Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, China.
J Invest Surg. 2021 Sep;34(9):947-956. doi: 10.1080/08941939.2020.1716894. Epub 2020 Jan 27.
The cytokine TNF-α-stimulated gene-6 (TSG-6) had been verified to have a certain inhibitory effect on the inflammation. During wound healing, fibroblasts increasingly proliferated and deposited collagen fibers, leading to the formation of pathological scars. We sought to elucidate the mechanism by which the TGF-β1/Smad pathway was mediated by TSG-6 in human keloid fibroblasts.
Human keloid fibroblast cells were isolated from keloid tissue by enzyme digestion and identified by immunocytochemistry. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed which were then transfected into human keloid fibroblasts. The mRNA and protein levels of TSG-6 were detected respectively by RT-PCR and western blot assay. The intracellular localization of TGF-β1-induced proteins and phosphorylated (p)-Smad2/3 in keloid fibroblasts were investigated using an immunofluorescence assay. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was detected by RT-PCR.
TSG-6 could effectively interfere the TGF-β1/Smad signal transduction pathway in keloid fibroblasts rather than in normal fibroblasts. The phosphorylation levels of Smad2/3 were notably reduced by TSG-6 treatment. TSG-6 blocked the complex formation of Smad2/3/4, and their nuclear translocation. However, it upregulated Smad7 expression, presenting dose dependence. PAI-1 was also suppressed by TSG-6 treatment.
TSG-6 inhibits proliferation by inducing apoptosis in keloid fibroblasts, which may be associated with TGF-β1/Smad pathway.
细胞因子 TNF-α 刺激基因-6（TSG-6）已被证实对炎症具有一定的抑制作用。在伤口愈合过程中，成纤维细胞增殖并沉积胶原蛋白纤维，导致病理性瘢痕形成。我们试图阐明 TSG-6 通过 TGF-β1/Smad 通路介导的机制在人瘢痕成纤维细胞中。
通过酶消化从瘢痕组织中分离出人瘢痕成纤维细胞，并通过免疫细胞化学进行鉴定。构建慢病毒载体 pLVX-puro-TSG-6 和 pLVX-shRNA1-TSG-6，然后转染人瘢痕成纤维细胞。分别通过 RT-PCR 和 Western blot 检测 TSG-6 的 mRNA 和蛋白水平。采用免疫荧光法研究 TGF-β1 诱导的蛋白和磷酸化（p）-Smad2/3 在瘢痕成纤维细胞中的细胞内定位。通过 RT-PCR 检测纤溶酶原激活物抑制剂-1（PAI-1）转录活性。
TSG-6 可有效干扰瘢痕成纤维细胞中的 TGF-β1/Smad 信号转导通路，而不是正常成纤维细胞。TSG-6 处理可显著降低 Smad2/3 的磷酸化水平。TSG-6 阻断了 Smad2/3/4 复合物的形成及其核转位，但上调了 Smad7 的表达，呈剂量依赖性。PAI-1 的表达也被 TSG-6 处理所抑制。
TSG-6 通过诱导瘢痕成纤维细胞凋亡抑制增殖，这可能与 TGF-β1/Smad 通路有关。
