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

【摘要】：類風(fēng)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(diǎn)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(xì)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(diǎn)的代謝性骨病,往往存在著OC的骨吸收活性與成骨細(xì)胞的骨形成活性之間的失衡。因此,這兩種疾病都存在著過(guò)強(qiáng)的破骨細(xì)胞活性。理論上,以破骨細(xì)胞作為靶點(diǎn)的治療策略,有望為RA或骨質(zhì)疏松患者帶來(lái)重大意義。MiR-146a與免疫反應(yīng)關(guān)系密切。MiR-146a的兩個(gè)靶蛋白：表皮生長(zhǎng)因子受體(EGFR)和TNF受體相關(guān)因子-6 (TRAF6)在破骨細(xì)胞的形成過(guò)程中具有重要作用。使用穩(wěn)定性高的轉(zhuǎn)運(yùn)系統(tǒng),使細(xì)胞內(nèi)miR-146a過(guò)表達(dá),通過(guò)下調(diào)EGFR和TRAF6的水平,實(shí)現(xiàn)對(duì)破骨細(xì)胞形成相關(guān)的信號(hào)通路的微調(diào)控,對(duì)于RA或骨質(zhì)疏松的治療有潛在的應(yīng)用價(jià)值。在本研究中,我們使用人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell, PBMC)作為破骨細(xì)胞前體,通過(guò)核因子κB受體活化因子配體(receptor activator of NF-kB ligand, RANKL)和巨噬細(xì)胞集落刺激因子(macrophage-colony stimulating factor, M-CSF)兩種細(xì)胞因子的刺激促使其向破骨細(xì)胞轉(zhuǎn)化；利用大腸桿菌原核表達(dá)系統(tǒng),經(jīng)異丙基-β-D硫代半乳糖苷(isopropy-p-D-thiogalactoside, IPTG)誘導(dǎo),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(jìn)行化學(xué)交聯(lián)后,VLPs獲得自主穿透細(xì)胞膜的能力。用不同劑量的MS2-miR-146a VLPs處理PBMCs后,伴隨著細(xì)胞內(nèi)miR-146a的升高,Western Blot檢測(cè)到miR-146a的兩個(gè)靶蛋白EGFR和TRAF6的表達(dá)水平下降；實(shí)時(shí)熒光反轉(zhuǎn)錄定量PCR提示破骨細(xì)胞的四個(gè)標(biāo)志基因：TRAP, PU.1CATK,CA2的表達(dá)水平明顯下調(diào)；通過(guò)抗酒石酸酸性磷酸酶(tartrate resistant acidphatase, TRAP)染色,發(fā)現(xiàn)破骨細(xì)胞的形成數(shù)量明顯減少；通過(guò)與骨片共培養(yǎng)并對(duì)骨片上的吸收陷窩進(jìn)行甲苯胺藍(lán)染色,發(fā)現(xiàn)破骨細(xì)胞的活性明顯受抑�？梢�(jiàn)細(xì)胞內(nèi)升高的miR-146a發(fā)揮了對(duì)破骨細(xì)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(jiǎn)單、高效、生物安全性好,具有良好的應(yīng)用前景。重組MS2 VLPs介導(dǎo)的miR-146a升高能否在動(dòng)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(jìn)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(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.22

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