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基于RNA干扰技术对热休克蛋白27在角膜上皮伤口愈合过程中功能的研究。

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing.

作者信息

Yoo Aeri, Park Hyun-Min, Kang Soon-Suk, Kim Eun-Soon, Tchah Hungwon, Kim Jae Yong

机构信息

Department of Ophthalmology, Saevit Eye Hospital.

Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center.

出版信息

J Vis Exp. 2016 Sep 27(115):54280. doi: 10.3791/54280.

DOI:10.3791/54280
PMID:27768052
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5092076/
Abstract

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

摘要

小干扰RNA（siRNA）是用于蛋白质编码基因短期沉默的最广泛使用的RNA干扰方法之一。siRNA是一种合成的RNA双链体，专门设计用于靶向mRNA转录本以诱导其降解，并且已被用于识别各种细胞过程中的新途径。关于磷酸化热休克蛋白27（HSP27）在角膜上皮伤口愈合中的作用的报道很少。在此，将培养的人角膜上皮细胞分为乱序对照-siRNA转染组和HSP27特异性siRNA转染组。然后进行划痕诱导的定向伤口测定、蛋白质印迹和流式细胞术。我们得出结论，HSP27在角膜上皮伤口愈合中起作用，可能涉及上皮细胞凋亡和迁移。在此提供样品制备和研究方案的逐步说明。

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本文引用的文献

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2
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Cornea. 2012 Jul;31(7):820-7. doi: 10.1097/ICO.0b013e31823f7685.
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Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and cornea.前列腺素类似物对结膜和角膜中细胞因子和基质金属蛋白酶的泪液蛋白质组学和表达的影响。
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Comparison of corneal epitheliotrophic capacity among different human blood-derived preparations.比较不同人血来源制剂的角膜上皮营养能力。
Cornea. 2011 Feb;30(2):208-14. doi: 10.1097/ICO.0b013e3181eadb67.
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Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP.分析HeLa细胞中基因表达谱对NASP过表达或siRNA介导的NASP缺失的反应。
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