【摘要】：人工關(guān)節(jié)無(wú)菌性松動(dòng)(以下簡(jiǎn)稱(chēng)無(wú)菌性松動(dòng))是導(dǎo)致關(guān)節(jié)置換術(shù)失敗的主要原因之一,其發(fā)病主要是多種類(lèi)型細(xì)胞和多種細(xì)胞因子共同作用引起骨溶解所導(dǎo)致的,但目前該疾病發(fā)生的病理機(jī)制尚未完全清楚。在磨損微粒誘導(dǎo)的小鼠顱骨骨溶解模型中,我們觀察到小鼠顱骨的成骨細(xì)胞中自噬斑點(diǎn)較對(duì)照組顯著增多,提示磨損微�？烧T導(dǎo)成骨細(xì)胞自噬水平的上升。為驗(yàn)證這一現(xiàn)象,我們?cè)隗w外用磨損微粒直接刺激成骨細(xì)胞,并采用western blot檢測(cè)自噬標(biāo)志蛋白 LC3(microtubule-associated protein 1 light chain 3)的表達(dá)、免疫熒光觀察自噬斑點(diǎn)的水平、透射電鏡直接觀察自噬體及自噬溶酶體的形成,結(jié)果證實(shí)磨損微粒顯著提高了成骨細(xì)胞自噬水平。鑒于成骨細(xì)胞凋亡是骨溶解的重要原因之一,而很多研究表明自噬與凋亡關(guān)系密切,我們探討了自噬在磨損微粒刺激成骨細(xì)胞過(guò)程中的作用。流式細(xì)胞術(shù)結(jié)果顯示,磨損微粒可濃度依賴(lài)性地增加成骨細(xì)胞凋亡,而自噬抑制劑3-MA(3-methyladenine)或干擾RNA(siATG5)可減低磨損微粒引起的細(xì)胞凋亡,這些結(jié)果說(shuō)明自噬參與了磨損微粒刺激的成骨細(xì)胞凋亡的發(fā)生。在分子機(jī)理研究中,應(yīng)用小分子抑制劑或干擾RNA等手段得到的數(shù)據(jù)提示,磨損微�？赡芡ㄟ^(guò)ERN1/MAPK8(endoplasmic reticulum to nucleus signaling 1/mitogen-activated protein kinase8)誘導(dǎo)自噬水平的升高,自噬通過(guò)調(diào)節(jié)BAX(BCL2-associated X protein)的表達(dá),促進(jìn)了成骨細(xì)胞凋亡。在動(dòng)物實(shí)驗(yàn)中,抑制自噬可減少磨損微粒引起的成骨細(xì)胞凋亡以及功能抑制,減輕骨溶解。通過(guò)調(diào)控自噬水平進(jìn)而影響成骨細(xì)胞的存活與功能,代表了一種全新的人工關(guān)節(jié)無(wú)菌性松動(dòng)治療的策略。骨細(xì)胞是骨骼系統(tǒng)中數(shù)量最多的細(xì)胞,可獨(dú)立地調(diào)控骨吸收和骨形成。過(guò)去對(duì)人工關(guān)節(jié)無(wú)菌性松動(dòng)中骨細(xì)胞的作用研究較少,在本實(shí)驗(yàn)中,我們對(duì)骨細(xì)胞在本疾病發(fā)病過(guò)程中作用進(jìn)行了探討。骨細(xì)胞上清液可抑制小鼠骨髓巨噬細(xì)胞(BMMs,bone marrow macrophages)向破骨細(xì)胞的分化,而磨損微粒刺激骨細(xì)胞后,其上清液的這種抑制破骨細(xì)胞分化作用減輕。進(jìn)一步研究顯示,磨損微粒可減少骨細(xì)胞分泌的破骨細(xì)胞分化抑制因子IFN-β(interferon-β),而功能模擬及抑制試驗(yàn)也證實(shí),骨細(xì)胞分泌IFN-β水平的下降是對(duì)破骨細(xì)胞分化抑制作用減低的關(guān)鍵因素。在機(jī)制研究中,實(shí)驗(yàn)發(fā)現(xiàn)自噬參與了 IFN-β水平的降低;抑制自噬可部分恢復(fù)骨細(xì)胞上清液對(duì)破骨細(xì)胞分化的抑制作用。本項(xiàng)研究提示了無(wú)菌性松動(dòng)發(fā)病的一種新的機(jī)制。過(guò)去的研究中,我們發(fā)現(xiàn)無(wú)菌性松動(dòng)患者界膜組織中內(nèi)質(zhì)網(wǎng)應(yīng)激(ER stress,endoplasmic reticulum stress)蛋白表達(dá)顯著上調(diào),考慮到成纖維細(xì)胞是界膜組織中的主要細(xì)胞類(lèi)型之一,我們?cè)隗w外和體內(nèi)實(shí)驗(yàn)中探討了成纖維細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激對(duì)無(wú)菌性松動(dòng)發(fā)病的影響。在人體界膜組織以及動(dòng)物模型的顱骨增生軟組織中,內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志蛋白均顯著上升。細(xì)胞實(shí)驗(yàn)中,磨損微粒刺激成纖維細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志蛋白上調(diào),并且內(nèi)質(zhì)網(wǎng)應(yīng)激下游蛋白XBPls(spliced X-box binding protein 1)也升高。采用小分子抑制劑和干擾RNA的實(shí)驗(yàn)結(jié)果顯示,ER stress/XBP1s參與了磨損微粒引起的成纖維細(xì)胞RANKL(receptor activator of nuclear factor(NF)-κB ligand)表達(dá)的上調(diào)。在動(dòng)物實(shí)驗(yàn)中,抑制 ER stress/XBP1s可減少磨損微粒刺激的破骨細(xì)胞形成,減輕骨溶解�？傊�,這部分結(jié)果揭示了無(wú)菌性松動(dòng)發(fā)生的一種新機(jī)制,即磨損微粒通過(guò)ER stress/XBP1s途徑引起成纖維細(xì)胞RANKL表達(dá)上調(diào),促進(jìn)骨溶解的發(fā)生,抑制ER stress/XBP1s可能成為治療無(wú)菌性松動(dòng)的新方法。
[Abstract]:The aseptic loosening of the artificial joint (hereinafter referred to as the aseptic loosening) is one of the main causes of the failure of the joint replacement. But the pathological mechanism of the disease is not yet completely clear. In the mouse skull bone lysis model induced by wear particles, we observed that the self-plaque point in the bone of the mouse's skull was significantly increased from the control group, suggesting that the wear particles could induce the increase in the autophagy level of the osteoblast. In order to verify this phenomenon, we directly stimulated the osteoblast with the wear particles in vitro, and the expression of the autophagy-associated protein 1 light chain 3 was detected by western blot. The level of the autophagy spot and the formation of autophagy were directly observed by the transmission electron microscope. The results confirmed that the wear particles significantly increased the self-phagocytic level of the osteoblast. In view of the fact that the apoptosis of osteoblasts is one of the important causes of osteolysis, many studies have shown that autophagy is closely related to apoptosis, and we have discussed the role of autophagy in the stimulation of osteoblasts in the