TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels.
TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels.
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小鼠原代硬脑膜细胞培养中的 TGF-β1 RNA 干扰：对 TGF-β1 受体、FGF-2 和 FGF-R1 mRNA 水平的下游影响。
DOI：
10.1097/prs.0b013e3181b98947
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发表时间：
2009
影响因子：
3.6
通讯作者：
Halligan,NadineLN
中科院分区：
文献类型：
--
作者：
Gosain,ArunK;Machol4th,JacquesA;Gliniak,Christy;Halligan,NadineLN
背景：转化生长因子(TGF)-β1和成纤维细胞生长因子(FGF)-2均被证明在小鼠颅骨缝合融合的调节中具有重要作用。降低这些细胞因子及其受体基因表达的方法已经建立，但由于副作用，临床应用受到限制。本研究检测了TGF-β1特异性小干扰RNA （siRNA）对小鼠硬脑膜细胞中TGF-β1及其受体TGF-βR1和TGF-βR2以及FGF-2及其R1受体信使RNA （mRNA）表达的影响。方法：建立CD-1小鼠硬脑膜原代细胞系。使用BLOCKiT检测Lipofectamine转染效率。用不同浓度的TGF-β1 siRNA转染硬脑膜细胞，确定最佳剂量。在随后的实验中，用16 nM TGF-β1 siRNA转染细胞，在转染后4、7、10和14天收获细胞，进行RNA分离和定量聚合酶链反应。结果：16 nM siRNA对TGF-β1 mRNA表达抑制效果最佳。转染后第4天，TGF-β1 mRNA水平显著降低，但在第14天恢复到基线水平。TGF-βR1 mRNA的表达在整个时间过程中不受转染的影响。然而，TGF-βR2、FGF-2和FGF-R1在转染后第4天表现出显著的mRNA表达抑制作用。结论：这些结果表明TGF-β1 siRNA在体外具有改变小鼠硬脑膜细胞因子的潜力，这些细胞因子负责缝线融合。使用siRNA技术操纵潜在的颅骨缝合生物学可能最终允许控制缝合融合。这种干预可能最终成为颅缝闭锁手术干预的有效辅助手段。
Background:Transforming growth factor (TGF)-β1 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-β1–specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-β1, its TGF-βR1 and TGF-βR2 receptors, and FGF-2 and its R1 receptor in murine dura cells.Methods: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-β1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-β1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction.Results:Optimal inhibition of TGF-β1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-β1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-βR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-βR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4.Conclusions:These results indicate that TGF-β1 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.