多发性硬化安全、有效、简易的细胞新疗法的临床前研究

In recent years, clinically diagnosed patients of multiple sclerosis (MS) are increasing, due to the rapid development of neuroimaging and molecular biology. In contrast, the treatment of MS is proceeding slowly. Our previous studies found that: ①Rho kinase (ROCK) inhibitor Fasudil can ameliorate clinical symptoms, and improve the demyelination in animal model of experimental autoimmune encephalomyelitis (EAE); ②clinical limitation in treating MS is also obvious, because of vasodilation effect, smaller safety window for long-term use, poor bioavailability and no oral drug. Thus, we used Fasudil in vitro to modify encephalitogenic spleen cells for cell therapy in EAE. The results demonstrated that Fasudil-modified splenic cells did not induce the development of passive EAE, but can decline the severity of active EAE in mice. This topic attempts to further understand the biological characteristics of encephalitogenic T or macrophages modified with Fasudil in vitro, observe clinical therapeutic effect of EAE mice by cell therapy of T cells or macrophages modified with Fasudil in vitro respectively, and explore the cellular and molecular mechanisms of Fasudil-modified T or macrophages in treating EAE by using western blot, flow cytometry, immunofluorescence and ELISA. We try to find a safe, effective and cheap cell therapy for the treatment of MS patients, and provide a theoretical basis and experimental data for the future clinical application.

近年来由于神经影像和分子生物学技术的迅速发展，临床上确诊的多发性硬化（MS）患者越来越多。相比之下，其治疗却举步维艰。前期研究发现：①Rho激酶抑制剂Fasudil可减轻MS动物模型实验性自身免疫性脑脊髓炎（EAE）小鼠的临床症状和脱髓鞘；②由于明显的血管扩张作用，长期使用安全窗较小，口服生物利用度差，且无口服剂型等局限性，我们曾用Fasudil体外修饰的致脑炎性脾细胞静脉回输，发现其不仅不能诱导被动EAE模型，而且可有效减轻主动EAE小鼠的临床症状和脱髓鞘。本课题试图进一步分别了解Fasudil体外修饰之T细胞/巨噬细胞的生物学特征，分别静脉回输观察其对EAE的疗效，同时利用Western blot、流式细胞术、免疫荧光、ELISA等方法，探索Fasudil修饰之T细胞/巨噬细胞体内细胞治疗的分子基础，为MS患者寻求一种安全、有效和廉价的细胞治疗选项，为拓宽其临床应用提供坚实的实验依据。

本课题以多发性硬化（MS）的经典动物模型—实验性自身免疫性脑脊髓炎（EAE）为研究对象，从体内外两方面探讨Fasudil修饰的免疫细胞—单个核细胞（MNCs）、巨噬细胞（Mφ）、T细胞治疗EAE的效果和机制。.体内实验结果：Fasudil修饰的免疫细胞推迟EAE小鼠发病，减轻临床症状，改善脱髓鞘，减少中枢炎性细胞浸润。Fasudil修饰的免疫细胞影响T细胞和Mφ分化，表现为抑制Th17细胞、促进Th2细胞产生；降低M1亚型CD16/32、IL-12、CD11c、CD40，增强M2亚型CD206、IL-10、CD14表达。Fasudil修饰的免疫细胞增强脊髓M2亚型Arg-1，抑制M1亚型iNOS表达。Fasudil修饰的免疫细胞抑制炎性细胞因子IL-17、IL-1β、INF-γ释放，增强抗炎性细胞因子IL-10分泌。Fasudil修饰的免疫细胞抑制脊髓p-NF-κB-P38信号通路激活和炎性细胞因子IL-17、IL-6、TNF-a水平，诱导MAP2和神经营养因子BDNF、NT-3表达。.体外实验结果：Fasudil抑制致脑炎性MNCs、Mφ、T细胞ROCK的活性。Fasudil减少MNCs和T细胞的Th1、Th17比例，增强Th2比例，抑制IFN-g、IL-17水平，促进IL-10分泌和Foxp3表达。Fasudil转换MNCs和Mφ的炎性M1表型至抗炎M2表型，即降低CD16/32、iNOS、IL-12水平，增强CD206、Arg-1、IL-10、CD14水平。Fasudil抑制Mφ的TLR-4、Myd88、COX-2、P38、ERK、JNK表达和炎性细胞因子IL-1β、IL-6、TNF-a水平。Fasudil抑制T细胞TLR-4-NF-κB信号通路激活。.Fasudil修饰的T细胞和Mφ相互作用实验结果：Fasudil修饰的Mφ降低致脑炎性T细胞IL-17、IFN-g表达，增强IL-10表达。Fasudil修饰的T细胞降低致脑炎性Mφ的CCR7、CD11c表达，增强IL-10、CD200、CD23表达。.Fasudil修饰的免疫细胞有治疗EAE的潜能，与Mφ由致炎的M1型转化为抗炎的M2型，降低Th17细胞分泌，促进Th2细胞表达，抑制TLRds-Myd88、p-NF-kB、MAPKs信号通路激活，促进神经营养因子表达，改善炎性微环境相关。