ASICs介导的酸性微环境抑制人髓核间充质干细胞增殖分化作用及其机制研究

Acid-sensing ion channels (ASICs) are voltage-insensitive cationic channels activated by extracellular protons. They play important roles in conditions associated with tissue-acidification such as cerebral ischemia or osteoarthritis. As the microenvironment of intervertebral disc would turn to high acid-environment during degeneration, and intervetebral disc and articular cartilage had similar biological bioactivities, whether ASICs regulated the degeneration of intervertebral disc? In our preliminary studies, we had found that human nucleus pulposus- mesenchymal stem cells (NP-MSCs) expressed ASICs, and low pH could inhibit the proliferation and the expression of “ stem cell related genes” of NP-MSCs. Therefore, we make a hypothesis that the pH may inhibit the proliferation and differentiation of the NP-MSCs by ASICs. In order to confirm the hypothesis, we designed the following studies: 1) to observe the effective of the pH on the bioactivities of human NP-MSCs which derived from different degeneration of intervertebral discs in vitro; 2) to record the activated currents of ASICs by whole-cell patch-clamp in vitro; 3) to observe the effective of the pH on the bioactivities of human NP-MSCs after ASICs over-expression, silenced by SiRNA or blocked by amiloride, PcTX1 or APETx2 in vitro; 4) to observe the effective of the amiloride for intervertebral disc degeneration in animal expriments.In general, based upon the possible mechanism of the NP-MSCs keep silence during the process of the degeneration might be clarified by these finds, and this study were also could provide a novel therapeutic strategies for intervertebral disc degeneration.

酸敏感离子通道（ASICs）是一类由胞外酸化激活的离子通道，在脑缺血、关节炎等酸化过程中起重要调控作用，而椎间盘退变与上述病变具有相似的酸化病理过程。我们前期研究已发现人髓核间充质干细胞（NP-MSCs）表达ASICs，且pH降低会抑制其增殖和“干性基因”表达。但酸环境如何调控NP-MSCs增殖分化？ASICs如何参与其中及其具体机制如何？因此，提出假设：酸环境改变可能通过ASICs调节NP-MSCs增殖分化。为此，拟在前期基础上：1）观察酸环境对人不同退变程度NP-MSCs增殖分化的影响；2）建立全细胞膜片钳记录H+激活ASICs电流；3）通过ASICs过表达、SiRNA、特异性及非特异性阻滞剂，观察酸环境对NP-MSCs增殖分化的影响；4）利用ASICs阻断剂体内观察酸环境对NP-MSCs的影响。旨在阐明椎间盘退变过程中NP-MSCs的“静默”机理，并为椎间盘退变生物学治疗提供新靶点。

在椎间盘退变过程（IVDD）中，伴随着微环境的改变，其中pH的改变对NP-MSCs生物学活性产生了极大影响，已经有研究证明了酸敏感离子通道（ASICs）在该过程中发挥了生物学功能，我们研究椎间盘退变患者中ASICs各亚基的表达情况，发现ASIC1和ASIC3随着退变程度的增加，表达水平呈现上升。揭示ASIC1和ASIC3可能是椎间盘退变过程中发挥作用的关键亚基，进一步地，通过在体外模拟椎间盘退变酸环境改变，发现酸环境会导致细胞增殖能力的下降，细胞周期发生停滞以及细胞提前衰老，于此同时，也伴随着炎性因子IL-6、IL-8的表达水平增加，加入ASICs抑制剂后，能缓解酸性条件下NP-MSCs的生物学活性改变，为了进一步探究ASICs在NP-MSCs中发挥作用的机制，根据ASICs的解构结构和功能特性，我们考虑可能与ASICs介导的Ca2+的内向流动有关，通过钙离子成像实验，我们发现，酸性条件下，会导致Ca2+的内流增加，而使用了ASICs抑制剂后，该效应在一定程度上被减弱了，这些结果提示ASICs抑制剂抑制了酸环境下的钙离子内流，进而抑制了钙离子作为第二信使传递的生物学改变。与此同时，考虑到NP-MSCs发生了提前衰老，我们检测衰老相关信号通路，发现p53、p21、p27、Rb1和p16蛋白水平的升高，以上结果提示ASIC1和ASIC3可能通过激活p53-p21/p27和p16-Rb1信号通路促进NP-MSCs细胞衰老及椎间盘退变。