, and the morphology of the cells has changed. It is not suitable for morphological observation and study of the lack of observation means. There is no study on the morphological changes of the complete lens epithelial cells and fibroblasts in [8-10]..
In this study, a method of observing the morphological changes of cells on the complete lens was obtained through the adjustment of the inverted phase contrast microscope ring aperture. The method was used to observe the proliferation, apoptosis and morphological changes of the epithelial cells of the lens, which provided the morphology for the physiological and pathological study of the crystalline cells. Information.
The morphological changes of intact lens epithelial cells were observed by inverted phase contrast microscope.
Objective To observe the morphological changes of mitotic proliferation and apoptosis in intact lens epithelial cells by inverted phase contrast microscope.
Methods the Wistar rat (200-250g) lens was cultured in vitro. The complete lens was placed under the inverted phase contrast microscope (Leica DMI3000) to adjust the annular aperture to obtain the best observation effect. The cultured lens was divided into three groups: the first group was group IGF-I, the rat lens incubated for 15 hours in the IGF-I of 20ng/ml, and the lens epithelial cells (LEC) were induced. S) split and proliferate; the second group was ultraviolet irradiation group. The 340-380nm ultraviolet stimulated luminescence of the phase microscope itself (the objective lens 40 x) irradiated the anterior surface of the lens for 10 minutes, and the apoptosis of the epithelial cells was induced. The third groups were blank control group and the rat lens was cultured in vitro for 7 days. The cell division of the epithelial cells was recorded by the inverted phase contrast microscope. The process of morphological changes of colonization and apoptosis. Hoechst33342/YO-PRO (?) -1/PI was used to stain the lens epithelial cells (LECs) and observe the apoptosis.
Results the best observation results were obtained by using the annular aperture PHO with high magnification lens (20 x, 40 x) to observe the epithelial cells of the whole lens. The anterior surface of the blank control group was evenly scattered in the small protuberance, the lens epithelial cells (LECs) could not be identified by the light microscope, and no obvious morphological changes were found after 7 days of cultured lens; 15 of the lenses were incubated in IGF-I. After hours, a large number of cell division phases appeared on the front surface of the lens, and the chromosomes and cell outlines were clearly visible. The number, position and phase of split cells could be observed directly. After the lens epithelial cells were irradiated with ultraviolet radiation, the nucleus moved to one side, and the circular "hole" structure was left in situ. The apoptosis Kit (Hoechst33342/YO -PRO (?) -1/PI detection revealed that all the epithelial cells in the UV irradiated area were completely apoptotic, and the epithelial cells outside the irradiation area were not affected.
Conclusion under the inverted phase contrast microscope, the intact lens epithelial cells show unique morphological changes during the process of division, proliferation and apoptosis.
In the second part, the morphological changes of intact lens fibroblasts were observed by inverted phase contrast microscope.
Objective To observe the morphological changes of intact lens fibroblasts after inverted phase contrast microscope.
Methods the lens of Wistar rats (200-250g) was divided into four groups in vitro: the first group was the ultraviolet irradiation group, and the 340-380nm ultraviolet luminescence (40 *) of the phase microscope was used to irradiate the posterior surface of the lens for 10 minutes. The second group was group H202, the rat lens was incubated in the H202 of 100 mu M, and the third group was the high concentration galactose group. The rat lens was incubated for 72 hours in 150 M galactose, and the fourth groups were blank control group and the rat lens was cultured in vitro for 7 days. The morphological characteristics of the lens fibroblast injury were recorded by inverted phase contrast microscope.
Results in the blank control group, the superficial fiber morphology and arrangement of the superficial layer of the posterior surface of the lens was clearly visible, and no obvious morphological changes were found after 7 days of culture. After the ultraviolet radiation of the fibroblasts, there were two forms of damage, and the fibrous deformation in the irradiated area was condensed into small balls. After incubation of the lens in 100 M H202 for 48 hours, the equatorial damage occurred and the lens was gradually pushed forward to the central area. After incubation of the lens in 150 u M galactose for 72 hours, the surface fiber was damaged by radiation.
Conclusion under the inverted phase contrast microscope, the morphological characteristics of intact lens fibers induced by different factors are different.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R776.1

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