間充質(zhì)干細(xì)胞抗肝臟纖維化作用及其機(jī)制的探索研究
發(fā)布時(shí)間：2018-08-13 21:04
【摘要】：間充質(zhì)干細(xì)胞(Mesenchymal stem cells, MSCs)由于其多向分化潛能、免疫調(diào)節(jié)和多因子分泌功能等諸多特性而成為干細(xì)胞研究領(lǐng)域的熱點(diǎn)。近年來(lái),越來(lái)越多研究顯示了MSCs在組織修復(fù)和疾病治療方面的應(yīng)用潛能。本研究重點(diǎn)探討了小鼠骨髓MSCs (mBM-MSCs)的抗肝臟纖維化作用,以及其中的細(xì)胞和分子機(jī)制。 首先我們證實(shí)了mBM-MSCs移植能向損傷肝臟遷移和整合,并且通過(guò)抑制纖維化過(guò)程中肝星狀細(xì)胞活化,有效降低小鼠肝臟纖維化程度。隨后在全基因芯片鑒定肝臟損傷相關(guān)因子時(shí),我們發(fā)現(xiàn)Delta-likel (Dlkl)在損傷肝臟中急劇表達(dá),提示其在肝臟損傷過(guò)程中可能發(fā)揮一定作用。因此我們對(duì)Dlkl在肝臟纖維化過(guò)程中的作用和機(jī)制做了進(jìn)一步探討。體內(nèi)外實(shí)驗(yàn)證實(shí)了Dlkl促肝臟纖維化的重要作用。研究結(jié)果提示肝損傷過(guò)程中實(shí)質(zhì)細(xì)胞表達(dá)Dlkl并通過(guò)旁分泌的方式作用于肝星狀細(xì)胞,促進(jìn)肝星狀細(xì)胞活化,其中50kDa的游離片段起主要作用,而非25kDa的小片段。 進(jìn)一步研究發(fā)現(xiàn)mBM-MSCs移植顯著抑制肝纖維化過(guò)程中Dlkl的表達(dá),而且主要在mRNA水平。免疫熒光雙標(biāo)以及后續(xù)"rescue"實(shí)驗(yàn)進(jìn)一步證實(shí)了mBM-MSCs對(duì)Dlk1表達(dá)的抑制在其抗纖維化過(guò)程中起關(guān)鍵性作用。隨后體外共培養(yǎng)等一系列實(shí)驗(yàn)表明,mBM-MSCs通過(guò)分泌因子調(diào)控Dlk1表達(dá),而成纖維細(xì)胞生長(zhǎng)因子2(FGF2)可能是其中重要因子之一。單獨(dú)使用18kDaFGF2在體內(nèi)外也能有效抑制Dlk1表達(dá)和肝星狀細(xì)胞活化、降低肝臟纖維化,從側(cè)面驗(yàn)證了FGF2在mBM-MSCs調(diào)控Dlk1過(guò)程中的作用,并提示了該因子抗肝臟纖維化新功能。 綜上所述,本研究證實(shí)了小鼠骨髓間充質(zhì)干細(xì)胞移植的抗肝臟纖維化效果,并展示了其中相關(guān)細(xì)胞和分子機(jī)制：肝實(shí)質(zhì)細(xì)胞接受損傷信號(hào)刺激表達(dá)Dlkl并通過(guò)旁分泌方式促進(jìn)肝星狀細(xì)胞活化,參與肝臟纖維化發(fā)生；mBM-MSCs移植以Dlk1為靶點(diǎn),通過(guò)分泌FGF2抑制其表達(dá)而降低肝星狀細(xì)胞活化水平,最終發(fā)揮抗肝臟纖維化功效。
[Abstract]:Mesenchymal stem cell (Mesenchymal stem cells, MSCs) has become a hotspot in stem cell research due to its multi-differentiation potential, immunomodulation and multi-factor secretory function. In recent years, more and more studies have shown the potential of MSCs in tissue repair and disease treatment. The aim of this study was to investigate the anti-hepatic fibrosis effect of mouse bone marrow MSCs (mBM-MSCs) and its cellular and molecular mechanisms. First, we confirmed that mBM-MSCs transplantation can migrate and integrate into the injured liver, and reduce the degree of liver fibrosis by inhibiting the activation of hepatic stellate cells during the process of fibrosis in mice. Subsequently, in the identification of liver injury related factors by whole gene chip, we found that Delta-likel (Dlkl) was expressed rapidly in the injured liver, suggesting that it may play a role in the process of liver injury. Therefore, we further explore the role and mechanism of Dlkl in the process of liver fibrosis. In vivo and in vitro experiments confirmed the important role of Dlkl in promoting liver fibrosis. The results suggest that parenchymal cells express Dlkl and act on hepatic stellate cells in paracrine manner during liver injury, and promote the activation of hepatic stellate cells. Free segments of 50kDa play a major role, not small fragments of 25kDa. Further studies showed that mBM-MSCs transplantation significantly inhibited the expression of Dlkl during hepatic fibrosis, and mainly at the level of mRNA. Immunofluorescence double labeling and subsequent "rescue" experiments further confirmed that the inhibition of Dlk1 expression by mBM-MSCs plays a key role in the process of anti-fibrosis. Subsequently, a series of experiments showed that mBM-MSCs regulated the expression of Dlk1 by secretory factor, and fibroblast growth factor 2 (FGF2) may be one of the important factors. 