【摘要】：白內(nèi)障是眾多致盲原因中最常見的一種,嚴(yán)重威脅人類的健康。硒是一個(gè)人體必需微量元素,以硒蛋白的形式發(fā)揮作用。其中,有研究表明小鼠15kDa硒蛋白(Sep15)基因敲除出生后1.5個(gè)月即出現(xiàn)晶狀體渾濁,可見Sep15在晶狀體生長(zhǎng)發(fā)育過程中發(fā)揮重要作用,然而其機(jī)理不清楚。探討Sep15與白內(nèi)障的關(guān)系,對(duì)于利用硒預(yù)防或推遲白內(nèi)障的發(fā)生具有重要的科學(xué)意義和潛在的應(yīng)用價(jià)值。基于晶狀體鈣黏蛋白(N-cadherin,CDH2)在晶狀體細(xì)胞分化中發(fā)揮不可或缺的作用,本研究以人晶狀體上皮細(xì)胞(SRA01/04)為研究對(duì)象,采用RNAi、實(shí)時(shí)熒光定量PCR、蛋白質(zhì)印跡等方法,探討了Sep15基因沉默和毒胡蘿卜素(Tg)對(duì)鈣黏蛋白表達(dá)的影響,以揭示Sep15在晶狀體細(xì)胞分化中的作用。主要研究結(jié)果如下:(1)Tg對(duì)晶狀體細(xì)胞的損傷作用呈現(xiàn)濃度依賴性,隨著濃度的增加,細(xì)胞凋亡率增加;1μM Tg可顯著誘發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激,增加GRP78的mRNA及蛋白質(zhì)表達(dá)水平;低濃度的Tg可誘導(dǎo)Cadherin的表達(dá)增加,但不影響Sep15蛋白質(zhì)水平;高濃度的Tg則明顯抑制Cadherin和Sep15的蛋白質(zhì)表達(dá)。(2)用堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)作用于晶狀體細(xì)胞,低濃度bFGF可明顯刺激細(xì)胞的增殖,高濃度的bFGF則促進(jìn)細(xì)胞的分化。Tg與b FGF共同作用于細(xì)胞后,bFGF的加入可以緩解由內(nèi)質(zhì)網(wǎng)應(yīng)激所造成的細(xì)胞損傷,減少Tg對(duì)Cadherin表達(dá)的影響;而Tg的加入又會(huì)妨礙到bFGF介導(dǎo)的細(xì)胞分化。(3)Tg和bFGF的加入,對(duì)Sep15基因沉默效果無顯著影響;而Sep15基因沉默不影響GRP78的表達(dá),也未能引起CDH2蛋白質(zhì)表達(dá)水平的顯著性變化,說明Sep15基因敲除導(dǎo)致白內(nèi)障的形成,與內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)傳導(dǎo)途徑和細(xì)胞分化信號(hào)分子CDH2沒有直接關(guān)系;但Sep15基因沉默明顯加劇了Tg誘發(fā)的內(nèi)質(zhì)網(wǎng)應(yīng)激,導(dǎo)致CDH2表達(dá)的增加。該結(jié)果表明Sep15在Tg誘發(fā)內(nèi)質(zhì)網(wǎng)應(yīng)激所致的CDH2表達(dá)紊亂發(fā)揮重要保護(hù)作用。
[Abstract]:Cataract is the most common cause of blindness, a serious threat to human health. Selenium is a necessary trace element in human body and plays a role in the form of selenoprotein. Among them, some studies have shown that the mouse 15kDa selenoprotein (Sep15) gene knockout 1. 5 months after birth appeared lens opacity, we can see that Sep15 plays an important role in lens growth and development, but its mechanism is not clear. To explore the relationship between Sep15 and cataract is of great scientific significance and potential application value to prevent or delay the occurrence of cataract by using selenium. Based on the indispensable role of lens cadherin (CDH 2) in lens differentiation, human lens epithelial cells (SRA01/04) were studied in this study. Real time quantitative PCR, (PCR,) was used to determine the expression of CDH 2 in human lens epithelial cells (LECs). The effects of Sep15 gene silencing and carotene (Tg) on the expression of cadherin were studied by Western blot to reveal the role of Sep15 in lens cell differentiation. The main results were as follows: (1) the damage effect of Tg on lens cells was concentration-dependent, and the apoptosis rate increased with the increase of concentration, 1the Tg induced endoplasmic reticulum stress significantly, and increased the expression of mRNA and protein of GRP78. Low concentration of Tg could increase the expression of Cadherin, but did not affect the level of Sep15 protein. High concentration of Tg significantly inhibited the protein expression of Cadherin and Sep15. (2) the lens cells were treated with basic fibroblast growth factor (bFGF), and the proliferation of lens cells was significantly stimulated by low concentration of bFGF. High concentration of bFGF promoted the differentiation of cells. After TG and bFGF co-acted on cells, the addition of bFGF could alleviate the cell damage caused by endoplasmic reticulum stress and reduce the effect of Tg on the expression of Cadherin. The addition of Tg could prevent bFGF-mediated cell differentiation. (3) the addition of Tg and bFGF had no significant effect on the silencing effect of Sep15 gene. The silencing of Sep15 gene did not affect the expression of GRP78, nor did it cause significant changes in the expression level of CDH2 protein, indicating that Sep15 gene knockout resulted in cataract formation. There was no direct relationship between endoplasmic reticulum stress signal transduction pathway and cell differentiation signal molecule CDH2. However, Sep15 gene silencing significantly aggravated the endoplasmic reticulum stress induced by Tg, which resulted in the increase of CDH2 expression. These results suggest that Sep15 plays an important role in the expression disorder of CDH2 induced by Tg-induced endoplasmic reticulum stress.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R776.1

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