Noggin short interfering RNA gene transfer enhances the in vivo ectopic bone formation induced by Bone Morphogenetic Protein-2

Noggin是骨形态发生蛋白（BMP）的主要胞外拮抗剂之一。有研究表明，在BMP诱导下，未分化间充质细胞和成肌细胞中noggin表达上调，这可能导致BMP在骨形成中的作用减弱。在本研究中，我们研究了短干扰RNA沉默noggin在体外成骨分化和体内异位骨形成中的作用。实时荧光定量RT-PCR检测BMP-4诱导成肌细胞系C2 C12表达Noggin。BMP-4刺激后Noggin mRNA表达呈剂量和时间依赖性增加。我们设计了针对noggin的双链短干扰RNA（siRNA）。将表达noggin和siRNA的质粒载体共转染入C2 C12细胞中，并通过Western blot证实成功特异性抑制noggin蛋白表达。将noggin siRNA转染入C2 C12细胞中也抑制了内源性noggin mRNA的表达。 ...更多信息 n，并增强BMP-4诱导的碱性磷酸酶活性达2倍。通过小鼠异位成骨实验检测noggin siRNA基因转染对rhBMP-2诱导的骨形成的影响。将Noggin siRNA质粒注射到背肌内，并对肌肉进行电刺激，然后将含有rhBMP-2的胶原盘植入肌肉内。术后第4天，从椎间盘周围的肌肉组织中提取总RNA用于真实的时间RT-PCR分析。电穿孔介导的noggin siRNA的转移显著降低了noggin mRNA的表达，其在植入含有rhBMP-2的胶原盘后增加。术后3周，取出种植体，用软X线机拍摄X线片，测量每个听小骨的骨矿含量（BMC）。noggin siRNA基因转染后，rhBMP-2诱导的新生骨体积和平均骨矿含量显著增加。结论：noggin siRNA基因转染能增强BMP-4诱导的成骨细胞分化和BMP-2诱导的成骨作用。虽然需要进一步的研究，这种方法可能是有用的工具，在临床应用。少

Noggin is one of major extracellular antagonists of bone morphogenetic proteins(BMPs). It is reported that noggin is upregulated in undifferentiated mesenchymal cells and myoblasts in response to BMPs, and this may cause diminish the performance of BMPs in bone formation. In this study, we investigated the effect of noggin silencing by short interfering RNA in osteogenic differentiation in vitro, and the ectopic bone formation in vivo. Noggin expression induced by BMP-4 in C2C12 cells, a myoblastic cell line, was comfirmed by real-time RT-PCR. Noggin mRNA expression was elevated by BMP-4 stimulation in dose-and time-dependent manner. We designed double-stranded short interfering RNA(siRNA) for targeting noggin. Plasmid vectors expressing noggin and siRNA were co-transfected into C2C12 cells, and successful specific suppression of noggin protein expression was confirmed by Western blot. Transfection of the noggin siRNA into C2C12 cell also suppressed the endogenous noggin mRNA expressio … More n induced by BMP-4, and enhanced BMP-4 induced alkaline phosphatase activity up to two fold. In vivo effect of noggin siRNA gene transfer on rhBMP-2 induced bone formation was examined by ectopic bone formation assay in mice. Noggin siRNA plasmid was injected into the dorsal muscle and electropolation procedures was applied to the muscle, and then collagen disk containing of rhBMP-2 was implanted into the muscle. On days 4 after surgery, total RNA was extracted from muscle tissue around the disk for real time RT-PCR analysis. Electroporation-mediated transfer of noggin siRNA markedly decreased expression of noggin mRNA, which increased after implantation of the collagen disk containing rhBMP-2. At 3 weeks after surgery, the implants were harvested and radiographed with a soft X-ray apparatus, and bone mineral content(BMC) of each ossicle was measured. The size of new bone induced by rhBMP-2 and the mean BMC were significantly increased by noggin siRNA gene transfer. Conclusively, these findings suggest that noggin siRNA gene transfer enhances the osteoblast differentiation induced by BMP-4 in vitro and the bone formation induced by BMP-2 in vivo. While further study is needed, this approach may be useful tool in clinical application. Less