经血源性子宫内膜干细胞对中枢神经系统自体免疫疾病的治疗和机制研究

Multiple sclerosis (MS) is an autoimmune inflammatory degenerative disease of the brain and spinal cord triggered by degradation of the myelin sheath. No curative treatment is known for MS. Our previous study demonstrates that a key aspect of the treatment of MS is the suppression of patients’ immune responses (Nature, 2014). Transfer of adult stem cells, through their immunomodulatory effects, represents a promising therapeutic approach for MS. However, it has been shown that this treatment is only effective during early/intermediate and not the late stages of the disease. More critically, the cellular and molecular mechanisms of the therapeutic effects are unknown. In recent years, a new type of adult stem cells, menstrual blood–derived stem cells (MenSC), has been found to display good stemness and these cells can be obtained in a non-invasive way. Thus, the aims of this project are to explore the impact of the transfer of MenSC on experimental autoimmune encephalomyelitis (EAE), the prime animal model of MS, and to identify the mechanisms underlying this treatment. The MenSC will be injected during the early, middle, and late stages of EAE to identify the therapeutic efficacy of MenSC at the different stages. Using an in vitro stimulation strategy to enhance the anti-inflammatory capacity of MenSC, we plan to make MenSC have stronger beneficial effects at early stages and provide therapeutic benefit at late stages of EAE. To investigate the cellular and molecular mechanisms, we will deplete in vivo key types of immune cells just before MenSC transfer to detect the responsible cell types of the recipients. In addition, we will re-isolate MenSC from the lymphoid organs a few days after transfer and compare their gene expression profile with that of primary MenSC to detect the main molecular players of the “effector” MenSC. According to the thus identified biological features (eg. cytokine , co-stimulatory ligand ) of the molecules of the “effector" MenSC and the responsible cell types of the host (eg. T cells, B cells, NK cells), we intend to build an interaction network between MenSC and the host immune system to explain the therapeutic effect of stem cells. The results of this project will profoundly improve our knowledge of how stem cells improve the health of patients of autoimmune neurodegenerative diseases and provide principal support for clinical application of stem cells.

多发性硬化症（MS）是自体免疫性神经退行性疾病，尚没有治愈方案。我们在MS的动物模型实验性自体免疫性脑脊髓炎（EAE）的研究发现增强抑制性免疫是治疗MS的关键。干细胞有免疫调节功能并对前中期MS有抑制，但对后期MS疗效甚微，另外其作用原理不明了。近年来鉴定的经血源性子宫内膜干细胞（MenSC）尚未对此病探索治疗。据此，本项目将MenSC运用到EAE的前，中，后期，检测其在不同时期的疗效；对MenSC体外刺激提升其免疫抑制力，增强治疗强度和广度。为探明机理：逐类剔除免疫细胞，检测宿主内起重要作用的细胞类型；Gene array比较移植前后MenSC分子表达的变化，探测其关键作用分子；根据具体的分子特性和细胞类型建立MenSC和宿主免疫系统的关系模式。本课题的实施将鉴定并提升MenSC对中枢神经自体免疫疾病的疗效，建立干细胞治疗的分子细胞机制，为临床治疗提供理论支持。

多发性硬化症（MS）是自体免疫性神经退行性疾病，目前临床上尚无有效的治愈方法。一些免疫抑制药物虽然能够减轻MS的症状，但是病人的病情仍然会反复，而且此类药物副作用大，缺乏对髓鞘的保护和再生作用。我们在对MS的动物模型实验性自体免疫性脑脊髓炎（EAE）的研究中发现增强抑制性免疫是治疗MS的关键。干细胞有免疫调节功能并对前中期MS有抑制作用，但对后期MS疗效甚微，另外其作用机制也不明了。近年来鉴定的经血源性子宫内膜干细胞（MenSC）具有较好的免疫调节作用，但将其应用于多发性硬化症的研究较少。. 本项目研究中首先对MenSC进行分离提取、表型分析、TLR表达谱分析和分化潜能的鉴定，然后将MenSC移植到不同发病阶段的EAE小鼠体内，检测其在不同时期的疗效；检测运用不同的TLR受体激动剂对MenSC体外刺激后MenSC分泌的蛋白质组，鉴定并比较原生MenSC（未经刺激的MenSC）和刺激后MenSC对EAE的治疗效果，分析TLR激动剂刺激后对MenSC免疫抑制作用的影响。为探明干细胞移植到EAE小鼠体内后，其作用的分子和细胞机制，我们从宿主角度鉴定了免疫细胞参与者，炎症性T细胞、调节性T细胞等在MenSC移植前后在EAE小鼠体内的变化情况以及在髓鞘内的浸润情况。. 我们的研究结果表明：在EAE小鼠不同发病时期（day-1，6，10和19）通过尾静脉注射或腹腔注射移植MenSC均能减轻EAE小鼠的症状，而且脐带间充质干细胞和子宫内膜干细胞具有类似的治疗效果。Th1和Th17细胞在静脉注射和腹腔注射移植MenSC后的小鼠体内都有明显的改变，浆细胞样树突状细胞（pDCs）的比例明显降低，pDCs、cDCs和B细胞表面的共刺激分子的表达也受到抑制。. 我们的数据表明MenSC显著降低移植后EAE的疾病严重程度。因此，它有潜力被开发成可方便使用的异体间充质干细胞用于多发性硬化等自体免疫性疾病的治疗。