分析人脂肪组织来源间充质干细胞成骨分化过程中微小RNA表达及功能的方法。

Methods for analyzing microRNA expression and function during osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

作者信息

Kim Yeon Jeong, Jung Jin Sup

机构信息

Department of Physiology, School of Medicine, Pusan National University, Gyeongsangnam-do, Beomeo-ri, Korea.

出版信息

Methods Mol Biol. 2011;702:401-18. doi: 10.1007/978-1-61737-960-4_29.

DOI:10.1007/978-1-61737-960-4_29

PMID:21082418

Abstract

MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length that regulate gene expression at the posttranscriptional level. They may play important roles during osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (hASC). In this chapter, we focus on the methods and strategies for elucidating miRNA function during osteogenic differentiation. We describe a miRNA expression analysis protocol, and a lentiviral vector strategy for the ectopic expression of miRNA in hASC to determine the role of miRNA during osteogenic differentiation. We also describe miRNA inhibition to further determine the role of miRNA during osteogenic differentiation, and a luciferase assay to demonstrate direct binding between a specific miRNA and its putative target.

摘要

微小RNA（miRNA）是长度为21 - 23个核苷酸的单链RNA分子，在转录后水平调节基因表达。它们可能在脂肪组织来源的间充质干细胞（hASC）的成骨分化过程中发挥重要作用。在本章中，我们重点介绍在成骨分化过程中阐明miRNA功能的方法和策略。我们描述了一种miRNA表达分析方案，以及一种用于在hASC中异位表达miRNA以确定miRNA在成骨分化过程中作用的慢病毒载体策略。我们还描述了miRNA抑制以进一步确定miRNA在成骨分化过程中的作用，以及一种荧光素酶测定法以证明特定miRNA与其假定靶标之间的直接结合。