18kDaFGF2 alone also inhibited the expression of Dlk1 and the activation of hepatic stellate cells in vivo and in vitro, and decreased hepatic fibrosis. The role of FGF2 in the regulation of Dlk1 by mBM-MSCs in vitro and in vivo was verified, and the new function of FGF2 in anti-hepatic fibrosis was suggested. In conclusion, this study confirmed the anti-fibrosis effect of bone marrow mesenchymal stem cell transplantation in mice. The related cellular and molecular mechanisms were also demonstrated: hepatic parenchymal cells were stimulated by injury signal to express Dlkl and promoted hepatic stellate cell activation through paracrine, which was involved in hepatic fibrosis. Dlk1 was used as the target for transplantation of mBM-MSCs. By secreting FGF2 to inhibit its expression, the activation of hepatic stellate cells was reduced, and the effect of anti-hepatic fibrosis was finally played.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
本文編號(hào)：2182183
[Abstract]:Mesenchymal stem cell (Mesenchymal stem cells, MSCs) has become a hotspot in stem cell research due to its multi-differentiation potential, immunomodulation and multi-factor secretory function. In recent years, more and more studies have shown the potential of MSCs in tissue repair and disease treatment. The aim of this study was to investigate the anti-hepatic fibrosis effect of mouse bone marrow MSCs (mBM-MSCs) and its cellular and molecular mechanisms. First, we confirmed that mBM-MSCs transplantation can migrate and integrate into the injured liver, and reduce the degree of liver fibrosis by inhibiting the activation of hepatic stellate cells during the process of fibrosis in mice. Subsequently, in the identification of liver injury related factors by whole gene chip, we found that Delta-likel (Dlkl) was expressed rapidly in the injured liver, suggesting that it may play a role in the process of liver injury. Therefore, we further explore the role and mechanism of Dlkl in the process of liver fibrosis. In vivo and in vitro experiments confirmed the important role of Dlkl in promoting liver fibrosis. The results suggest that parenchymal cells express Dlkl and act on hepatic stellate cells in paracrine manner during liver injury, and promote the activation of hepatic stellate cells. Free segments of 50kDa play a major role, not small fragments of 25kDa. Further studies showed that mBM-MSCs transplantation significantly inhibited the expression of Dlkl during hepatic fibrosis, and mainly at the level of mRNA. Immunofluorescence double labeling and subsequent "rescue" experiments further confirmed that the inhibition of Dlk1 expression by mBM-MSCs plays a key role in the process of anti-fibrosis. Subsequently, a series of experiments showed that mBM-MSCs regulated the expression of Dlk1 by secretory factor, and fibroblast growth factor 2 (FGF2) may be one of the important factors. 18kDaFGF2 alone also inhibited the expression of Dlk1 and the activation of hepatic stellate cells in vivo and in vitro, and decreased hepatic fibrosis. The role of FGF2 in the regulation of Dlk1 by mBM-MSCs in vitro and in vivo was verified, and the new function of FGF2 in anti-hepatic fibrosis was suggested. In conclusion, this study confirmed the anti-fibrosis effect of bone marrow mesenchymal stem cell transplantation in mice. The related cellular and molecular mechanisms were also demonstrated: hepatic parenchymal cells were stimulated by injury signal to express Dlkl and promoted hepatic stellate cell activation through paracrine, which was involved in hepatic fibrosis. Dlk1 was used as the target for transplantation of mBM-MSCs. By secreting FGF2 to inhibit its expression, the activation of hepatic stellate cells was reduced, and the effect of anti-hepatic fibrosis was finally played.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro[J];World Journal of Gastroenterology;2005年22期
，本文編號(hào)：2182183
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