多种类型细胞参与人工关节无菌性松动发病机制的研究

发布时间：2018-11-10 21:15

【摘要】：人工关节无菌性松动(以下简称无菌性松动)是导致关节置换术失败的主要原因之一,其发病主要是多种类型细胞和多种细胞因子共同作用引起骨溶解所导致的,但目前该疾病发生的病理机制尚未完全清楚。在磨损微粒诱导的小鼠颅骨骨溶解模型中,我们观察到小鼠颅骨的成骨细胞中自噬斑点较对照组显著增多,提示磨损微粒可诱导成骨细胞自噬水平的上升。为验证这一现象,我们在体外用磨损微粒直接刺激成骨细胞,并采用western blot检测自噬标志蛋白 LC3(microtubule-associated protein 1 light chain 3)的表达、免疫荧光观察自噬斑点的水平、透射电镜直接观察自噬体及自噬溶酶体的形成,结果证实磨损微粒显著提高了成骨细胞自噬水平。鉴于成骨细胞凋亡是骨溶解的重要原因之一,而很多研究表明自噬与凋亡关系密切,我们探讨了自噬在磨损微粒刺激成骨细胞过程中的作用。流式细胞术结果显示,磨损微粒可浓度依赖性地增加成骨细胞凋亡,而自噬抑制剂3-MA(3-methyladenine)或干扰RNA(siATG5)可减低磨损微粒引起的细胞凋亡,这些结果说明自噬参与了磨损微粒刺激的成骨细胞凋亡的发生。在分子机理研究中,应用小分子抑制剂或干扰RNA等手段得到的数据提示,磨损微粒可能通过ERN1/MAPK8(endoplasmic reticulum to nucleus signaling 1/mitogen-activated protein kinase8)诱导自噬水平的升高,自噬通过调节BAX(BCL2-associated X protein)的表达,促进了成骨细胞凋亡。在动物实验中,抑制自噬可减少磨损微粒引起的成骨细胞凋亡以及功能抑制,减轻骨溶解。通过调控自噬水平进而影响成骨细胞的存活与功能,代表了一种全新的人工关节无菌性松动治疗的策略。骨细胞是骨骼系统中数量最多的细胞,可独立地调控骨吸收和骨形成。过去对人工关节无菌性松动中骨细胞的作用研究较少,在本实验中,我们对骨细胞在本疾病发病过程中作用进行了探讨。骨细胞上清液可抑制小鼠骨髓巨噬细胞(BMMs,bone marrow macrophages)向破骨细胞的分化,而磨损微粒刺激骨细胞后,其上清液的这种抑制破骨细胞分化作用减轻。进一步研究显示,磨损微粒可减少骨细胞分泌的破骨细胞分化抑制因子IFN-β(interferon-β),而功能模拟及抑制试验也证实,骨细胞分泌IFN-β水平的下降是对破骨细胞分化抑制作用减低的关键因素。在机制研究中,实验发现自噬参与了 IFN-β水平的降低;抑制自噬可部分恢复骨细胞上清液对破骨细胞分化的抑制作用。本项研究提示了无菌性松动发病的一种新的机制。过去的研究中,我们发现无菌性松动患者界膜组织中内质网应激(ER stress,endoplasmic reticulum stress)蛋白表达显著上调,考虑到成纤维细胞是界膜组织中的主要细胞类型之一,我们在体外和体内实验中探讨了成纤维细胞内质网应激对无菌性松动发病的影响。在人体界膜组织以及动物模型的颅骨增生软组织中,内质网应激标志蛋白均显著上升。细胞实验中,磨损微粒刺激成纤维细胞内质网应激标志蛋白上调,并且内质网应激下游蛋白XBPls(spliced X-box binding protein 1)也升高。采用小分子抑制剂和干扰RNA的实验结果显示,ER stress/XBP1s参与了磨损微粒引起的成纤维细胞RANKL(receptor activator of nuclear factor(NF)-κB ligand)表达的上调。在动物实验中,抑制 ER stress/XBP1s可减少磨损微粒刺激的破骨细胞形成,减轻骨溶解。总之,这部分结果揭示了无菌性松动发生的一种新机制,即磨损微粒通过ER stress/XBP1s途径引起成纤维细胞RANKL表达上调,促进骨溶解的发生,抑制ER stress/XBP1s可能成为治疗无菌性松动的新方法。
[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.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687.4

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