Department of Plastic Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Mol Cell Biochem. 2011 Jun;352(1-2):25-33. doi: 10.1007/s11010-011-0736-2. Epub 2011 Feb 2.
Hypertrophic scars result from a dysregulated process in wound healing. Although the basic mechanism is unclear, increased proliferation and decreased cell apoptosis are noticed in the development of hypertrophic scar. In previous study, we found that secreted frizzled-related protein 2 (SFRP2), which was associated with cell proliferation, apoptosis, and differentiation, was dramatically upregulated in hypertrophic scar (HS) tissue. In this study short hairpin RNA (shRNA) targeting SFRP2 was employed to characterize SFRP2 function in hypertrophic scar-derived fibroblasts (HSFb). Cell proliferation was assessed by MTT, dynamic growth curves, and BRDU assays. Meanwhile, Cell apoptosis was detected using fluorescence-activated cell sorting (FACS). Caspase-3 activity was assayed by spectrophotometry. Fibroblast populated collagen lattice (FPCL) model was employed to evaluate the contractility of HSFb. Further, real-time PCR and western blot were used to measure the mRNA and protein expressions of α-SMA in HSFb. In addition, mRNA levels of type I and III procollagen were assayed by quantitative real-time PCR. The results revealed that shRNA targeting SFRP2 significantly promoted the apoptosis of HSFb, while it had no effect on the cell proliferation. Decreased synthesis of a-smooth muscle actin (α-SMA) in HSFb and reduced contraction of fibroblasts in the FPCL model were observed. Quantitative RT-PCR suggested that the mRNAs of type I and III procollagen were significantly downregulated. In conclusion, as a novel anti-apoptosis gene, SPRP2 was present in hypertrophic scars. Importantly, shRNA targeting SFRP2 may provide a new approach to preventing the formation of HS.
增生性瘢痕是创伤愈合过程中失调的结果。虽然基本机制尚不清楚，但在增生性瘢痕的发展过程中，注意到细胞增殖增加和细胞凋亡减少。在以前的研究中，我们发现与细胞增殖、凋亡和分化相关的分泌卷曲相关蛋白 2（SFRP2）在增生性瘢痕（HS）组织中显著上调。在这项研究中，靶向 SFRP2 的短发夹 RNA（shRNA）被用于表征增生性瘢痕衍生成纤维细胞（HSFb）中的 SFRP2 功能。通过 MTT、动态生长曲线和 BRDU 测定评估细胞增殖。同时，使用荧光激活细胞分选（FACS）检测细胞凋亡。通过分光光度法测定半胱天冬酶-3 活性。采用纤维母细胞填充胶原晶格（FPCL）模型评估 HSFb 的收缩性。进一步，实时 PCR 和蛋白质印迹用于测量 HSFb 中α-SMA 的 mRNA 和蛋白表达。此外，通过定量实时 PCR 测定 I 型和 III 型前胶原的 mRNA 水平。结果表明，靶向 SFRP2 的 shRNA 显著促进 HSFb 的凋亡，而对细胞增殖没有影响。在 FPCL 模型中观察到 HSFb 中α-平滑肌肌动蛋白（α-SMA）的合成减少和成纤维细胞的收缩减少。定量 RT-PCR 表明 I 型和 III 型前胶原的 mRNA 显著下调。总之，作为一种新的抗凋亡基因，SFRP2 存在于增生性瘢痕中。重要的是，靶向 SFRP2 的 shRNA 可能为预防 HS 的形成提供一种新方法。
