Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells
Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells
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DOI：
10.3892/etm.2015.2378
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发表时间：
2015-06-01
影响因子：
2.7
通讯作者：
Wang, Xudong
中科院分区：
文献类型：
--
作者：
Sun, Hao;Liu, Zhixu;Wang, Xudong
远端基因（DLX）在调节生物体发育中发挥重要作用。 DLX2对于原基的分化和发育至关重要，决定了颌面骨骼模式的后续发育和表型，是调节第一鳃弓发育的主要候选基因。本研究的目的是探讨DLX2过表达对MC3T3-E1细胞体外成骨分化的影响。通过用鼠干细胞病毒（MSCV）亚克隆构建DLX2表达逆转录病毒载体并通过测序验证。用pMSCV-DLX2转染MC3T3-E1细胞，并用嘌呤霉素选择稳定克隆。分别使用 q 定量聚合酶链反应 (PCR) 和蛋白质印迹分析测定 DLX2 的 mRNA 和蛋白表达水平。此外，通过定量 PCR 评估了成骨生物标志物、碱性磷酸酶 (ALP)、骨钙素 (OCN)、runt 相关转录因子 (RUNX)2 和 Msh 同源盒 (MSX)2 的表达水平。通过ALP检测和茜素红染色评价DLX2过表达对成骨分化的影响。使用Student-Newman-Keuls方法通过方差分析来分析数据。通过直接测序验证，成功的 pMSCV-DLX2 构建使 DLX2 能够在体外过表达。与对照组相比，MC3T3-E1-DLX2 细胞中 ALP 活性和茜素红染色增强。在成骨诱导过程中，DLX2过表达可以上调早期ALP和MSX2的表达，以及后期OCN的表达，而RUNX2表达与对照组相比没有观察到统计学上的显着差异。因此，DLX2在体外过表达通过上调骨形成相关基因（例如ALP和MSX2）诱导MC3T3-E1细胞的成骨分化。
Distal-less genes (DLX) play important roles in regulating organism development. DLX2 is crucial for the differentiation and development of the primordium, which determines the subsequent development and phenotype of the maxillofacial skeletal patterns, and is the primary candidate gene that regulates the development of the first branchial arch. The aim of the present study was to investigate the effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells in vitro. A DLX2-expression retrovirus vector was constructed by subcloning with a murine stem cell virus (MSCV) and verified by sequencing. MC3T3-E1 cells were transfected with pMSCV-DLX2 and stable clones were selected with puromycin. The mRNA and protein expression levels of DLX2 were determined using qqantitative polymerase chain reaction (PCR) and western blot analysis, respectively. In addition, the expression levels of the osteogenic biomarkers, alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor (RUNX)2 and Msh homeobox (MSX)2, were assessed by quantitative PCR. ALP detection and Alizarin red staining were conducted to evaluate the effect of DLX2 overexpression on osteogenic differentiation. The data were analyzed by analysis of variance using the Student-Newman-Keuls method. Successful pMSCV-DLX2 construction, as verified by direct sequencing, enabled DLX2 overexpression in vitro. Enhanced ALP activity and Alizarin red staining were observed in the MC3T3-E1-DLX2 cells when compared with the control group. During osteogenic induction, DLX2 overexpression was demonstrated to upregulate ALP and MSX2 expression at the early stage and OCN expression at the late stage, while no statistically significant difference was observed in RUNX2 expression when compared with the control group. Therefore, DLX2 overexpression in vitro induced the osteogenic differentiation of MC3T3-E1 cells via upregulating bone formation-associated genes, such as ALP and MSX2.