本文選題：晶狀體 + 形態(tài)變化��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：前言 晶狀體是由晶狀體上皮細(xì)胞(lens epithelial cells, LECs)和晶狀體纖維細(xì)胞構(gòu)成的透明器官,其獨(dú)特的細(xì)胞形態(tài)和規(guī)律的排列結(jié)構(gòu)對(duì)維持晶狀體的透明性具有重要作用[1,3]。白內(nèi)障的發(fā)生正是由于兩種細(xì)胞發(fā)生了形態(tài)結(jié)構(gòu)的改變,增加了對(duì)光的散射和吸收,導(dǎo)致晶狀體的透明度下降[1,2,4]。晶狀體細(xì)胞形態(tài)學(xué)的研究,對(duì)了解晶狀體的生理功能和白內(nèi)障的病理過(guò)程具有重要意義。 傳統(tǒng)的晶狀體細(xì)胞的形態(tài)學(xué)研究大多是將晶狀體固定切片后進(jìn)行光鏡、電鏡觀察,然而在樣本固定切片的過(guò)程中,化學(xué)及物理因素會(huì)引起晶狀體細(xì)胞形態(tài)結(jié)構(gòu)的改變破壞,并且該方法不能對(duì)同一樣本進(jìn)行連續(xù)觀察[5-7]。離體培養(yǎng)的晶狀體上皮細(xì)胞脫離了原來(lái)的解剖位置,細(xì)胞的形態(tài)發(fā)生了變化,并不適合于形態(tài)學(xué)的觀察研究[8-10]。由于觀察手段的缺乏,目前尚無(wú)研究對(duì)完整晶狀體上皮細(xì)胞及纖維細(xì)胞的形態(tài)變化特征進(jìn)行觀察和描述。 本研究通過(guò)對(duì)倒置相差顯微鏡環(huán)形光闌的調(diào)整,得到了一種觀察完整晶狀體上細(xì)胞形態(tài)變化的方法,并用該方法觀察記錄了晶狀體上皮細(xì)胞的增殖、凋亡及纖維細(xì)胞受損后的形態(tài)變化過(guò)程,為晶狀體細(xì)胞的生理病理研究提供了形態(tài)學(xué)資料。 第一部分倒置相差顯微鏡觀察完整晶狀體上皮細(xì)胞的形態(tài)變化 目的倒置相差顯微鏡觀察完整晶狀體上皮細(xì)胞分裂增殖及凋亡活動(dòng)的形態(tài)變化特征。 方法體外培養(yǎng)Wistar大鼠(200-250g)晶狀體。將完整晶狀體置于倒置相差顯微鏡(Leica DMI3000)下,調(diào)整環(huán)狀光闌以獲得最佳的觀察效果。培養(yǎng)的晶狀體分為三組：第一組為IGF-I組,大鼠晶狀體在20ng/ml的IGF-I中孵育15小時(shí),誘導(dǎo)晶狀體上皮細(xì)胞(LECs)分裂增殖；第二組為紫外照射組,利用相差顯微鏡本身的340-380nm紫外激發(fā)光(物鏡40×)照射晶狀體前表面10分鐘,誘導(dǎo)上皮細(xì)胞發(fā)生凋亡；第三組為空白對(duì)照組,大鼠晶狀體體外培養(yǎng)7天。應(yīng)用倒置相差顯微鏡觀察記錄上皮細(xì)胞分裂增殖和凋亡的形態(tài)變化過(guò)程。應(yīng)用凋亡試劑盒(Hoechst33342/YO-PRO(?)-1/PI)對(duì)晶狀體上皮細(xì)胞(LECs)進(jìn)行染色,觀察測(cè)定細(xì)胞凋亡 結(jié)果采用環(huán)狀光闌PHO搭配高倍物鏡(20×,40×)觀察完整晶狀體的上皮細(xì)胞,能獲得最佳的觀察效果；空白對(duì)照組晶狀體前表面存在均勻散在小隆起,晶狀體上皮細(xì)胞(LECs)光鏡下無(wú)法分辨,培養(yǎng)7天后未見(jiàn)明顯的形態(tài)改變；在IGF-I中孵育15小時(shí)后,晶狀體前表面出現(xiàn)大量細(xì)胞分裂相,染色體及細(xì)胞輪廓清晰可見(jiàn),分裂細(xì)胞的數(shù)量、位置及分裂時(shí)相可以直接觀察得到；晶狀體上皮細(xì)胞受到紫外線照射后,細(xì)胞核移向一邊,原位留下圓形“空穴”結(jié)構(gòu)。凋亡試劑盒(Hoechst33342/YO-PRO(?)-1/PI)檢測(cè)發(fā)現(xiàn),紫外照射區(qū)的上皮細(xì)胞全部凋亡,照射區(qū)以外的上皮細(xì)胞未受影響。 結(jié)論倒置相差顯微鏡下,完整晶狀體上皮細(xì)胞在分裂增殖和凋亡過(guò)程中表現(xiàn)出了獨(dú)特的形態(tài)學(xué)變化。 第二部分倒置相差顯微鏡觀察完整晶狀體纖維細(xì)胞的形態(tài)變化 目的倒置相差顯微鏡觀察完整晶狀體纖維細(xì)胞損傷后的形態(tài)變化特征。 方法體外培養(yǎng)Wistar大鼠(200-250g)晶狀體,分為四組：第一組為紫外照射組,利用相差顯微鏡本身的340-380nm紫外激發(fā)光(物鏡40×)照射晶狀體后表面10分鐘；第二組為H202組,大鼠晶狀體在100μM的H202中孵育48小時(shí)；第三組為高濃度半乳糖組,大鼠晶狀體在150μM的半乳糖中孵育72小時(shí)；第四組為空白對(duì)照組,大鼠晶狀體體外培養(yǎng)7天。應(yīng)用倒置相差顯微鏡觀察記錄晶狀體纖維細(xì)胞損傷后的形態(tài)特征。 結(jié)果空白對(duì)照組晶狀體后表面淺層纖維形態(tài)及排列結(jié)構(gòu)清晰可見(jiàn),培養(yǎng)7天后未見(jiàn)明顯的形態(tài)改變；纖維細(xì)胞受到紫外線照射后,出現(xiàn)兩種形態(tài)的損傷,受照部位纖維變形凝聚成小球,附近的纖維雖然本身未出現(xiàn)改變,但其間距變寬；晶狀體在100μM的H202中孵育48小時(shí)后,赤道部出現(xiàn)環(huán)形損傷,并逐漸向中央推進(jìn)；晶狀體在150μM的半乳糖中孵育72小時(shí)后,表層纖維的出現(xiàn)放射樣損傷。 結(jié)論倒置相差顯微鏡下,不同因素誘導(dǎo)的完整晶狀體纖維損傷的形態(tài)學(xué)表現(xiàn)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R776.1

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