Lentivirus-mediated TNF-α gene silencing and overexpression of osteoprotegerin inhibit titanium particle-induced inflammatory response and osteoclastogenesis in vitro
Lentivirus-mediated TNF-α gene silencing and overexpression of osteoprotegerin inhibit titanium particle-induced inflammatory response and osteoclastogenesis in vitro
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DOI：
10.3892/mmr.2015.4620
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发表时间：
2016-01-01
影响因子：
3.4
通讯作者：
Liu, Ping
中科院分区：
文献类型：
--
作者：
Peng, Li;Wang, Hongzhi;Liu, Ping
巨噬细胞和破骨细胞在磨损颗粒吞噬后释放促炎因子，促进破骨细胞生成。在这一病理过程中，核因子-kappaB受体配体(RANKL)和肿瘤坏死因子-α(TNF-α)是导致骨吸收和炎症反应的关键因子。本研究旨在构建携带肿瘤坏死因子α小干扰(Si)RNA和骨保护素(OPG)基因的重组慢病毒载体，并在钛(Ti)颗粒诱导的炎症反应细胞模型中观察Lenti-siTNFα-OPG对磨损颗粒诱导的炎症反应和破骨细胞形成的影响。构建Lenti-siTNFα-OPG载体，并在颗粒刺激前分别导入RAW264.7和MC3T3-E1细胞。用Western印迹和酶联免疫吸附试验检测肿瘤坏死因子-α、成骨细胞生长因子和RANKL的蛋白水平，用逆转录-定量聚合酶链式反应检测炎症因子肿瘤坏死因子-α、白介素1β和白介素6以及成骨细胞生长因子和RANKL的mRNA表达水平。用碱性磷酸酶试剂盒检测碱性磷酸酶(ALP)活性。在Lenti-siTNFa-OPG载体的存在下，炎症因子和RANKL的mRNA表达水平以及肿瘤坏死因子-α的蛋白水平均被下调。OPG的mRNA表达和蛋白水平上调，ALP活性升高。提示Lenti-siTNFα-OPG基因可抑制磨粒诱导的炎症反应和破骨细胞的形成，值得进一步研究以预防和/或治疗磨粒诱导的骨溶解。
Macrophages and osteoclasts release proinflammatory factors and promote osteoclastogenesis following the phagocytosis of wear particles. During this pathological process, receptor of nuclear factor kappa B ligand (RANKL) and tumor necrosis factor (TNF)-alpha are critical factors contributing to resorption and the inflammatory response. The present study aimed to construct recombination lentivirus vectors carrying TNF-alpha small interfering (si)RNA and osteoprotegerin (OPG) cDNA, and to examine the effects of Lenti-siTNF alpha-OPG on the wear particle-induced inflammatory response and osteoclastogenesis in a titanium (Ti) particle-induced-inflammatory response cell model. Lenti-siTNF alpha-OPG vectors were constructed and transnfected into RAW264.7 and MC3T3-E1 cells, respectively, prior to particle stimulation. The protein levels of TNF-alpha, OPG and RANKL were evaluated using western blot analysis and enzyme-linked immunosorbent assays, and the mRNA expression levels of the inflammatory factors, TNF-alpha, interleukin (IL)-1 beta and IL-6, as well as OPG and RANKL, were measured using reverse transcription-quantitative polymerase chain reaction analysis. The activity of alkaline phosphatase (ALP) was examined using an ALP kit. In the presence of the Lenti-siTNFa-OPG vector, the mRNA expression levels of the inflammatory factors and RANKL were downregulated, as were the protein levels of TNF-alpha. The mRNA expression and protein levels of OPG were upregulated, and ALP activity was increased. These findings suggested that Lenti-siTNF alpha-OPG transfection inhibited the wear particle-induced inflammatory response and osteoclastogenesis, which warrants further investigation for the prevention and/or treatment of wear particle-induced osteolysis.