阻断STAT3调控Th17/Treg平衡抑制cGVHD的实验研究

Chronic graft-versus-host disease (cGVHD) is the most serious complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is the syndrome resembling autoimmune disorders, and significant negative impacts on patient survival and quality of life. The effectiveness of management strategies beyond systemic corticosteroids is controversial. Those non-targeted therapies lead to malignancy relapse, severe infection and secondary tumor, so explore target therapies for cGVHD is urgent in the future. The precise mechanism responsible for chronic GVHD remains unclear. Emerging evidences demonstrated the turbulence of Th17 and Treg cells on the development of chronic GVHD.. Regulatory T cell (Treg) play important role for the development of chronic graft-versus-host disease by inhibiting effector T cells. Th17 cells, newly demonstrated distinct from other CD4+ T helper cells (Th1- or Th2-cells), are implicated in mediating auto-immune diseases and allograft rejection. It is well known that Treg and Th17 cells are main orchestra leaders in pathogenesis symphony of CD4+ T cells-mediated autoimmunity. STAT3 plays a central role in polarizing CD4+T cell differentiate to Th17 or Treg, which modulates the balance of Th17/Treg. However, no studies have addressed the functional role of Th17/Treg banance in development of chronic GVHD. As a preliminary study, we discovered that the expression of STAT3 upregulated in cGVHD patients compared with non-cGVHD patients, along with the proportion of Th17 cells increased and Treg cells decreased in cGVHD group. Our studies suggest that blocking STAT3 signal may ameliorate cGVHD by modulating the balance of Th17/Treg, which may provide a new approach for GVHD treatment.. Understanding the role of Th17/Treg balance in the pathogensis of chronic GVHD and target blocking STAT3 to modulate Th17/Treg balance may allow us to develop new targeted therapeutic approaches that limit global immune suppression and improve both disease-related and treatment-related outcomes.

调节性T细胞（Treg）制约效应T细胞的失衡，是慢性移植物抗宿主病（cGVHD）发生的重要机制。Th17细胞是新发现的一种效应性T细胞亚群，在自身免疫性疾病和器官移植排斥反应中发挥极其重要的作用，并与Treg细胞在分化和功能上有密切关联。JAK/STAT信号转导途径中的STAT3是诱导Th细胞向Th17或Treg分化的"支点"，是调控Th17/Treg平衡的关键。我们前期研究发现cGVHD患者Th17↑/Treg↓、STAT3基因表达上调，故此提出阻断STAT3→调控Th17↓/Treg↑平衡→抑制cGVHD的研究思路。本研究拟以cGVHD小鼠模型为研究对象，采用RNAi等技术，探讨阻断STAT3信号对诱导Th17、Treg细胞分化及其相互调控的作用及机制，阐明Th17/Treg细胞"漂移"在cGVHD发生发展过程中的作用，为cGVHD的免疫治疗新策略提供实验依据。

调节性T细胞（Treg）制约效应T细胞的失衡，是慢性移植物抗宿主病（cGVHD）发生的重要机制。Th17细胞在自身免疫性疾病和器官移植排斥反应中发挥极其重要的作用，并与Treg细胞在分化和功能上密切关联。JAK/STAT信号转导途径中的STAT3是诱导Th细胞向Th17或Treg分化的“支点”，是调控Th17/Treg平衡的关键。本研究提出阻断STAT3→调控Th17↓/Treg↑平衡→抑制cGVHD的研究思路。本研究体内外实验应用STAT3-shRNA抑制小鼠STAT3基因表达均可出现Th17 ↓/Treg↑平衡的漂移，符合本研究的预期设想。体外抑制小鼠naïveCD4+Th细胞STAT3基因的表达，干扰组中IL-2细胞因子的表达明显上升、Treg细胞比例增高、Th17细胞比例降低，与阴性对照组比较，差异有统计学意义；证实在IL-2分泌增多的状态下，可促进Treg细胞，而抑制Th17细胞的产生；但STAT3抑制后如何促进IL2的表达尚需进一步研究。与阴性对照组比较，Th1、2细胞亚群的比例并无显著改变，提示阻断STAT3靶点，主要效应细胞为Th17/Treg。干扰组IL-9细胞因子的分泌明显增多，推测STAT3的阻断可能带来Th9细胞分化的倾斜，这一类新的Th细胞在cGVHD中扮演的角色尚需进一步探讨。CD117+早期干祖细胞的STAT3基因下调，对其增殖、凋亡及分化为各系造血集落的能力并无显著影响，验证STAT3-shRNA慢病毒载体的安全性，也是本研究的创新点之一。通过供受鼠之间主要组织相容性复合物相合，次要组织相容性抗原不合的异基因骨髓移植，在照射剂量为700 cGy的清髓预处理后，输注骨髓细胞数为8×10＊6、脾细胞数为8×10＊6 的小鼠成功诱导出cGVHD的临床和病理表现，可作为临床研究cGVHD的理想动物模型。STAT3-shRNA活体内治疗小鼠cGVHD取得初步疗效，临床表现为脱毛减少、气促好转、活力恢复、体重上升；治疗的主要效应器官为肺脏，其作用机制与抑制p-STAT3蛋白表达、纠正Th17/Treg失衡密切相关。临床研究中亦发现移植后发生cGVHD的患者存在Th17/Treg失衡、STAT3基因mRNA水平的表达增高。因此，本研究揭示STAT3是cGVHD的免疫治疗新靶点，对于推进临床疾病的精准治疗具有重要的意义。

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