Cleveland, Ohio; and Milwaukee, Wis. From the Department of Plastic Surgery, Case Western Reserve University School of Medicine, and the Department of Plastic Surgery, Medical College of Wisconsin.
Plast Reconstr Surg. 2009 Nov;124(5):1466-1473. doi: 10.1097/PRS.0b013e3181b98947.
Transforming growth factor (TGF)-beta1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 receptors, and FGF-2 and its R1 receptor in murine dura cells.
A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction.
Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4.
These results indicate that TGF-beta1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.
转化生长因子（TGF）-β1 和成纤维细胞生长因子（FGF）-2 都被证明在调节小鼠颅骨缝合融合中具有重要作用。已经建立了降低这些细胞因子及其各自受体基因表达的方法，但由于副作用，临床应用受到限制。在这项研究中，作者研究了 TGF-β1 特异性小干扰 RNA（siRNA）对小鼠硬脑膜细胞中 TGF-β1、其 TGF-βR1 和 TGF-βR2 受体以及 FGF-2 和其 R1 受体的 mRNA 表达的影响。
从 CD-1 小鼠中建立了原代硬脑膜细胞系。使用 BLOCKiT 测定了 Lipofectamine 的转染效率。用不同浓度的 TGF-β1 siRNA 转染硬脑膜细胞，以确定最佳剂量。在后续实验中，将细胞用 16 nM TGF-β1 siRNA 转染，并在转染后第 4、7、10 和 14 天收获 RNA 进行分离和定量聚合酶链反应。
在 16 nM siRNA 时，TGF-β1 mRNA 表达的最佳抑制效果。在转染后第 4 天，TGF-β1 mRNA 水平显著降低，但在第 14 天恢复到基线。整个时间过程中，TGF-βR1 mRNA 表达不受转染影响。然而，TGF-βR2、FGF-2 和 FGF-R1 的 mRNA 表达在转染后第 4 天显著抑制。
这些结果表明，TGF-β1 siRNA 有可能改变体外负责颅骨缝合融合的小鼠硬脑膜细胞因子。用 siRNA 技术操纵潜在的颅骨缝合生物学最终可能控制缝合融合。这种干预措施最终可能成为颅缝早闭手术干预的有效辅助手段。
