凝血酶通过调控MIF抑制MSCs归巢在SSc-ILD患者治疗中的作用及其机制研究

Systemic sclerosis (SSc) is an autoimmune disease involving the skin and many other internal organs. Interstitial lung disease (ILD) is a frequent complication of SSc that often has a poor prognosis. Mesenchymal stem cells (MSCs) have increasingly become the major stem cells for cell therapy, with their multilineage differentiation, self-renewal, immune-regulation and other abilities. But it has not achieved the expected efficiency in the MSCs transplantation for the treatment of SSc-ILD patients. Our clinical data showed that anticoagulation therapy before transplantation was correlated with efficacy of transplantation. Only those patients who received anticoagulant treatment got good outcome after MSCs transplantation. It suggested that maybe there were some coagulation factors interefering with effects of MSCs. Previous and our preliminary studies have shown that, thrombin (Thro) levels in serum and bronchoalveolar lavage fluid increased remarkably in patients with SSc-ILD. Our histopathology results also have found that expression of Thro in lung tissue of SSc-ILD patients increased significantly. Other recent researches have indicated that Thro could induce the secretion of macrophage migration inhibitory factor (MIF), while MIF could inhibit homing of MSCs to the damaged tissues. This research will further explore the effect and mechanisms of Thro/MIF on MSCs homing. The aim of this study is to find the causes of poor outcome of MSCs transplantation in the treatment of SSc-ILD patients and to provide theoretical foundation for optimization of transplantation program.

系统性硬化症（SSc）是一种以纤维化为特征的自身免疫性疾病，其并发症间质性肺疾病（ILD）与高死亡率密切相关，治疗效果差。MSCs是一种多能祖细胞，具有多向分化、自我更新、支持造血、组织修复、免疫调节等特征, 已成为细胞治疗的新型靶细胞，但在移植治疗SSc-ILD患者时却未取得预期的有效率。统计后发现，移植前是否进行抗凝治疗与移植疗效有相关性，提示可能存在某些凝血相关的因子影响了MSCs的移植疗效。国外研究和我们的前期研究表明，SSc-ILD患者的血清和支气管肺泡灌洗液中凝血酶（Thro）的水平增高，患者肺组织中Thro表达也升高。亦有研究证实，Thro能诱导MIF分泌增加，而MIF能抑制MSCs归巢。本研究将进一步探讨Thro/MIF对MSCs归巢的影响及机制，分析临床上MSCs移植治疗SSc-ILD患者疗效差的原因，为优化MSCs移植方案提供理论依据。

系统性硬化症（SSc）是一种以纤维化为特征的自身免疫性疾病，其并发症间质性肺疾病（ILD）与高死亡率密切相关，治疗效果差。MSCs是一种具有免疫调控功能的多能祖细胞，已被报道可有效治疗多种疾病，但在移植治疗SSc-ILD患者时却未取得预期的有效率。前期实验提示可能存在某些凝血相关的因子影响了MSCs的移植疗效。国外研究和我们的前期研究表明，SSc-ILD患者的血清和支气管肺泡灌洗液中凝血酶（Thro）的水平增高，患者肺组织中Thro表达也升高。亦有研究证实，Thro能诱导MIF分泌增加，而MIF能抑制MSCs归巢。本研究则进一步探讨了Thro/MIF对MSCs归巢的影响及机制。从以下几个方面展开研究：1. 确定SSc-ILD患者与血清Thro水平的相关性；2. 揭示Thro/MIF对MSCs 归巢的影响机制；3. miRNA在SSc-ILD发病中的意义。 37例SSc-ILD患者的平均血清Thrombin浓度为92.95±70.34 ng/ml，而20例健康对照组的平均血清Thrombin浓度为22.62±2.35 ng/ml，两组有Thr水平有显著性差异。SSc-ILD患者的Thr水平较正常对照组明显升高，而高水平的Thr与皮肤损害、肺动脉高压、ACA和Scl70抗体阳性存在相关性。验证七种血清miRNA(miR-140-5p，miR-21，miR-142-3p，miR-29a，miR-92a，miR-151-5p，let-7g）在SSC患者中的表达，主成分分析(PCA)从这些数据中提取最重要和最具代表性的元素，发现miR-151-5p是主要成分，并且主要代表其他6个miRNAs，但并没有发现其与ILD有潜在相关性。体外实验证明，Thro显示出一定的抑制胞膜CXCR4表达的现象，而Thro抑制剂、MIF、CD74-Ab以及PI3K-AKT-mTOR抑制剂均能使MSCs胞膜及胞浆的CXCR4表达增加，表明MSCs的趋化作用可能是通过促进PI3K-AKT-mTOR信号通路起作用。MSCs粘附实验证明MIF可通过PI3K-AKT-mTOR信号通路抑制MSCs粘附到内皮细胞。所以本项目初步发现Thro/MIF在MSCs归巢中的影响及机制，有助于分析临床上MSCs移植治疗SSc-ILD患者疗效差的原因，为优化MSCs移植方案、提高SSc-ILD患者的疗效提供理论依据。