Landim de Barros Thamine, Brito Victor Gustavo Balera, do Amaral Caril Constante Ferreira, Chaves-Neto Antonio Hernandes, Campanelli Ana Paula, Oliveira Sandra Helena Penha
School of Dentistry of Araçatuba, UNESP - Univ Estadual Paulista, Campus Araçatuba, Department of Basic Sciences, São Paulo, Brazil; Programa de Pós-graduação Multicêntrico em Ciências Fisiológicas - SBFIS/FOA - Araçatuba, Department of Basic Sciences School of Dentistry of Araçatuba, UNESP - Univ Estadual Paulista, Araçatuba, Department of Basic Sciences, São Paulo, Brazil.
School of Dentistry of Araçatuba, UNESP - Univ Estadual Paulista, Campus Araçatuba, Department of Basic Sciences, São Paulo, Brazil.
Life Sci. 2016 Feb 1;146:174-83. doi: 10.1016/j.lfs.2016.01.015. Epub 2016 Jan 13.
Spontaneously hypertensive rats (SHR) and normotensive rats (W) has significant changes in bone metabolism. The purpose of this study was to investigate whether, the genetic predisposition, is sufficient to induce changes in the osteoblast differentiation and osteogenic markers in the BMSCs or in the femoral bone. For this we use young SHR rats without hypertension, but, with genetic predisposition in compared with young W.
BMSCs were cultured in a proliferation medium (MEM) or osteogenic medium. Osteogenic differentiation was analyzed by proliferation, total protein, alkaline phosphatase, mineralization, and the mRNA expression of RUNX-2, β-cathenin, osterix, bone morphogenetic protein-2(BMP-2), osteocalcin (OCN), bone sialoprotein (BSP), collagen type I (Col I), and osteopontin (OPN).
Osteoblast differentiation in SHR BMSCs (SHRC) had an increased proliferation compared with W BMSCs (WC). After osteogenic induction, there was greater reduction in proliferation in SHR (SHROM) than in W, in the same condition (WOM). On day 7, although no significant difference in the ALP activity was observed between SHROM and WOM, poor mineralization and osteoblast differentiation was noted in SHROM. The Osterix and β-catenin are involved in the reduced osteoblast differentiation in SHROM. The decreased expression of osteoblast-associated proteins such as OCN, BSP, COL I and OPN revealed poor quality of extracellular matrix (ECM) in SHROM. In the femoral bone, the immunostaining of COL1, BALP, OPN and OCN in SHR was decreased compared with the W. TRAP-positive immunoreactions were observed in major extension in the SHR femur.
This study is the first to compare osteoblast differentiation in vitro and femoral bone from SHR and W rats. Our results demonstrated that young SHR (4weeks old), without hypertension, but with genetic predisposition, had alterations in osteoblast differentiation of BMSCs and in the femoral bone when compared with their progenitor strain, W.
自发性高血压大鼠（SHR）和正常血压大鼠（W）的骨代谢有显著变化。本研究的目的是调查遗传易感性是否足以诱导骨髓间充质干细胞（BMSCs）或股骨中骨细胞分化和成骨标志物的变化。为此，我们使用无高血压但有遗传易感性的年轻SHR大鼠，并与年轻的W大鼠进行比较。
将BMSCs培养于增殖培养基（MEM）或成骨培养基中。通过增殖、总蛋白、碱性磷酸酶、矿化以及RUNX-2、β-连环蛋白、osterix、骨形态发生蛋白-2（BMP-2）、骨钙素（OCN）、骨唾液蛋白（BSP）、I型胶原蛋白（Col I）和骨桥蛋白（OPN）的mRNA表达来分析成骨分化。
与W大鼠的BMSCs（WC）相比，SHR大鼠的BMSCs（SHRC）中骨细胞分化的增殖增加。在成骨诱导后，在相同条件下（WOM），SHR（SHROM）的增殖减少比W大鼠更明显。在第7天，虽然SHROM和WOM之间未观察到碱性磷酸酶活性有显著差异，但在SHROM中发现矿化和成骨细胞分化较差。Osterix和β-连环蛋白与SHROM中骨细胞分化减少有关。OCN、BSP、COL I和OPN等成骨细胞相关蛋白的表达降低表明SHROM中细胞外基质（ECM）质量较差。在股骨中，与W大鼠相比，SHR大鼠中COL1、BALP、OPN和OCN的免疫染色减少。在SHR大鼠股骨的主要延伸部位观察到抗酒石酸酸性磷酸酶（TRAP）阳性免疫反应。
本研究首次比较了SHR和W大鼠的体外骨细胞分化和股骨情况。我们的结果表明，4周龄无高血压但有遗传易感性的年轻SHR大鼠与其亲代品系W大鼠相比，其BMSCs的成骨细胞分化和股骨存在改变。
