本文選題：miR-146a　切入點：MS2噬菌體病毒樣顆粒　出處：《北京協(xié)和醫(yī)學院》2015年碩士論文　論文類型：學位論文

【摘要】：類風濕性關(guān)節(jié)炎(rheumatoid arthritis, RA)是一種以慢性關(guān)節(jié)炎癥為特點的全身性自身免疫病,其主要臨床表現(xiàn)是關(guān)節(jié)內(nèi)軟骨和骨的破壞、關(guān)節(jié)腫脹和關(guān)節(jié)功能障礙。其中,破骨細胞被證明在RA的關(guān)節(jié)損害中發(fā)揮著關(guān)鍵作用。骨質(zhì)疏松(osteoporosis)是一種多病因的、以單位體積內(nèi)骨組織量減少為特點的代謝性骨病,往往存在著OC的骨吸收活性與成骨細胞的骨形成活性之間的失衡。因此,這兩種疾病都存在著過強的破骨細胞活性。理論上,以破骨細胞作為靶點的治療策略,有望為RA或骨質(zhì)疏松患者帶來重大意義。MiR-146a與免疫反應關(guān)系密切。MiR-146a的兩個靶蛋白：表皮生長因子受體(EGFR)和TNF受體相關(guān)因子-6 (TRAF6)在破骨細胞的形成過程中具有重要作用。使用穩(wěn)定性高的轉(zhuǎn)運系統(tǒng),使細胞內(nèi)miR-146a過表達,通過下調(diào)EGFR和TRAF6的水平,實現(xiàn)對破骨細胞形成相關(guān)的信號通路的微調(diào)控,對于RA或骨質(zhì)疏松的治療有潛在的應用價值。在本研究中,我們使用人外周血單個核細胞(peripheral blood mononuclear cell, PBMC)作為破骨細胞前體,通過核因子κB受體活化因子配體(receptor activator of NF-kB ligand, RANKL)和巨噬細胞集落刺激因子(macrophage-colony stimulating factor, M-CSF)兩種細胞因子的刺激促使其向破骨細胞轉(zhuǎn)化；利用大腸桿菌原核表達系統(tǒng),經(jīng)異丙基-β-D硫代半乳糖苷(isopropy-p-D-thiogalactoside, IPTG)誘導,獲得基于MS2噬菌體的裝載miR-146a的重組病毒樣顆粒(MS2-miR-146a VLPs);與Tat47-57進行化學交聯(lián)后,VLPs獲得自主穿透細胞膜的能力。用不同劑量的MS2-miR-146a VLPs處理PBMCs后,伴隨著細胞內(nèi)miR-146a的升高,Western Blot檢測到miR-146a的兩個靶蛋白EGFR和TRAF6的表達水平下降；實時熒光反轉(zhuǎn)錄定量PCR提示破骨細胞的四個標志基因：TRAP, PU.1CATK,CA2的表達水平明顯下調(diào)；通過抗酒石酸酸性磷酸酶(tartrate resistant acidphatase, TRAP)染色,發(fā)現(xiàn)破骨細胞的形成數(shù)量明顯減少；通過與骨片共培養(yǎng)并對骨片上的吸收陷窩進行甲苯胺藍染色,發(fā)現(xiàn)破骨細胞的活性明顯受抑�？梢娂毎麅�(nèi)升高的miR-146a發(fā)揮了對破骨細胞形成和活性的抑制作用。這種基于MS2噬菌體病毒樣顆粒的遞送方法簡單、高效、生物安全性好,具有良好的應用前景。重組MS2 VLPs介導的miR-146a升高能否在動物水平控制RA軟骨和骨破壞、關(guān)節(jié)炎癥或骨質(zhì)疏松尚有待進一步研究。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R593.22

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