透明质酸活化CD44与HIF-1α信号通路协同促进髓核组织再生的机理

The study of biological regeneration therapy for disc degeneration of is a research hotspot. Using hyaluronic acid, we have developed one kind of bionic nucleus pulposus, which was verified the effect on treatment of lumbar disc degeneration on rhesus monkey model. However, its mechanism is unclear. There are stem/progenitor cells in the degenerated nucleus pulposus. Our studies shown that bionic nucleus pulposus promoted the differentiation of mesenchymal stem cells into nucleus pulposus cells under the hypoxia microenvironment. Hyaluronic acid could activate CD44, which could promote HIF-1α expression through MEK / ERK, PI3K / Akt pathway. We also demonstrated that HIF-1α could transcriptionally activate CD44. Based on these evidences, we put forward the scientific hypothesis: hyaluronic acid can activate endogenous stem / progenitor cells to promote degenerative nucleus regeneration via the crosstalk of the activity of the CD44 and HIF-1α signaling pathways. In this study, the degenerative nucleus pulposus cells will be cultured in the bionic nucleus pulposus under hypoxia environment to verify the effects of the proliferation and the secretion of extracellular matrix; the mechanism of regeneration effect on the degenerative disc via the activities of the endogenous stem/progenitor cells by the crosstalk between the CD44 and HIF-1α signaling pathways will be explained well, which will be verified by knock-out mice. Finally, we will put forward the mechanism of the regenerative mechanism of degenerative disc repair by the bionic nucleus pulposus to provide experimental evidences for clinical transformation of bionic nucleus pulposus..degenerative lumbar disc via cross-talk between the CD44 pathways activated by hyaluronic acid and HIF-1α signal pathways irritated by hypoxia. This project aims to research on the regenerative effect of the bionic nucleus on degenerative nucleus pulposus by providing comfortable micro-environment for remnants of cell survival, activating of CD44 pathway to proliferate and secret the extracellular matrix; reveal the mechanism of activation of endogenous MSCs via cross-talk between the CD44 pathways activated by hyaluronic acid and HIF-1α signal pathways irritated by hypoxia; and elucidate the theory of degenerative lumbar intervertebral disc regeneration under the micro-environment of the bionic nucleus pulposus and hypoxia, which provide theoretic basis and experimental data for the clinical transformation of the bionic nucleus pulposus.

腰椎间盘退变的生物再生治疗是研究热点。课题组应用透明质酸等制备了仿生髓核，治疗恒河猴腰椎间盘退变效果良好，但其作用机制不清。退变髓核中含有干/祖细胞；我们研究表明仿生髓核与低氧协同促进间充质干细胞向髓核细胞分化；透明质酸激活CD44，可通过MEK/ERK、PI3K/Akt通路促进HIF-1α表达；同时，我们证实了HIF-1α可转录激活CD44。综上提出科学假设：透明质酸活化的CD44与低氧环境中HIF-1α信号通路交叉串话可激活内源性干/祖细胞、促进退变髓核再生修复。本项目拟从临床获取退变髓核样本，低氧下仿生髓核立体培养，研究其对退变椎间盘髓核残存细胞增殖和分泌细胞外基质的作用；上调或阻断信号通路研究CD44与HIF-1α信号通路交叉串话激活内源性干/祖细胞向髓核细胞分化、促进髓核再生的规律，采用基因敲除鼠验证；阐明透明质酸基仿生髓核修复椎间盘退变的机理，为仿生髓核临床转化提供实验依据。

腰椎间盘退变的生物再生治疗是研究热点。课题组应用透明质酸等制备了仿生髓核，治疗恒河猴腰椎间盘退变效果良好，但其作用机制不清。.本项目首先从临床及幼猴获取退变椎间盘组织，分离和培养髓核细胞并连续扩增传代；建立了动态力学培养下椎间盘退变器官培养模型。上述研究为椎间盘退变的进一步研究建立了基础。.本项目研究了椎间盘的低氧微环境下，透明质酸(HA)对促进退变髓核的再生修复的作用及机制。研究发现，在缺氧条件下，在HA存在下，髓核细胞(NPC)中细胞外基质基因和蛋白质的表达增强。此外，与单独存在HA和缺氧相比，CD44的表达增加。通过用氯化钴或二甲基乙二酰甘氨酸处理，CD44表达显着增加。NPC中HIF-1α的过表达显着上调CD44的表达。研究表明缺氧条件正向增强在HA存在下NPC基质合成的能力，这与HIF-1α转录激活导致CD44表达增加有关。.我们利用一击加载椎间盘退变器官培养模型来探索椎间盘(IVD)对机械应力的反应。IVD经受40%椎间盘高度的应变一秒钟，然后在生理负荷下培养。将甲磺酸米托醌(MitoQ)或其他抑制剂注入IVD。仅进行生理负荷培养的IVD用作对照。机械应力后线粒体膜电位立即显着降低（P<0.01）。ROS阳性细胞的百分比在机械应力后的前12小时内显着增加，然后在48小时内下降到低水平。MitoQ或鱼藤酮预处理显着降低了ROS阳性细胞的比例（P<0.01）。机械应力后12小时，髓核(NP)细胞活力急剧下降，并在48小时后达到稳定状态。MitoQ预处理增加了NP细胞活力，并在机械应力后12小时缓解了标志物变化。上述研究结果表明，线粒体ROS在机械应力后的早期时间点在IVD中作为程序性NP细胞死亡和ECM变性的重要调节剂。

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