miR-146a抑制PBMC源性破骨细胞形成的实验研究

发布时间：2018-03-10 00:01

本文选题：miR-146a　切入点：MS2噬菌体病毒样颗粒　出处：《北京协和医学院》2015年硕士论文　论文类型：学位论文

【摘要】：类风湿性关节炎(rheumatoid arthritis, RA)是一种以慢性关节炎症为特点的全身性自身免疫病,其主要临床表现是关节内软骨和骨的破坏、关节肿胀和关节功能障碍。其中,破骨细胞被证明在RA的关节损害中发挥着关键作用。骨质疏松(osteoporosis)是一种多病因的、以单位体积内骨组织量减少为特点的代谢性骨病,往往存在着OC的骨吸收活性与成骨细胞的骨形成活性之间的失衡。因此,这两种疾病都存在着过强的破骨细胞活性。理论上,以破骨细胞作为靶点的治疗策略,有望为RA或骨质疏松患者带来重大意义。MiR-146a与免疫反应关系密切。MiR-146a的两个靶蛋白：表皮生长因子受体(EGFR)和TNF受体相关因子-6 (TRAF6)在破骨细胞的形成过程中具有重要作用。使用稳定性高的转运系统,使细胞内miR-146a过表达,通过下调EGFR和TRAF6的水平,实现对破骨细胞形成相关的信号通路的微调控,对于RA或骨质疏松的治疗有潜在的应用价值。在本研究中,我们使用人外周血单个核细胞(peripheral blood mononuclear cell, PBMC)作为破骨细胞前体,通过核因子κB受体活化因子配体(receptor activator of NF-kB ligand, RANKL)和巨噬细胞集落刺激因子(macrophage-colony stimulating factor, M-CSF)两种细胞因子的刺激促使其向破骨细胞转化；利用大肠杆菌原核表达系统,经异丙基-β-D硫代半乳糖苷(isopropy-p-D-thiogalactoside, IPTG)诱导,获得基于MS2噬菌体的装载miR-146a的重组病毒样颗粒(MS2-miR-146a VLPs);与Tat47-57进行化学交联后,VLPs获得自主穿透细胞膜的能力。用不同剂量的MS2-miR-146a VLPs处理PBMCs后,伴随着细胞内miR-146a的升高,Western Blot检测到miR-146a的两个靶蛋白EGFR和TRAF6的表达水平下降；实时荧光反转录定量PCR提示破骨细胞的四个标志基因：TRAP, PU.1CATK,CA2的表达水平明显下调；通过抗酒石酸酸性磷酸酶(tartrate resistant acidphatase, TRAP)染色,发现破骨细胞的形成数量明显减少；通过与骨片共培养并对骨片上的吸收陷窝进行甲苯胺蓝染色,发现破骨细胞的活性明显受抑。可见细胞内升高的miR-146a发挥了对破骨细胞形成和活性的抑制作用。这种基于MS2噬菌体病毒样颗粒的递送方法简单、高效、生物安全性好,具有良好的应用前景。重组MS2 VLPs介导的miR-146a升高能否在动物水平控制RA软骨和骨破坏、关节炎症或骨质疏松尚有待进一步研究。
[Abstract]:Rheumatoid arthritis (RAA) is a systemic autoimmune disease characterized by chronic arthritis. Its main clinical manifestations are the destruction of articular cartilage and bone, joint swelling and joint dysfunction. Osteoclasts have been shown to play a key role in the joint damage of RA. Osteoporosis is a metabolic osteopathy characterized by reduced bone mass per unit volume. There is often an imbalance between OC's bone resorption activity and osteoblast's osteogenesis activity. Therefore, both diseases have excessive osteoclast activity. In theory, osteoclasts are targeted at therapeutic strategies. Two target proteins, Epidermal growth factor receptor (EGFR) and TNF receptor-associated factor -6 (-6), are expected to be of great significance to RA or osteoporosis patients. MiR-146a plays an important role in the formation of osteoclasts. Using a stable transit system, Overexpression of miR-146a in cells and microregulation of the signal pathway associated with osteoclast formation by down-regulating the levels of EGFR and TRAF6 may be of potential value in the treatment of RA or osteoporosis. We used human peripheral blood mononuclear cells, peripheral blood mononuclear cells, as precursors of osteoclasts. The osteoclasts were transformed into osteoclasts by stimulation of nuclear factor 魏 B receptor activator of NF-kB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). Induced by isopropy-p-D-thiogalactoside (IPTG) of isopropy-p-D-thiogalactoside (IPTG), the recombinant virus-like particles loaded with MS2 phage, MS2-miR-146a VLPsN, were chemically crosslinked with Tat47-57 to obtain the ability of autonomic penetration of PBMCs. With the increase of intracellular miR-146a, the expression level of EGFR and TRAF6 of two target proteins of miR-146a were detected by Western Blot, and the expression of four marker genes of osteoclasts, namely, the expression of EGFR and TRAF6 in osteoclasts, was down-regulated by real-time fluorescence reverse transcription quantitative PCR, and the expression of the four marker genes of osteoclasts was down-regulated. By tartrate resistant acidphatase (trips) staining, it was found that the number of osteoclasts was significantly reduced, and toluidine blue staining was performed on the resorption lacunae of bone slices by co-culture with osteoclasts. It was found that the activity of osteoclasts was significantly inhibited. It was found that the increased miR-146a played an inhibitory role on the formation and activity of osteoclasts. The delivery method based on MS2 phage like virus particles was simple, efficient and safe. The recombinant MS2 VLPs mediated miR-146a elevation can control RA cartilage and bone destruction at animal level, arthritis or osteoporosis remains to be further studied.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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