Summary

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

Published: July 08, 2020
doi:

Summary

microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型(HIGA小鼠)肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。

Abstract

免疫球蛋白A(IgA)肾病是一种原发性肾小球肾炎,其特征是IgA沉积异常,导致终末期肾衰竭。近年来,在IgA肾病的发病机制中报道了microRNA(miRNA)的参与。然而,没有使用小动物模型分析IgA肾病中miRNA的既定方法。因此,我们开发了一种可靠的方法来分析IgA小鼠模型(HIGA小鼠)肾脏中的miRNA。该协议的目标是检测与对照小鼠肾脏中的水平相比,HIGA小鼠肾脏中miRNA的表达水平改变。简而言之,该方法包括四个步骤:1)从HIGA小鼠获得肾脏样本;2)从肾脏样本中纯化总RNA;3)从总RNA合成互补DNA;4)miRNA的定量逆转录聚合酶链反应(qRT-PCR)。使用这种方法,我们成功地检测了HIGA小鼠肾脏中几种miRNA(miR-155-5p,miR-146a-5p和miR-21-5p)的表达水平。这种新方法可以应用于其他分析IgA肾病中miRNA的研究。

Introduction

免疫球蛋白A(IgA)肾病是一种原发性肾小球肾炎,其特征是IgA在肾小球系膜区域1,2中的异常沉积。它是最常见的原发性肾小球肾炎,在20%-40%的患者中导致终末期肾衰竭2。病因尚不清楚,但与持续性黏膜感染有关1,3。皮质类固醇、免疫抑制剂和肾素-血管紧张素系统抑制剂已被提出作为治疗方法1,3但尚未完全确立3。因此,需要进一步研究明确治疗IgA肾病的病因和治疗方法。

microRNA(miRNA)是小的非编码RNA,在调节基因表达中起重要作用4,5。据报道,miRNA参与各种疾病的发病机制,有些已被确定为疾病生物标志物和治疗剂4,5。近年来,miRNA与IgA肾病发病机制之间的关联也有报道2,6,7例如,miR-148b被证明与IgA肾病2,6,7患者的IgA结构异常有关而miR-148b和let-7b被记录为检测IgA肾病新型生物标志物7。尽管了解miRNA对IgA肾病的影响可能有助于进一步阐明病因和治疗2,但尚未建立使用小动物模型分析IgA肾病中miRNA的标准方法2

我们在此开发了一种简单可靠的方法来测量IgA肾病小鼠模型(HIGA小鼠)肾脏中的miRNA表达水平。HIGA小鼠是一种特征性的ddY菌株,显示血清IgA水平特别高,肾小球8,9,10,11中IgA的异常沉积。因此,HIGA小鼠可用作IgA肾病小鼠模型8,9,10,11。我们的方法包括四个主要步骤:首先,通过手术从HIGA小鼠获得肾脏样本;其次,使用基于硅胶膜的离心柱均质化样品并纯化总RNA;第三,使用逆转录从总RNA合成互补DNA(cDNA);第四,通过定量逆转录聚合酶链反应(qRT-PCR)检测miRNA的表达水平。这种方法的基本原理和结果的可靠性基于以前的报告12,13。我们表明,这是一种准确测量IgA肾病小鼠模型中miRNA表达水平的有用技术,并且可用于促进未来对IgA肾病中miRNA的研究。

Protocol

动物实验经吉智医科大学动物伦理委员会批准,并符合《吉智医科大学实验动物指南》中的实验动物使用和护理指南。 1. 从HIGA小鼠中获取肾脏样本 注意:HIGA小鼠在8,9,10,11岁25周后显示出稳定的IgA肾病表型。应选择Balb/c小鼠作为对照组8、<sup c…

Representative Results

我们研究了miRNA在HIGA小鼠肾脏中的表达水平(n = 10)。该结果完全基于所描述的协议获得。选择Balb / c小鼠的肾脏作为对照(n = 10)。在两组中,均选择25周龄。供应商仅提供雌性HIGA小鼠。三种miRNA(miR-155-5p、miR-146a-5p和miR-21-5p)的表达水平;图1)被检测到,之前报道与 IgA 肾病有关15,16。采用t检验比较两组相对miRNA表达水平,P<0.05?…

Discussion

我们能够使用这种新方法测量IgA肾病小鼠模型(HIGA小鼠)肾脏中miRNA的表达水平。IgA肾病是一种无法解释的疾病,需要进一步研究以阐明其病因和治疗靶点1,3。然而,获取人类肾脏样本是高度侵入性的。这种新技术的优点在于它允许使用小动物研究IgA肾病,并且应该有助于未来对IgA肾病和miRNA的研究。在未来的研究中,该方法可以应用于治疗IgA肾病的新…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢Edanz Group(www.edanzediting.com)的Sarah Williams博士编辑了这份手稿的草稿。

Materials

BALB/cCrSlc (25-week-old, female) Japan SLC, Inc. none Mouse for control
HIGA/NscSlc (25-week-old, female) Japan SLC, Inc. none IgA nephropathy mouse model
MicroAmp Optical 96 well reaction plate for qRT-PCR Thermo Fisher Scientific 4316813 96-well reaction plate
MicroAmp Optical Adhesive Film Thermo Fisher Scientific 4311971 adhesive film for 96-well reaction plate
miScript II RT kit Qiagen 218161 Experimental kit for synthesis of cDNA
miRNeasy Mini kit Qiagen 217004 Experimental kit fot extraction of total RNA
miScript Primer Assay (RNU6-2) Qiagen MS00033740 miRNA-specific primer
miScript Primer Assay (miR-155-5p) Qiagen MS00001701 miRNA-specific primer
miScript Primer Assay (miR-146a-5p) Qiagen MS00001638 miRNA-specific primer
miScript Primer Assay (miR-21-5p) Qiagen MS00009079 miRNA-specific primer
miScript SYBR Green PCR kit Qiagen 218073 Experimental kit for real-time PCR
QIA shredder Qiagen 79654 biopolymer-shredding spin column
QuantStudio 12K Flex Flex Real-Time PCR system Thermo Fisher Scientific 4472380 real-time PCR instrument
QuantStudio 12K Flex Software version 1.2.1. Thermo Fisher Scientific 4472380 real-time PCR instrument software
takara biomasher standard Takara Bio 9790B silicon homogenizer

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Kaneko, S., Yanai, K., Ishii, H., Aomatsu, A., Ito, K., Hirai, K., Ookawara, S., Ishibashi, K., Morishita, Y. Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice. J. Vis. Exp. (161), e61535, doi:10.3791/61535 (2020).

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