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小鼠骨骼中骨细胞基因表达分析：激光辅助显微切割方案的优化

Osteocyte gene expression analysis in mouse bone: optimization of a laser-assisted microdissection protocol.

作者信息

Palmier Mathilde, Maître Marlène, Doat Hélène, Lesté-Lasserre Thierry, Maurel Delphine B, Boiziau Claudine

机构信息

Inserm, University of Bordeaux, BioTis Laboratory UMR 1026, Bordeaux, France.

Inserm, University of Bordeaux, Neurocentre Magendie UMR 1215, Bordeaux, France.

出版信息

JBMR Plus. 2024 Jun 24;8(8):ziae078. doi: 10.1093/jbmrpl/ziae078. eCollection 2024 Aug.

DOI:10.1093/jbmrpl/ziae078
PMID:39045129
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11264292/
Abstract

Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (, , , , ). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.

摘要

在骨细胞中，骨细胞数量最多，但也是最难研究的，因为它们位于致密的矿化基质内部。由于它们参与骨稳态，需要多种工具来了解它们在骨生理和病理中的作用。这项工作旨在建立一种激光辅助显微切割方案，以分离骨细胞并分析其基因表达。目标是克服目前最常用技术的局限性：从整块骨中提取RNA。为了进行激光显微切割和后续的基因表达分析，该方案的五个主要步骤已针对骨组织进行了调整。在测试了许多参数后，我们发现最佳选择是：（1）采用未固定的速冻组织；（2）如有必要，使用支持带系统进行冷冻切片以改善组织形态；（3）显微切割感兴趣的区域；（4）如果可行，通过弹射回收骨片，或者通过重力回收。最后，（5）采用沉淀法进行RNA提取效率最高，并且能够对已充分描述的骨细胞基因（……）的表达进行定量。这项工作描述了针对股骨和颅骨优化的两种方案，并概述了在激光显微切割遇到困难时可以尝试的许多优化选项。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/d71991d4b8a0/ziae078f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/e7f948f0f6f0/ziae078ga1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/ff1ce69026ce/ziae078f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/099f5a4e7dcd/ziae078f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/428e78a82f4e/ziae078f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/d71991d4b8a0/ziae078f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/e7f948f0f6f0/ziae078ga1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/ff1ce69026ce/ziae078f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/099f5a4e7dcd/ziae078f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/428e78a82f4e/ziae078f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e4/11264292/d71991d4b8a0/ziae078f4.jpg

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3
Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamoto's Film Method (2020).用川本氏薄膜法制备非固定和未脱钙硬组织的薄冰冻切片（2020）。
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4
The Osteocyte: New Insights.成骨细胞：新的认识。
Annu Rev Physiol. 2020 Feb 10;82:485-506. doi: 10.1146/annurev-physiol-021119-034332.
5
A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones.一种可从新鲜冷冻小鼠骨骼中获取高质量RNA的激光捕获显微切割方案。
J Vis Exp. 2019 Sep 16(151). doi: 10.3791/60197.
6
Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells.基于皮质骨细胞转录组和外泌体蛋白质组分析的骨老化特征。
J Orthop Surg Res. 2019 May 10;14(1):129. doi: 10.1186/s13018-019-1163-4.
7
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8
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9
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