自噬作用對源於骨髓的間葉幹細胞功能影響之探討

Study on the Effects of Autophagy on the Functions of Bone-Marrow Derived Mesenchymal Stem Cell

邱宜婕(Yi-Chieh Chiu)

指導教授 ： 江伯倫

國立臺灣大學/醫學院/免疫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102409

摘要

間葉幹細胞（Mesenchymal stem cells, MSCs）是具有免疫調節特性的多能性幹細胞，已被應用於多種疾病的治療。自噬作用（Autophagy）是細胞去除細胞內不必要的成分並為自身提供生存信號的機制。先前的研究表明，間葉幹細胞中自噬作用的調節改變了間葉幹細胞的衰老現象、分化潛能和免疫抑制功能。然而，對於自噬作用改善間葉幹細胞免疫抑制的潛在機制尚不清楚。在這項初步研究中，我們自BALB/c小鼠的骨髓沖出骨髓性細胞進行體外培養及純化出間葉幹細胞，並測試其具有該有的間葉幹細胞性質。接著，我們研究了通過3-甲基腺嘌呤（3-methyladenine, 3-MA）和雷帕黴素（Rapamycin）自噬調節後，間葉幹細胞的存活力和分化能力。結果表明透過3-甲基腺嘌呤下調間葉幹細胞的自噬作用降低了細胞存活力，並且兩種調節均降低了間葉幹細胞的硬骨分化能力。對於活化的T細胞增殖以及細胞因子（cytokines）和趨化因子（chemokines）產生的抑制功能，我們發現由雷帕黴素上調的自噬作用增強了間葉幹細胞的抑制功能，並減少了活化T細胞分泌促炎性細胞因子和趨化因子，包括IFN-γ、IL-4、IL-6、IL-17、CCL2和CCL3。我們揭示了自噬作用轉化由間葉幹細胞調節的免疫抑制的潛在機制可能是通過細胞外調節蛋白激酶（extracellular regulated protein kinases, ERK）1/ 2途徑而不是ILK/ AKT/ GSK3途徑所調控的。ERK1/ 2途徑改變了間葉幹細胞表面的血管細胞黏附分子-1（Vascular cell adhesion protein -1, VCAM-1）表達。之後，我們用脾臟細胞刺激間葉幹細胞模擬以間葉幹細胞進行治療時細胞在體內的情形，我們觀察到在雷帕黴素預處理下間葉幹細胞上OX40配體（OX-40 ligand, OX-40L）的表達升高，而3-甲基腺嘌呤預處理的間葉幹細胞則降低了OX-40L表達。因此我們得出結論，由雷帕黴素誘導的自噬作用可透過活化ERK1/2途徑和上調OX-40L增強間葉幹細胞的免疫抑制功能。

關鍵字

間葉幹細胞 ； 自噬作用 ； 細胞外調節蛋白激酶1/2 ； 血管細胞黏附分子-1 ； OX-40配體

並列摘要

Mesenchymal stem cells (MSCs) are multipotent stem cells that have immunomodulatory characteristics and have been applied to various diseases. Autophagy is a mechanism that cells remove unnecessary intracellular components and provide survival signaling for cells. Previous studies indicated that autophagy modulation in MSCs altered the senescence, differentiation potential, and immunosuppressive functions of MSCs. The underlying mechanisms which improve the immunosuppression of MSCs, however, remain elusive. In this study, we eluted myeloid cells from the bone marrow of BALB/c mice, cultured and purified MSCs in vitro, and featured the adequate properties of MSCs. Next, we investigated the viability, differentiation ability of MSCs after autophagy modulations through 3-methyladenine (3-MA) and rapamycin. The results showed that downregulation of autophagy by 3-MA decreased the cell viability and both modulations lowered the osteogenesis of MSCs. For the suppressive functions on T cell proliferation, as well as cytokines and chemokines production, we discovered that upregulating autophagy by rapamycin reinforced the suppressive functions of MSCs and reduced proinflammatory cytokines and chemokines secretion from activated T cells, including IFN-, IL-4, IL-6, IL-17, CCL2, and CCL3. We revealed the underlying mechanisms of autophagy converting the MSC-mediated immunosuppression might be regulated via the ERK1/2 pathway but not the ILK/ AKT/ GSK3β pathway. The ERK1/2 pathway altered VCAM-1 expression on the surface of MSCs. Afterward, we primed MSCs with splenocytes to mimic the MSC-based therapies in vivo, and we observed that the expression of OX-40L on MSCs was elevated under rapamycin pretreatment, whereas 3-MA pretreated MSCs reduced the OX-40L expression. We thus concluded the rapamycin-induced autophagy enhanced the immunosuppressive functionality of MSCs through ERK1/2 activation and OX-40L upregulation.

並列關鍵字

Mesenchymal stem cells ； Autophagy ； ERK1/2 ； VCAM-1 ； OX-40L

參考文獻

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