[1]杨静. 人原代晶状体上皮细胞中PEDF基因下调引起波形蛋白和αB-晶状体蛋白含量的变化[J].眼科新进展,2015,35(8):713-717.[doi:10.13389/j.cnki.rao.2015.0195]
　YANG Jing. Reduction of PEDF regulating expression of vimentin and αB-crystallin in human lens epithelial cells[J].Recent Advances in Ophthalmology,2015,35(8):713-717.[doi:10.13389/j.cnki.rao.2015.0195]
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 人原代晶状体上皮细胞中PEDF基因下调引起波形蛋白和αB-晶状体蛋白含量的变化
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
35卷
期数:
2015年8期
页码:
713-717
栏目:
实验研究
出版日期:
2015-08-05

文章信息/Info

Title:
 Reduction of PEDF regulating expression of vimentin and αB-crystallin in human lens epithelial cells
作者:
 杨静
 510060　广东省广州市，中山大学中山眼科中心
Author(s):
 YANG Jing
 Zhongshan Ophthalmic Center , Sun Yat-sen University , Guangzhou 510060 , Guangdong Province . China
关键词:
 ; 色素上皮衍生因子; RNA干扰; 波形蛋白; αB-晶状体蛋白; 人晶状体上皮细胞
Keywords:
 ; pigment epithelium-derived factor ;  RNAi;  vimentin ;  αB-crystallin ;  human lens epithelial cells
DOI:
10.13389/j.cnki.rao.2015.0195
文献标志码:
A
摘要:
 目的　研究人原代晶状体上皮细胞中，下调色素上皮衍生因子（ｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ－ｄｅｒｉｖｅｄｆａｃｔｏｒ，ＰＥＤＦ）的表达对波形蛋白和αＢ－晶状体蛋白表达的影响。方法　取２０～３５岁青年人晶状体上皮细胞进行原代培养。构建三种短发卡ＲＮＡ（ｓｈｏｒｔｈａｉｒｐｉｎＲＮＡ，ｓｈＲＮＡ）以特异性下调原代人晶状体上皮细胞中的ＰＥＤＦ基因ＲＮＡ表达水平。感染细胞后，用Ｒｅａｌ－ｔｉｍｅＰＣＲ检测ＰＥＤＦ基因ｍＲＮＡ的表达水平，确定ＲＮＡ干扰效率。人原代晶状体上皮细胞ＰＥＤＦ基因ＲＮＡ干扰４８ｈ后，用ＷｅｓｔｅｒｎＢｌｏｔ检测其中波形蛋白以及αＢ－晶状体蛋白表达水平的变化，以ｓｈＲＮＡ－ｓｃｒａｍｂｌｅ组为阴性对照组。结果　人原代晶状体上皮细胞被成功分离培养，带有绿色荧光蛋白的表达ＰＥＤＦｓｈＲＮＡ的病毒载体对人原代晶状体上皮细胞的感染效率都在８０％左右。Ｒｅａｌ－ｔｉｍｅＰＣＲ结果显示ＰＥＤＦ基因ｍＲＮＡ的表达明显被三种ｓｈＲＮＡ下调。ＷｅｓｔｅｒｎＢｌｏｔ结果显示ＰＥＤＦ基因的表达下调引起了波形蛋白的表达下降（在三个实验组中分别下降了７２％、７９％和９３％）以及αＢ－晶状体蛋白的表达增加（在三个实验组中分别增加了２１９％、２０４％、９３％）。结论　人原代晶状体上皮细胞中ＰＥＤＦ基因下调可引起维持晶状体透明性的两种重要蛋白的改变，提示ＰＥＤＦ与白内障形成有一定的联系。
Abstract:
 Objective To explore the effect of pigment epithelium-derived factor ( PEDF) down-regulation on the expression of vimentin and αB-crystallin in human lens epithelial cells. Methods Human lens epithelial cells adhering to anterior capsules collected from young donor eyes aged from 20 - 35 years were cultured. Three short hairpin RNA ( shRNA) constructs were designed to specifically down-regulate the expression of PEDF in human lens epithelial cells. Real time PCR was performed to confirm the down regulation of PEDF gene expression. In order to detect whether reducing the gene expression of PEDF would affect the expression of vimentin or αB-crystallin, Western Blot was used at 48 hours after expression of the PEDF directed shRNA. Results Primary human lens epithelial cells were isolated and cultured successfully. 80% cells were infected with lentiviral PEDF shRNA particles. PEDF gene expression in the human lens epithelial cells was strongly down-regulated by the three separate shRNA constructs. Western blot results revealed that the down-regulation of PEDF expression resulted in a concomitant decrease in expression of vimnentin (72% ,7g% and 93% decrease in three experimental group respectively) and an increase in αB-crystallin protein (2lg% ,204% and 93% increase in three experimental group respectively) . Conclrision Decreased expression of PEDF in primary human lens epithelial cells results in alteration of vimentin and αB-crystallin ,which are two critical proteins for maintairung lens clarity. The result suggest a tight relationship between PEDF expression and cataract formation.

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更新日期/Last Update: 2015-07-31