【摘要】：目的:骨性關(guān)節(jié)炎(Osteoarthritis,OA)是一種常見的慢性關(guān)節(jié)病。其發(fā)病率隨著年齡增長逐漸增加。其病理特征為原發(fā)或繼發(fā)性的關(guān)節(jié)軟骨病變,具體表現(xiàn)為關(guān)節(jié)軟骨膠原蛋白降解、蛋白多糖降解和關(guān)節(jié)軟骨結(jié)構(gòu)破壞,同時(shí)也會(huì)導(dǎo)致一定程度的滑膜炎癥。WHO數(shù)據(jù)顯示,10%男性及18女性%以及65%年齡超過60歲的人都患有不同程度的骨關(guān)節(jié)炎。雖然OA的病因是比較復(fù)雜的(遺傳,體質(zhì),生物力學(xué)因素),但我們對(duì)OA的發(fā)病機(jī)制的認(rèn)識(shí)正不斷加深。骨橋蛋白(Osteopontin,OPN)是一種廣泛分布于人體組織中,具有多種功能的磷酸化蛋白。OPN可由多種細(xì)胞分泌,包括巨噬細(xì)胞、淋巴細(xì)胞、上皮細(xì)胞、血管平滑肌細(xì)胞、滑膜細(xì)胞以及軟骨細(xì)胞。在骨組織中,OPN可調(diào)節(jié)細(xì)胞基質(zhì)和細(xì)胞間的活動(dòng),參與骨折修復(fù)中軟骨向骨的轉(zhuǎn)化以及破骨細(xì)胞對(duì)骨基質(zhì)的附著。骨橋蛋白(Osteopontin,OPN)與骨關(guān)節(jié)炎的嚴(yán)重程度及疾病的發(fā)生進(jìn)展密切相關(guān),但OPN在OA發(fā)病機(jī)制中的作用尚未明確。本研究目的旨在探討骨橋蛋白在膝骨關(guān)節(jié)炎(Osteoarthritis,OA)中的調(diào)控作用以及涉及的信號(hào)轉(zhuǎn)導(dǎo)通路。方法:1.選取15名OA患者其中男性7例,女性8例,平均年齡61.2±8.5歲。選取同期正常對(duì)照組15例,為不伴有骨關(guān)節(jié)炎的半月板損傷或韌帶損傷。對(duì)照組男性9例,女性6例,平均年齡42.4±7.8歲。Ⅱ型膠原酶一步消化法處理OA滑膜組織,分離培養(yǎng)OA軟骨細(xì)胞并傳代,取第一代OA軟骨細(xì)胞繼續(xù)培養(yǎng)。2.根據(jù)文獻(xiàn)報(bào)道siRNA序列,合成OPN-siRNA,與OPN組作為實(shí)驗(yàn)組,無意義序列Con-siRNA為陰性對(duì)照組,將兩組序列進(jìn)行脂質(zhì)體轉(zhuǎn)染,采用Western Blotting檢測(cè)NF-κB(nuclear factor-KappaB)p65、p-NF-κB p65的蛋白水平,將得到的Western blot圖片,用IPP軟件處理,得出灰密度值,進(jìn)行目的蛋白NF-κB/p-NF-κB的均一化處理和內(nèi)參蛋白β-actin的均一化處理,將濃度組得到的NF-κB/p-NF-κB p65的均一化結(jié)果除以β-actin的結(jié)果,得到各組的相對(duì)定量值。3.以轉(zhuǎn)染Con-siRNA的OA滑膜細(xì)胞為對(duì)照組,轉(zhuǎn)染OPN-siRNA、OPN、PDTC及PDTC聯(lián)合OPN處理細(xì)胞為實(shí)驗(yàn)組。用Western blot檢測(cè)MMP13表達(dá)情況。用上述方法得到各組相對(duì)定量值。結(jié)果:相比對(duì)照組,經(jīng)OPN干預(yù)后,P65含量及P65磷酸化顯著增加(P=0.0025,P=0.0019)。然而,OPN-siRNA處理后的P65含量及P65磷酸化相比對(duì)照組顯著降低(P=0.0024,P=0.0176)。OPN可顯著提高M(jìn)MP13蛋白含量(P=0.0075),而OPN-siRNA可顯著降低蛋白含量(P=0.0021),NF-κB抑制劑(PDTC)可明顯拮抗OPN對(duì)MMP13蛋白的調(diào)控(P=0.0231)。結(jié)論:OPN可促進(jìn)OA滑膜細(xì)胞NF-κB p65、p-NF-κB p65的蛋白表達(dá),并通過激活NF-κB信號(hào)通路,促進(jìn)MMP13蛋白表達(dá)。這可能是OA發(fā)病機(jī)理的重要組成部分。
[Abstract]:Objective: osteoarthritis (Osteoarthritis,OA) is a common chronic arthropathy. The incidence rate increases with age. The pathological features are primary or secondary articular cartilage lesions, including collagen degradation of articular cartilage, degradation of proteoglycan and destruction of articular cartilage structure, as well as synovitis to a certain extent. WHO data show that, Ten percent of men and 18 percent of women and 65 percent of people over 60 had varying degrees of osteoarthritis. Although the etiology of OA is complex (genetic, physical, biomechanical factors), our understanding of the pathogenesis of OA is deepening. Osteopontin (Osteopontin,OPN) is a phosphorylated protein widely distributed in human tissues. OPN can be secreted by a variety of cells, including macrophages, lymphocytes, epithelial cells, vascular smooth muscle cells. Synovial cells and chondrocytes. In bone tissue, OPN can regulate the activity of cell matrix and cells, participate in the transformation of cartilage to bone and the attachment of osteoclasts to bone matrix in fracture repair. Osteopontin (Osteopontin,OPN) is closely related to the severity and progression of osteoarthritis, but the role of OPN in the pathogenesis of OA is not clear. The aim of this study was to investigate the regulatory role of osteopontin in knee osteoarthritis (Osteoarthritis,OA) and the signal transduction pathways involved. Methods: 1. 15 patients with OA were selected, including 7 males and 8 females, with an average age of 61.2 鹵8.5 years. 15 cases of normal control group were selected as meniscus injury or ligament injury without osteoarthritis. There were 9 males and 6 females in the control group, with an average age of 42.4 鹵7.8.Type 鈪，

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