| Arkadia和UCH37表达载体的构建及其对TGF-β1/Smads信号通路的影响 |
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| 引用本文： | 廖芳芳,苑嗣纯,张中文,吴国娟.Arkadia和UCH37表达载体的构建及其对TGF-β1/Smads信号通路的影响[J].中国农业科学,2012,45(9):1848-1856. |
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| 作者姓名： | 廖芳芳 苑嗣纯 张中文 吴国娟 |
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| 作者单位： | 北京农学院动物科技学院，北京 102206 |
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| 基金项目： | 国家自然科学甚金项目，北京市自然科学基金，北京市属高校人才强教i计划项目(PHR) |
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| 摘 要： | 【目的】构建泛素启动酶Arkadia和泛素羧基末端水解酶-5（亦称UCH37）的表达载体，并探讨Smad7蛋白的泛素化修饰对马兜铃酸肾病中TGF-β1/Smads信号通路的影响。【方法】通过观察绿色荧光的表达量及real-time PCR反应优化转染条件，转染Arkadia和UCH37 siRNA，real-time PCR检测mRNA表达抑制率。分别构建Arkadia和UCH37的表达载体pGPU6/GFP/Neo，经鉴定后转染细胞，real-time PCR和Western blotting检测Arkadia、UCH37和Smad7的表达变化。【结果】①30 pmol siRNA和1.5 μL LipofectamineTM 2000为最佳转染条件；②Arkadia-1490和UCH37-721的干扰效果最明显；③载体的插入序列完全正确，且pGPU6/GFP/Neo-Arkadia能上调马兜铃酸肾病模型中Smad7的表达，而pGPU6/GFP/Neo-UCH37使Smad7表达下降。【结论】成功构建了表达载体pGPU6/GFP/Neo-Arkadia和pGPU6/GFP/Neo-UCH37，且Arkadia和UCH37可分别放大、抑制TGF-β1/Smads信号通路。
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| 关 键 词： | Arkadia基因 UCH37基因 转染 表达载体 TGF-β1/Smads信号通路 |
| 收稿时间： | 2011-11-15 |
Constructions of Arkadia and UCH37 Expression Vectors and Effects on TGF-β1/Smad7 Signal Pathway |
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| LIAO Fang-fang
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YUAN Si-chun
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ZHANG Zhong-wen
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WU Guo-juan.Constructions of Arkadia and UCH37 Expression Vectors and Effects on TGF-β1/Smad7 Signal Pathway[J].Scientia Agricultura Sinica,2012,45(9):1848-1856. |
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| Authors: | LIAO Fang-fang YUAN Si-chun ZHANG Zhong-wen WU Guo-juan |
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| Institution: | (College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206) |
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| Abstract: | 【Objective】The objective of the study is to construct expression vectors of ubiquitination starting enzyme Arkadia and ubiquitin COOH-terminal hydmlase-L5(also known as UCH37),and to investigate the effect of Smad7 modified by ubiquitination on TGF-β1/Smad7 signal pathway in aristolochic acid nephropathy(AAN).【Method】The transfection condition was optimized by observing expressions of green fluorescences and undertaking real-time PCR reactions.After transferring the siRNA of Arkadia and UCH37,mRNA inhibition ratios were detected by real-time PCR.Then the expression vectors pGPU6/GFP/Neo of Arkadia and UCH37 were constructed,and transferred into cells after evaluations,and the expression changes of Arkadia,UCH37 and Smad7 were detected by real-time PCR and western blotting.【Result】Results showed that 30 pmol siRNA and 1.5 μL LipofectamineTM 2000 were the best transfection conditions.The interferences of Arkadia-1490 and UCH37-721 were most obvious.The inserted sequences of vectors were found to be correct,and pGPU6/GFP/Neo-Arkadia could increase Smad7 expression in AAN,while pGPU6/GFP/Neo-UCH37 was the opposite.【Conclusion】Expression vectors pGPU6/GFP/Neo-Arkadia and pGPU6/GFP/ Neo-UCH37 were constructed successfully,and it was found that Arkadia and UCH37 could separately enlarge,and inhibit TGF-β1/ Smad7 signal pathway. |
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| Keywords: | Arkadia gene UCH37 gene transfection expression vector TGF-β1/Smad7 signal pathway |
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