基底硬度变化下干细胞与肝内细胞间的相互作用影响其分化方向及细胞行为的力-化学机制探求

The Therapeutic potential of stem cells in liver fibrosis have been paid more close attention currently; However, reports on the differentiation and behavior of stem cells in this process are still contradictory now. We believe that under the variation of matrix stiffness in liver, the interactions between stem cells and liver origin cells are the key factors which determine stem cells differentiation and other cell behavior in liver fibrosis. Meanwhile, signals pathway such as HGF，Wnt，TGF-beta1-Smads, integrins -RhoA, PI3K-Akt etc., also play important roles in this process. These mechanical-chemical signals can cross-talk between each other through the variation of cell contractility and result in being integrated, and then impact on the differentiation of stem cells consequently. To verify this hypothesis, this project intends to use traction force microscope and micropillars array which could be loaded by magnetic force to research interactions between bone marrow mesenchymal stem cells, hepatic cells and hepatic stellate cells; comparing the performance of cell contractility in those processes as well as the relationship between the state of stem cell differentiation and cell contractility; fluorescent protein FRET would be applied to investigate the mutual relationship between the activation or inhibition of those signals pathways aforementioned and the variation of cell contractility. As this project was carried out, it would help to deepen the understanding of the differentiation behavior of stem cells and the interaction mechanisms between mechanical and chemical signals in these process. Furthermore, it would help to develop suitable mechanical-chemical conditions for stem cells therapeusis of liver fibrosis, avoiding its negative effects as well

干细胞在治疗肝纤维化中的作用目前备受关注，然而对此过程中干细胞分化及行为却存在相互矛盾的报道。我们认为在基质硬度变化的前提下，干细胞与肝内细胞之间的相互作用，是影响干细胞分化方向和行为的关键因素，HGF，Wnt，TGF-b1-Smad，整联蛋白-RhoA，PI3K-Akt等信号亦起重要作用。这些力-化学信号可共同影响细胞收缩力而整合和互调，最终决定干细胞的分化和行为。为验证此假设，本项目拟利用牵引力显微镜和可磁力加载的微柱阵列研究不同基底硬度条件下，骨髓间充质干细胞与肝细胞、肝星状细胞间的相互作用，比较此过程中细胞收缩力的状态，以及干细胞分化状态与其细胞收缩力之间的关系，结合荧光蛋白FRET研究前述信号通路的激活和抑制与细胞收缩力变化之间的双向关系。本项目研究的开展可深化对干细胞分化行为以及其中力化学信号交互作用机理的认识，帮助找寻干细胞治疗肝纤维化的适宜力-化学条件，避免其负面作用的发生

肝纤维化是多种慢性肝病或肝损伤所引发的病理愈创反应，可发展为肝硬化乃至肝癌，严重威胁人类健康。干细胞治疗肝纤维化是较有希望的治疗手段。鉴于干细胞是治疗还是加重肝纤维化存在相互矛盾的报道。本研究提出了干细胞治疗肝纤维化所表现出的结果差异，是由于肝内力化学环境的差异所导致的，并基于该假说，构建了在不同硬度基底上单独或共培养骨髓间充质干细胞和肝内细胞的培养模型，利用RT-PCR，转录组学分析，磷酸化组学分析，牵引力显微镜，FRET技术，流式细胞仪，免疫荧光等技术，研究了不同基底硬度下，干细胞与肝内细胞间的相互作用，及其可能的力化学信号机制。结果表明：软基底上共培养作用下，干细胞倾向于肝样分化，促进细胞外基质的降解，肝细胞增殖，有利于肝脏功能恢复，但同时炎症反应增强，激活后肝星状细胞的存活增强。而在较高硬度基底上则表现为促进了肝星状细胞的代谢和蛋白表达，干细胞失去肝样分化倾向，故而不利于干细胞对纤维化的治疗。当硬度更高（37kpa）时，细胞相互作用表现为与软基底趋同的趋势，仅干细胞没有类似软基底的肝样分化，却有一定促进激活后肝星状细胞凋亡的表现。细胞在所研究的4.5kpa-37kpa的区间，对基底硬度的响应，主要与HIF-1信号通路相关，而在共培养中的响应，却更多和TNF-α信号通路相关，不同硬度上这些信号通路的激活有较大的差异，是导致细胞做出差异响应的可能机制。进而通过建立细胞收缩力相关指标与胞内信号蛋白激活，以及不同因子作用对细胞收缩力的影响，验证了细胞收缩力可作为整合细胞力化学信号的重要形式的假说。同时本研究自行编制了应用于牵引力显微镜的计算程序Matlab模块，设计的模拟渐变基底硬度的微柱阵列，具有一定创新性和实用性。本研究的结果，在一定程度上揭示了干细胞在肝纤维化治疗中产生不同表现的可能机制，增进了对于细胞力化学信号转换与整合的理解。对于肝纤维化的治疗，人工肝构建等具有一定的知识积累和机理阐述的作用。所积累的研究模型和程序亦可用于后续研究