GSK-3β通过调控Treg/Th17分化及功能减轻肝移植排斥反应的机制研究

Liver transplantation has become a lifesaving procedure for patients with chronic end-stage liver disease, however, rejection is still a major cause of early graft loss and a risk factor for long-term recipient post-transplant survival. Recently, Th17 and regulatory T (Treg) cells are thought to be critically involved in mediating and regulating immunological response of transplantation. We have conducted a preliminary study and found that the proportion of Treg in peripheral blood was lower in patients with acute rejection when compared with that of non-acute-rejection patients. Meanwhile, higer GSK-3β activity of Treg was found in patients with acute rejection. Moreover, we also found GSK-3β inhibitor significantly enhanced Treg differentiation and inhibited Th17 cell differentiation. We hypothesize that inhibition of GSK-3β alleviates the rejection of liver transplantation via regulating Treg/Th17 cell differentiation and enhancing Treg function. To verify this hypothesis, flow cytometry, Western Blot, lentiviral vector transduction and RNA interference were applied to investigate the role of GSK-3β in liver transplantation in vivo and vitro. This research could provide new ideas and methods to improve the long-term outcome of liver transplantation.

肝移植手术日趋成熟，手术相关并发症明显减少，但术后排斥仍然严重影响患者生存率，目前调控Treg/Th17分化已成为减轻排斥反应的研究热点。申请者发现肝移植急性排斥患者Treg明显减少而Th17增多，并且Treg的GSK-3β活性升高，体外抑制GSK-3β活性使CD4+T细胞向Treg分化增多而向Th17分化减少，并增强Treg功能。为此我们提出假说：抑制GSK-3β活性促进Treg/Th17向Treg分化及增强Treg功能，减轻肝移植排斥反应。为验证此假说，我们将通过CD4+T细胞体外分化培养和大鼠肝移植模型，采用流式细胞仪、Westernblot、慢病毒载体转染、RNA干扰等手段，从分子、细胞、组织以及动物整体水平等多方面探讨GSK-3β在肝移植排斥反应中的作用，明确GSK-3β通过调控Treg/Th17分化及功能的分子机制。本项目的成功实施将为提高肝移植长期疗效提供新的思路和方法。

肝移植手术日趋成熟，已成为治疗终末期肝病最有效的治疗方案，手术相关并发症明显减少，但术后排斥仍然严重影响患者生存.最近研究发现Treg和Th17在器官移植免疫平衡中起重要作用。本课题组研究人naïve CD4+ T细胞体外诱导Treg和Th17时，通过抑制GSK-3β活性是否可以促进Treg/Th17向Treg分化及增强Treg功能，减轻肝移植排斥反应。.本课题的主要内容分两部分：一、抑制GSK-3β活性促进体外Treg分化及其机制。分离健康志愿者外周血的Naïve CD4+ T细胞，加入10ng/ml TGF-β和100U/ml IL-2，在第1、2、4、6天用流式细胞仪分析CD4+ CD25+Foxp3+ 细胞数量，并收集相应时间点的细胞检测相关指标，结果提示人iTreg分化促进了GSK3β的Ser9磷酸化，并且贯穿整个iTreg的分化过程，也就是说人iTreg分化过程中GSK3β活性下降，其机制是iTreg分化激活了GSK3β的上游ERK通路。随后，观察改变GSK3β的活性后iTreg分化情况，选用了两种GSK3β的选择性抑制剂SB216763和TDZD-8，以及用慢病毒转染GSK3β质粒，使Naïve CD4+ T细胞过表达GSK3β。结果提示，抑制GSK3β活性促进Treg的分化，而增强GSK3β活性减少Treg分化，并且抑制GSK3β活性上调了Treg相关的效应因子和增强Treg的抑制功能。相关机制研究提示：抑制GSK3β活性促进iTreg的分化部分是通过TGF-β/ Smad3通路实现的。.二、抑制GSK-3β活性抑制Th17分化及其机制。Naïve CD4+ T细胞，加入1 ng/ml TGF-β和10 U/ml IL-6和10 U/ml IL-1β，培养6天后检测相应指标。抑制GSK3β活性减少Th17的分化，而增强GSK3β活性促进Th17的分化。其机制是，GSK3β通过调节STAT3活性影响Th17分化。.上述的体外研究证实了我们提出的假说，即抑制GSK-3β活性促进人Treg的分化而抑制Th17分化，在数量上实现了Treg/Th17平衡向Treg倾斜，同时抑制GSK-3β活性还增强Treg功能，在质量上提高Treg的免疫调节功能。本项目的研究结果将为器官移植探寻新的免疫治疗方案提供实验支持和理论基础。