虫草素对人脂肪干细胞成骨分化作用的实验研究

发布时间：2018-01-16 22:10

  本文关键词：虫草素对人脂肪干细胞成骨分化作用的实验研究　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 脂肪间充质干细胞 骨病 虫草素 炎症

【摘要】：背景和目的慢性炎症疾病的患者比普通人群有更多的骨丢失及更高的骨折发生率,骨吸收增加而骨形成明显受到抑制。间充质干细胞(mesenchymal stem cells,MSCs)作为具有多向分化潜能的始祖细胞,能在相关细胞因子影响下分化为成骨细胞而促进骨形成,减少骨丢失及改善临床症状。但处于炎症微环境中的间充质干细胞成骨分化能力却受到了明显的抑制。TNF-α能抑制成骨细胞分化成熟的主要通路,且大量体外研究及动物模型显示过量的TNF-α抑制了成骨细胞的增殖及分化,增加了成骨细胞及其前体细胞的凋亡。虫草素(3-脱氧腺苷)是从稀有珍贵的传统中药冬虫夏草中分离出来的有效成分。它在很多不同的细胞及动物模型中表现出较强的抗炎作用。然而,虫草素对于TNF-α诱导的人脂肪间充质干细胞成骨分化抑制的作用仍然不清楚。方法从类风湿性关节炎患者腹部脂肪组织中分离出人脂肪间充质干细胞并进行传代培养,在成骨培养液中加入不同浓度的TNF-α及虫草素,通过碱性磷酸酶染色,茜素红染色,RT-PCR,westen-blotting等方法检测ALP活性,钙沉积含量,成骨标志基因Runx2和Osx mRNA表达水平等相关指标来评估脂肪干细胞增殖及成骨分化的能力。结果本研究中发现20,40ug/ml的虫草素能诱导细胞死亡。有趣的是10ug/ml的虫草素却能抵消由20ng/ml TNF-α引起的细胞死亡。同时虫草素对人脂肪干细胞成骨分化的影响显示出剂量依赖性:10ug/ml的虫草素能促进脂肪干细胞成骨分化,而40ug/ml的虫草素却抑制了脂肪干细胞的成骨分化。同时我们发现10ug/ml的虫草素能保护TNF-α诱导人脂肪间充质干细胞成骨分化抑制的作用。本研究还提示10ug/ml的虫草素能恢复在成骨分化中受TNF-α显著抑制的Runx2和Osx mRNA表达水平。TNF-α能通过抑制ⅠκB α磷酸化及随后P65释放和易位到细胞核而激活NF-κB信号通路,本研究中发现10ug/ml的虫草素能抑制TNF-α的这种作用。结论本研究揭示了炎症细胞因子抑制骨形成的初步机制,凸显出虫草素在细胞治疗炎性骨病中促脂肪干细胞骨形成的巨大潜力及临床意义。
[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- 伪 could inhibit the main pathway of osteoblast differentiation and maturation. A large number of in vitro studies and animal models showed that excessive TNF- 伪 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- 伪 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- 伪 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- 伪. This study also suggests that 10ugrml Cordycepin can recover the Runx2 and Osx significantly inhibited by TNF- 伪 during osteogenic differentiation. MRNA expression level. TNF- 伪 can activate NF- 魏 B signaling pathway by inhibiting phosphorylation of 鈪，

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