Ikegawa Naoshi, Sasho Takahisa, Yamaguchi Satoshi, Saito Masahiko, Akagi Ryuichiro, Muramatsu Yuta, Akatsu Yorikazu, Fukawa Taisuke, Nakagawa Koichi, Nakajima Arata, Suzuki Takane, Takahashi Kazuhisa
a Department of Orthopaedic Surgery , Graduate School of Medicine, Chiba University , Chiba , Japan.
b Department of Orthopaedic Surgery , Sakura Medical Center, Toho University , Sakura , Japan.
Connect Tissue Res. 2016 May;57(3):190-9. doi: 10.3109/03008207.2015.1121250. Epub 2015 Dec 31.
Our previous study showed that partial-thickness articular cartilage defects (PTCDs) created in immature rats spontaneously healed to resemble normal hyaline cartilage, but that of mature rats did not. To identify molecules involved in the spontaneous cartilage repair observed in this model, gene expression was compared between PTCD and sham-operated cartilage of immature and mature rats.
Six sets of gene comparisons were made at 12, 24, and 48 hours after the creation of PTCDs in immature and mature rats using microarrays. All the genes upregulated in immature cartilage at 12 hours were selected for further analysis if their expression pattern was not irregular such that diminished at 24 hours and re-upregulated at 48 hours. Relationships among genes selected through the above steps were analyzed using Ingenuity Pathway Analysis (IPA) software. After deriving networks, important molecules were further narrowed down by location within a network. Genes were regarded as central if they had relationships with more than 10 molecules in a network. Protein localization in tissues was confirmed by immunohistochemistry.
Five networks were identified. Their functional annotations were gene expression, cell cycle, growth and proliferation, and cell signaling. Transforming growth factor-beta (TGF-β) was centrally located in the network with the highest IPA score and mothers against decapentaplegic homolog-3 (Smad3) were centrally located in the second highest ranking network. Phosphorylated Smad3 was detected in the nuclei of chondrocytes in immature cartilage.
Our data suggest the possible importance of Smad3 in the TGF-β signaling in the spontaneous healing of PTCDs in immature rats.
我们之前的研究表明，在未成熟大鼠中制造的部分厚度关节软骨缺损（PTCD）可自发愈合，类似于正常透明软骨，但成熟大鼠的则不会。为了确定该模型中观察到的自发软骨修复所涉及的分子，比较了未成熟和成熟大鼠PTCD与假手术软骨之间的基因表达。
使用微阵列在未成熟和成熟大鼠制造PTCD后12、24和48小时进行六组基因比较。如果未成熟软骨中在12小时上调的所有基因的表达模式没有异常，即不在24小时降低并在48小时重新上调，则选择进行进一步分析。使用 Ingenuity Pathway Analysis（IPA）软件分析通过上述步骤选择的基因之间的关系。得出网络后，通过网络内的位置进一步缩小重要分子的范围。如果基因在一个网络中与超过10个分子有关系，则将其视为核心基因。通过免疫组织化学确认组织中的蛋白质定位。
确定了五个网络。它们的功能注释是基因表达、细胞周期、生长和增殖以及细胞信号传导。转化生长因子-β（TGF-β）位于IPA评分最高的网络中心，而抗五肢瘫同源蛋白3（Smad3）位于排名第二高的网络中心。在未成熟软骨的软骨细胞核中检测到磷酸化的Smad3。
我们的数据表明Smad3在未成熟大鼠PTCD自发愈合的TGF-β信号传导中可能具有重要作用。