presence of wear particles. The flow cytometry results show that the wear particles can increase the apoptosis of the osteoblast in a concentration-dependent manner, and the autophagy inhibitor 3-MA (3-methylenedienine) or the interfering RNA (siATG5) can reduce the apoptosis of the cells caused by the wear particles, which result in the occurrence of the apoptosis of the osteoblast participating in the stimulation of the wear particles. In the study of molecular mechanism, it is suggested that the wear particles may induce the increase of autophagy by ERN1/ MAPK8 (endoplasmic reticulum to nadus signaling 1/ mitgen-activated protein kinase8), and the autophagy promotes the apoptosis of the osteoblast by regulating the expression of BAX (BCL2-associated X protein). In animal experiments, the inhibition of autophagy can reduce the apoptosis of the osteoblast and the function inhibition caused by the wear particles, and the osteolysis is reduced. By controlling the autophagy level and thus the survival and function of the osteoblast, a new strategy for aseptic loosening treatment of the artificial joint is represented. Osteoclasts are the largest number of cells in the bone system, and the bone resorption and bone formation can be regulated independently. In the past, the role of the bone cells in the aseptic loosening of the artificial joint is less, and in this experiment, we have discussed the role of the bone cells in the pathogenesis of the disease. The supernatant of the bone cells can inhibit the differentiation of the bone marrow macrophages (BMMs, bone marrow macroPages) to the osteoclast, and the osteoclast differentiation is relieved after the wear particles stimulate the bone cells. Further studies have shown that the wear particles can reduce the osteoclast differentiation inhibitory factor, IFN-1, which is secreted by the bone cells, and the functional simulation and the inhibition test also confirm that the decrease in the level of the activity of the osteoclasts is a key factor in the reduction of the inhibition of osteoclast differentiation. In the mechanism study, it was found that autophagy was involved in the reduction of the level of IFN-osteoclast; inhibition of autophagy could partially restore the inhibitory effect of the supernatant on the differentiation of osteoclasts. This study suggests a new mechanism for aseptic loosening. In the past studies, we have found that the expression of ER stress, endoplasmic reticulatum stress, in a sterile loose patient-bound membrane tissue, is up-regulated, taking into account that fibroblasts are one of the main cell types in the membrane tissue, The effects of endoplasmic reticulum stress on aseptic loosening were discussed in vitro and in vivo. The endoplasmic reticulum stress marker protein increased significantly in the human body membrane tissue and in the soft tissue of the skull of the animal model. In the cell experiment, the endoplasmic reticulum stress marker protein of the fibroblast was upregulated, and the expression of the endoplasmic reticulum stress downstream protein XBPls (splaned X-box binding protein 1) was also increased. The results of small molecule inhibitor and interfering RNA show that ER stress/ XBP1s is involved in the up-regulation of the expression of the fibroblast RANKL (NF)-In animal experiments, the inhibition of ER stress/ XBP1s can reduce the formation of osteoclasts stimulated by the wear particles and reduce the osteolysis. In conclusion, this part of the results revealed a new mechanism of aseptic loosening, that is, the increased expression of RANKL in the fibroblasts by the ER stress/ XBP1s pathway, promoting the occurrence of osteolysis, and the inhibition of ER stress/ XBP1s as a new method for the treatment of aseptic loosening.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R687.4

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