Sep15基因沉默和毒胡萝卜素对人晶状体上皮细胞钙黏蛋白表达的影响
发布时间：2019-03-27 21:29
【摘要】：白内障是众多致盲原因中最常见的一种,严重威胁人类的健康。硒是一个人体必需微量元素,以硒蛋白的形式发挥作用。其中,有研究表明小鼠15kDa硒蛋白(Sep15)基因敲除出生后1.5个月即出现晶状体浑浊,可见Sep15在晶状体生长发育过程中发挥重要作用,然而其机理不清楚。探讨Sep15与白内障的关系,对于利用硒预防或推迟白内障的发生具有重要的科学意义和潜在的应用价值。基于晶状体钙黏蛋白(N-cadherin,CDH2)在晶状体细胞分化中发挥不可或缺的作用,本研究以人晶状体上皮细胞(SRA01/04)为研究对象,采用RNAi、实时荧光定量PCR、蛋白质印迹等方法,探讨了Sep15基因沉默和毒胡萝卜素(Tg)对钙黏蛋白表达的影响,以揭示Sep15在晶状体细胞分化中的作用。主要研究结果如下:(1)Tg对晶状体细胞的损伤作用呈现浓度依赖性,随着浓度的增加,细胞凋亡率增加;1μM Tg可显著诱发内质网应激,增加GRP78的mRNA及蛋白质表达水平;低浓度的Tg可诱导Cadherin的表达增加,但不影响Sep15蛋白质水平;高浓度的Tg则明显抑制Cadherin和Sep15的蛋白质表达。(2)用碱性成纤维细胞生长因子(bFGF)作用于晶状体细胞,低浓度bFGF可明显刺激细胞的增殖,高浓度的bFGF则促进细胞的分化。Tg与b FGF共同作用于细胞后,bFGF的加入可以缓解由内质网应激所造成的细胞损伤,减少Tg对Cadherin表达的影响;而Tg的加入又会妨碍到bFGF介导的细胞分化。(3)Tg和bFGF的加入,对Sep15基因沉默效果无显著影响;而Sep15基因沉默不影响GRP78的表达,也未能引起CDH2蛋白质表达水平的显著性变化,说明Sep15基因敲除导致白内障的形成,与内质网应激信号传导途径和细胞分化信号分子CDH2没有直接关系;但Sep15基因沉默明显加剧了Tg诱发的内质网应激,导致CDH2表达的增加。该结果表明Sep15在Tg诱发内质网应激所致的CDH2表达紊乱发挥重要保护作用。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R776.1
本文编号：2448578
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R776.1
【参考文献】
相关期刊论文 前8条
1 刘红梅;黄开勋;徐辉碧;;内质网硒蛋白的研究进展[J];中国科学:化学;2014年04期
2 周雪琼;贺凌飞;余日安;;内质网应激及未折叠蛋白反应通路研究进展[J];中国职业医学;2012年03期
3 曹莉;熊正文;;神经型钙黏附蛋白与肿瘤关系的研究进展[J];实用医学杂志;2012年03期
4 张琼;张昌菊;;宫颈鳞癌组织和细胞系中P120ctn及E-cad的表达及生物学意义[J];现代妇产科进展;2007年05期
5 黄才国;张军;李闻捷;毛积芳;;大鼠老化进程中晶状体蛋白水溶性向水不溶性的转变[J];国际眼科杂志;2007年01期
6 刘静,刘冰;后发性白内障9例的手术治疗[J];国际眼科杂志;2003年03期
7 马鹏鹏,薛社普,韩代书;RNA干扰技术的原理与应用[J];中国组织化学与细胞化学杂志;2003年02期
8 孙红,惠延年,王琳,韩泉洪,杜红俊,马吉献;晶状体上皮细胞凋亡与白内障发生的分子生物学机制[J];中国临床康复;2003年03期
相关博士学位论文 前4条
1 徐佳;先天性白内障相关的βB2晶状体蛋白突变的致病机制研究[D];浙江大学;2013年
2 易建华;上皮-间充质转变（EMT）在百草枯中毒大鼠肺纤维化中的作用及机制探讨[D];浙江大学;2012年
3 柳珑;GEFT调节晶状体上皮细胞分化的机制研究[D];湖南师范大学;2011年
4 周军;过氧亚硝酸根对胰岛素信号转导的调控作用及其机理[D];华中科技大学;2009年
，本文编号：2448578
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2448578.html
最近更新
教材专著
