内源性IL-6介导MSCs移植治疗HIBD的神经保护作用及其机制探讨

Immunomodulation of MSCs is more efficient at restoring biological function of injured tissue than on trans-differentiation during transplantation. However, the exact mechanisms and characteristics of MSCs on immunoregulation are still unknown, especially in the damaged niche after hypoxic-ischemic insult. It is a study hotspot on the immunomodulation of MSCs transplantation to treatment brain damage resulting in neural disorders till now. In the last funding of NSFC, we found that MSCs endogenously secreted lots of IL-6 and suppressed apoptosis of the damaged PC12 cells following H2O2 treatment in order to improving neural functions. However, the specific targets and exact mechanism involved in IL-6 regulating damaged microenvironment following MSCs transplantation remain unknown. In our previous studies, we have already built the animal model of hypoxic-ischemic brain damage (HIBD), as well as cell models of oxygen-glucose deprivation (OGD) and coculture. Meanwhile, we have gotten several recombinant adenovirus of siTLR2, Ad-IL-6. On the basis of the front new discoveries from the last project, we will take endogenous IL-6 of MSCs as the keypoint and HIBD as therapy target. The current study will determine the molecular mechanism of IL-6 secretion from MSCs in vitro using associated recombinant adenovirus of siTLR2, Ad-IL-6 and siIL-6 and some agonists, and then test IL-6 immunomodulatory function after MSCs transplantation in vivo through HIBD model, at last find the targets and the key proteins in associated signaling pathway of IL-6 with OGD model and coculture technique. The aim is to reveal the targets and regulatory mechanisms of IL-6 immunomodulatory functions for the treatment of HIBD following MSCs transplantation through the three different levels of the mechanism study in vitro to determinate effects in vivo, and then to discovery the detail targets in vitro. The study can provide reliable experimental basis for cytotherapy nervous diseases and offer a new treatment for improving the clinic therapy of HIBD.

MSCs移植治疗脑损伤等神经性疾病的免疫调节作用已成为目前研究热点，本课题组在前一个NSFC项目的研究中发现，MSCs内源性分泌大量IL-6的同时，对损伤细胞具有抗凋亡的作用，促进H2O2损伤的PC12细胞神经功能的恢复。然而，MSCs移植过程中IL-6在损伤微环境中的具体作用靶点和调节机理目前尚不清楚。本项目拟在前一个研究课题新发现的基础上，以MSCs内源性分泌的IL-6为切入点，通过已成功建立HIBD动物模型、ODG模型和共培养技术，以siTLR2、siIL-6和AdIL-6重组腺病毒为工具，从MSCs分泌IL-6分子机制-MSCs移植治疗HIBD中IL-6的调节作用-MSCs来源的IL-6作用靶点及其信号通路开展研究。通过体外机制研究、体内机制验证、体外作用靶点探索三个不同层面深入研究，旨在揭示MSCs移植治疗HIBD中IL-6的免疫调节机制，为促进细胞治疗神经性疾病提供新的途径。

缺氧缺血性脑损伤（HIBD）是新生儿高发神经性疾病，是导致儿童神经系统伤残的主要原因之一。大量研究表明，骨髓间充质干细胞（MSCs）移植治疗对HIBD损伤具有神经保护作用。我们的研究表明MSCs的移植治疗可提高HIBD大鼠的学习记忆功能，并且发现MSCs可内源性分泌大量IL-6，那么这种MSCs来源的IL-6分泌机制是什么？它的作用靶点是什么？其作用机制又是怎样？该项目针对上述问题展开研究，发现MSCs共培养上调IL-6分泌水平，抑制凋亡因子Bax的表达。上调/下调TLR2/NFκB信号通路可相应增加/减少MSCs中IL-6的分泌水平，而siIL-6-MSCs的共培养增加OGD损伤PC12细胞中的Bax表达水平和静息膜电位。表明MSCs内源性IL-6的分泌是受到TLR2/NFκB信号通路调控的，其生物学功能是抑制OGD损伤的PC12细胞凋亡。利用稳定表达siIL-6的MSCs细胞进行体内移植，发现MSCs移植可明显改善新生HIBD大鼠的学习记忆功能，促进LTP；而MSCs的这种神经保护作用随着siIL-6-MSCs的移植降低海马内的IL-6分泌水平而相应减弱。体外研究表明，MSCs分离共培养不仅可以上调培养体系中IL-6的分泌水平，而且可激活OGD损伤原代神经元或星形胶质细胞中的IL-6R/STAT3信号通路。上调OGD损伤的星形胶质细胞Bcl-2/Bax比值，增强抗凋亡作用。升高OGD损伤神经元中SOCS1表达水平，抑制HIBD损伤后炎性因子IFNr、IP-10的分泌。而siIL-6-MSCs分离共培养，抑制IL-6R/STAT3信号通路活化，降低OGD损伤的星形胶质细胞Bcl-2/Bax比值，减少OGD损伤神经元中SOCS1表达水平，增加炎性因子IFNr、IP-10的分泌。表明MSCs内源性IL-6可促进损伤星形胶质细胞抗凋亡，减少损伤神经元分泌炎性因子，从而对HIBD发挥神经保护作用。.目前该项目已发表SCI学术论文7篇，CSCD中文核心期刊文章2篇；获得重庆市卫生和计划生育委员会医学成果二等奖1项，参加国际国内学术会议10人次，申请专利1项，培养毕业研究生6名，其中4名博士研究生，2名硕士研究生。