Zhang Ya-Dong, Zhao Shi-Chang, Zhu Zhong-Sheng, Wang Yi-Fei, Liu Jian-Xiang, Zhang Zhi-Cai, Xue Feng
Department of Orthopaedics, Shanghai Fengxian Central Hospital, South Campus of Shanghai Sixth People's Hospital, Shanghai Jiaotong University, Shanghai, China.
Department of Orthopaedics, Shanghai Sixth People's Hospital, Shanghai Jiaotong University, Shanghai, China.
Cell Physiol Biochem. 2017;42(4):1277-1293. doi: 10.1159/000478957. Epub 2017 Jul 11.
BACKGROUND/AIMS: The aim of this study was to investigate the influence of Cx43- and Smad-mediated TGF-β/BMP signaling pathway on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cartilage and inhibition of ossification.
BMSCs of Wistar rats were cultured and assigned into 5 groups for transfection with adenoviruses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were employed to detect mRNA and protein expressions of target genes. The condition of cartilage and ossification were measured by a series of staining methods. Subcutaneous injection of mesenchymal stem cells (MSCs) into nude rats was performed.
After transfection, compared to the AdGFP group, the corresponding target mRNAs were overexpressed in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups, and overexpression of BMP2 at the mRNA and protein expression was observed in the AdSmad1 and AdCx43 + AdSmad1 groups. The mRNA expressions of aggrecan (ACAN) and collagen type II alpha 1 (Col2a1), the glycosaminoglycan content of the extracellular matrix and the expression of type II collagen, Col2a1, osteopontin (OPN) and osteocalcin (OC) were higher in the AdBMP2, AdSmad1, AdCx43 + AdSmad1 and AdCx43 + AdSmad1 + AdBMP2 groups than in the AdGFP group; alkaline phosphatase (ALP) activity and mRNA and protein expressions of Runx2 were also higher in these groups than in the AdGFP group. Heterotopic osteogenesis tests demonstrated evident cartilage differentiation ability in the AdCx43 + AdSmad1 + AdBMP2 groups. In comparison, the AdCx43 + AdSmad1 and AdSmad1 groups exhibited weaker cartilage differentiation abilities.
Cx43 and Smad1 promote BMP-induced cartilage differentiation of BMSCs and inhibit osteoblast differentiation, which provide a new strategy for cartilage tissue engineering using exogenous Cx43 and Smad1.
背景/目的：本研究旨在探讨Cx43和Smad介导的TGF-β/BMP信号通路对骨髓间充质干细胞（BMSCs）向软骨分化及骨化抑制的影响。
培养Wistar大鼠的BMSCs，并将其分为5组进行腺病毒转染。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测靶基因的mRNA和蛋白表达。通过一系列染色方法检测软骨和骨化情况。将间充质干细胞（MSCs）皮下注射到裸鼠体内。
转染后，与AdGFP组相比，AdBMP2、AdSmad1、AdCx43 + AdSmad1和AdCx43 + AdSmad1 + AdBMP2组中相应的靶mRNA过表达，且在AdSmad1和AdCx43 + AdSmad1组中观察到BMP2在mRNA和蛋白表达水平上的过表达。AdBMP2、AdSmad1、AdCx43 + AdSmad1和AdCx43 + AdSmad1 + AdBMP2组中聚集蛋白聚糖（ACAN）和Ⅱ型胶原α1（Col2a1）的mRNA表达、细胞外基质的糖胺聚糖含量以及Ⅱ型胶原、Col2a1、骨桥蛋白（OPN）和骨钙素（OC）的表达均高于AdGFP组；这些组中的碱性磷酸酶（ALP）活性以及Runx2的mRNA和蛋白表达也高于AdGFP组。异位成骨试验表明AdCx43 + AdSmad1 + AdBMP2组具有明显的软骨分化能力。相比之下，AdCx43 + AdSmad1和AdSmad1组的软骨分化能力较弱。
Cx43和Smad1促进BMP诱导的BMSCs向软骨分化并抑制成骨细胞分化，这为利用外源性Cx43和Smad1进行软骨组织工程提供了新策略。
