A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.
A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.
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使用小干扰 RNA 沉默快速评估骨骼基因功能的便捷体内模型
DOI：
10.1371/journal.pone.0167222
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Song C
中科院分区：
文献类型：
--
作者：
Zhang W;Liu C;Hai B;Du G;Wang H;Leng H;Xu Y;Song C
在体外研究骨骼是很困难的，因为它含有各种参与串扰的细胞类型。骨在生物学上连接着各种器官，因此越来越明显的是，必须在完整的动物中以综合的方式研究骨骼生理学。我们开发了一种模型，使用局部骨内小干扰RNA（siRNA）注射，以快速评估靶基因对局部骨骼环境的影响。在该模型中，对160-g雄性Sprague-Dawley大鼠处理1-2周。左胫骨接受骨内注射的甲状旁腺激素1受体（Pth 1 r）或胰岛素样生长因子1受体（Igf-1 r）siRNA转染复合物装载在泊洛沙姆407水凝胶，和右胫骨接受相同体积的对照siRNA。所有动物均接受骨内注射重组人甲状旁腺激素（1-34）（rhPTH（1-34））或胰岛素样生长因子-1（IGF-1）。分别在安乐死前6天和2天注射钙黄绿素绿色和茜素红。采用RT-PCR和免疫组化方法检测IGF-1 R和PTH 1 R的表达水平。骨密度（BMD），微观结构，矿物质沉积率（MAR），并通过双能X线吸收测定，显微CT，组织学和生物力学测试的强度。RT-PCR和免疫组化结果显示，与右胫骨相比，siRNA处理的左胫骨中IGF-1 R和PTH 1 R表达水平显著降低（均p<0.05）。使用泊洛沙姆407水凝胶作为控释系统延长了单剂量siRNA的沉默效果; IGF-1 R的mRNA表达水平在两周时低于一周时（p<0.01）。与右胫骨相比，左胫骨的BMD、骨微结构参数、MAR和骨强度显著降低（均p<0.05）。这种简单方便的局部骨内siRNA注射模型在很短的治疗期（≤7天）内用非常少量的siRNA实现了基因沉默。
It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA) injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1–2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r) or insulin-like growth factor 1 receptor (Igf-1r) siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1–34) (rhPTH (1–34)) or insulin-like growth factor-1 (IGF-1). Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD), microstructure, mineral apposition rates (MARs), and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05). Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01). The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05). This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of siRNA over a short treatment period (≤7 days).
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影响因子：
64.5
作者：
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