无标记定量蛋白质组学分析抗坏血酸诱导的乳牙和恒牙牙髓干细胞中差异表达的成骨细胞相关蛋白。

Label-free Quantitative Proteomic Analysis of Ascorbic Acid-induced Differentially Expressed Osteoblast-related Proteins in Dental Pulp Stem Cells from Deciduous and Permanent Teeth.

机构信息

Centre for Research and Graduate Studies, University of Cyberjaya, 63000 Cyberjaya, Selangor, Malaysia.

Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.

出版信息

Curr Stem Cell Res Ther. 2023;18(3):417-428. doi: 10.2174/1574888X17666220627145424.

DOI:10.2174/1574888X17666220627145424

PMID:35762553

Abstract

BACKGROUND

Proteomic is capable of elucidating complex biological systems through protein expression, function, and interaction under a particular condition.

OBJECTIVE

This study aimed to determine the potential of ascorbic acid alone in inducing differentially expressed osteoblast-related proteins in dental stem cells via the liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) approach.

METHODS

The cells were isolated from deciduous (SHED) and permanent teeth (DPSC) and induced with 10 μg/mL of ascorbic acid. Bone mineralisation and osteoblast gene expression were determined using von Kossa staining and reverse transcriptase-polymerase chain reaction. The label-free protein samples were harvested on days 7 and 21, followed by protein identification and quantification using LC-MS/MS. Based on the similar protein expressed throughout treatment and controls for SHED and DPSC, overall biological processes followed by osteoblast-related protein abundance were determined using the PANTHER database. STRING database was performed to determine differentially expressed proteins as candidates for SHED and DPSC during osteoblast development.

RESULTS

Both cells indicated brownish mineral stain and expression of osteoblast-related genes on day 21. Overall, a total of 700 proteins were similar among all treatments on days 7 and 21, with 482 proteins appearing in the PANTHER database. Osteoblast-related protein abundance indicated 31 and 14 proteins related to SHED and DPSC, respectively. Further analysis by the STRING database identified only 22 and 11 proteins from the respective group. Differential expressed analysis of similar proteins from these two groups revealed ACTN4 and ACTN1 as proteins involved in both SHED and DPSC. In addition, three (PSMD11/RPN11, PLS3, and CLIC1) and one (SYNCRIP) protein were differentially expressed specifically for SHED and DPSC, respectively.

CONCLUSION

Proteome differential expression showed that ascorbic acid alone could induce osteoblastrelated proteins in SHED and DPSC and generate specific differentially expressed protein markers.

摘要

背景

蛋白质组学能够通过特定条件下的蛋白质表达、功能和相互作用来阐明复杂的生物系统。

目的

本研究旨在通过液相色谱-质谱/质谱（LC-MS/MS）方法确定抗坏血酸单独诱导牙源性干细胞中差异表达的成骨相关蛋白的潜力。

方法

从乳牙（SHED）和恒牙（DPSC）中分离细胞，并以 10μg/mL 的抗坏血酸诱导。通过 Von Kossa 染色和逆转录聚合酶链反应测定骨矿化和成骨基因表达。在第 7 天和第 21 天收集无标记蛋白样品，然后使用 LC-MS/MS 进行蛋白鉴定和定量。基于 SHED 和 DPSC 整个治疗和对照中表达相似的蛋白，使用 PANTHER 数据库确定整体生物过程以及成骨相关蛋白丰度。STRING 数据库用于确定在成骨细胞发育过程中作为 SHED 和 DPSC 候选的差异表达蛋白。

结果

两种细胞在第 21 天都显示出棕褐色的矿物质染色和成骨相关基因的表达。总体而言，第 7 天和第 21 天所有处理之间共有 700 种相似蛋白，其中 482 种蛋白出现在 PANTHER 数据库中。成骨相关蛋白丰度分别显示 31 种和 14 种蛋白与 SHED 和 DPSC 相关。STRING 数据库的进一步分析仅从各自的组中鉴定出 22 种和 11 种蛋白。对这两组相似蛋白的差异表达分析表明，ACTN4 和 ACTN1 是参与 SHED 和 DPSC 的蛋白。此外，PSMD11/RPN11、PLS3 和 CLIC1 三种蛋白和 SYNCRIP 蛋白分别是 SHED 和 DPSC 特异性差异表达的蛋白。