本文關鍵詞：蟲草素對人脂肪干細胞成骨分化作用的實驗研究　出處：《南京醫(yī)科大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 脂肪間充質(zhì)干細胞 骨病 蟲草素 炎癥

【摘要】：背景和目的慢性炎癥疾病的患者比普通人群有更多的骨丟失及更高的骨折發(fā)生率,骨吸收增加而骨形成明顯受到抑制。間充質(zhì)干細胞(mesenchymal stem cells,MSCs)作為具有多向分化潛能的始祖細胞,能在相關細胞因子影響下分化為成骨細胞而促進骨形成,減少骨丟失及改善臨床癥狀。但處于炎癥微環(huán)境中的間充質(zhì)干細胞成骨分化能力卻受到了明顯的抑制。TNF-α能抑制成骨細胞分化成熟的主要通路,且大量體外研究及動物模型顯示過量的TNF-α抑制了成骨細胞的增殖及分化,增加了成骨細胞及其前體細胞的凋亡。蟲草素(3-脫氧腺苷)是從稀有珍貴的傳統(tǒng)中藥冬蟲夏草中分離出來的有效成分。它在很多不同的細胞及動物模型中表現(xiàn)出較強的抗炎作用。然而,蟲草素對于TNF-α誘導的人脂肪間充質(zhì)干細胞成骨分化抑制的作用仍然不清楚。方法從類風濕性關節(jié)炎患者腹部脂肪組織中分離出人脂肪間充質(zhì)干細胞并進行傳代培養(yǎng),在成骨培養(yǎng)液中加入不同濃度的TNF-α及蟲草素,通過堿性磷酸酶染色,茜素紅染色,RT-PCR,westen-blotting等方法檢測ALP活性,鈣沉積含量,成骨標志基因Runx2和Osx mRNA表達水平等相關指標來評估脂肪干細胞增殖及成骨分化的能力。結(jié)果本研究中發(fā)現(xiàn)20,40ug/ml的蟲草素能誘導細胞死亡。有趣的是10ug/ml的蟲草素卻能抵消由20ng/ml TNF-α引起的細胞死亡。同時蟲草素對人脂肪干細胞成骨分化的影響顯示出劑量依賴性:10ug/ml的蟲草素能促進脂肪干細胞成骨分化,而40ug/ml的蟲草素卻抑制了脂肪干細胞的成骨分化。同時我們發(fā)現(xiàn)10ug/ml的蟲草素能保護TNF-α誘導人脂肪間充質(zhì)干細胞成骨分化抑制的作用。本研究還提示10ug/ml的蟲草素能恢復在成骨分化中受TNF-α顯著抑制的Runx2和Osx mRNA表達水平。TNF-α能通過抑制ⅠκB α磷酸化及隨后P65釋放和易位到細胞核而激活NF-κB信號通路,本研究中發(fā)現(xiàn)10ug/ml的蟲草素能抑制TNF-α的這種作用。結(jié)論本研究揭示了炎癥細胞因子抑制骨形成的初步機制,凸顯出蟲草素在細胞治療炎性骨病中促脂肪干細胞骨形成的巨大潛力及臨床意義。
[Abstract]:Background and objective patients with chronic inflammatory diseases have more bone loss and higher fracture rates than the general population. Mesenchymal stem cells (MSCs) were the progenitor cells with multiple differentiation potential. It can differentiate into osteoblasts under the influence of related cytokines and promote bone formation. The osteogenic differentiation ability of mesenchymal stem cells in inflammatory microenvironment was significantly inhibited. TNF- 偽 could inhibit the main pathway of osteoblast differentiation and maturation. A large number of in vitro studies and animal models showed that excessive TNF- 偽 inhibited the proliferation and differentiation of osteoblasts. Increased apoptosis of osteoblasts and their precursors. Cordycepin 3-deoxyadenosine). Cordyceps sinensis is a rare and precious traditional Chinese medicine Cordyceps sinensis isolated from the active ingredients. It has a strong anti-inflammatory effect in many different cell and animal models. However. The inhibitory effect of cordycepin on osteogenic differentiation of human adipose mesenchymal stem cells induced by TNF- 偽 is still unclear. Methods Human adipose mesenchymal stem cells were isolated from abdominal adipose tissue of patients with rheumatoid arthritis. Subculture. Different concentrations of TNF- 偽 and Cordycepin were added to the osteoblast culture medium. Then alkaline phosphatase staining and alizarin red staining were used to detect RT-PCR. The activity of ALP and the content of calcium deposition were detected by westen-blotting. The expression level of osteoblastic marker genes Runx2 and Osx mRNA were used to evaluate the proliferation and osteogenic differentiation of adipose stem cells. 40ugrml of Cordycepin can induce cell death. Interestingly, 10ugrml of Cordycepin can counteract 20ng / ml of Cordycepin. At the same time, the effect of Cordycepin on osteogenic differentiation of human adipose stem cells showed that Cordycepin could promote osteogenic differentiation of adipose stem cells in a dose-dependent manner. At the same time, we found that 10ug- / ml Cordycepin could protect human adipose mesenchymal stem cells from osteogenic differentiation induced by TNF- 偽. This study also suggests that 10ugrml Cordycepin can recover the Runx2 and Osx significantly inhibited by TNF- 偽 during osteogenic differentiation. MRNA expression level. TNF- 偽 can activate NF- 魏 B signaling pathway by inhibiting phosphorylation of 鈪，

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