Gene expression profile during osteoblaslic differentiation of human mesenchymal cells

脊柱后纵韧带骨化症（OPLL）是日本脊髓病的主要病因，通过椎旁韧带异位成骨来诊断。OPLL是一种具有强烈遗传背景的全身性高骨量疾病。为了检测与OPLL发病机制相关的基因，我们用cDNA微阵列分析了OPLL患者韧带细胞成骨分化过程中系统的基因表达谱，（OPLL细胞）、黄韧带骨化患者和非OPLL对照，以及用成骨分化培养基（OS）刺激后的人间充质干细胞（hMSC）。29个基因在OPLL细胞成骨分化过程中表达上调。锌指蛋白145（PLZF）是成骨细胞分化过程中高表达的基因之一。我们研究了PLZF对hMSCs和C2 C12细胞成骨分化的调控作用。siRNA介导的PLZF基因沉默导致成骨细胞特异性基因如碱性磷酸酶（ALP）、胶原蛋白1A 1（COL 1A 1）、Runx 2/cbfa 1（CBFA 1）和骨钙素（OCN）的减少，即使在hMSC中存在OS的情况下也是如此。PLZF的表达不受骨形态发生蛋白2（BMP-2）的影响，BMP-2的表达不受PLZF的影响。在C2 C12细胞中，PLZF的过表达增加了Cbfal和Collal的表达，而CBFA 1的过表达不影响Plzf的表达。这些结果表明，PLZE作为CBFA 1的上游调节因子在早期成骨细胞分化中起重要作用，从而可能在促进OPLL患者脊柱韧带细胞骨化中发挥作用。

Ossification of the posterior longitudinal ligament of the spine (OPLL) is the leading cause of myelopathy in Japan, and is diagnosed by ectopic bone formation in the paravertebral ligament OPLL is a systemic high bone mass disease with a strong genetic background To detect genes relevant to the pathogenesis of OPLL, we performed cDNA microarray analysis of systematic gene expression profiles during osteoblastic differentiation of ligament cells from OPLL patienis (OPLL cells), ossification of yellow ligament patients, and non-OPLL controls, and human mesenchymal stem cells (hMSCs) after stimulating with osteogenic differentiation medium (OS). Twenty-nine genes were up-regulated during osteoblastic differentiation in OPLL cells. Zinc finger protein 145 (PLZF) was one of the highly expressed genes during osteoblastic differentiation in all the cells examined. We investigated the roles of PLZF in the regulation of osteoblastic differentiation ofhMSCs and C2C12 cells. siRNA mediated gene-silencing of PLZF resulted in the reduction of osteoblast-specific genes such as alkaline phosphatase (ALP), collagen lAl (COL1A1), Runx2/cbfa1 (CBFA 1), and osteocalcin (OCN), even in the presence of OS in hMSCs. The expression of PLZF was unaffected by addition of bone morphogenetic protein 2 (BMP-2) and the expression of BMP-2 was not affected by PLZF in hMSCs. In C2C12 cells, overexpression of PLZF increased the expression of Cbfal and Collal, on the other hand, overexpression of CBFA1 did not affect the expression of Plzf. These findings indicate that PLZE plays important roles in early osteoblastic differentiation as an upstream regulator of CBFA1 and thereby might pailicipate in promoting ossification of spinal ligament cells in OPLL patients.