EPC联合胸腺肽β4 抗造血干细胞移植后HVOD的作用及机制研究

Hepatic veno-occlusive disease (HVOD) is one of the major complications of hematopoietic stem cell transplantation (HSCT). Injury of hepatic sinusoidal endothelial cell (HSEC) plays a key role in the occurrence of HVOD, which not only promotes the release of inflammatory factors and aggravate self-injury, but also leads to capillarization of hepatic sinusoids, leading to the occurrence of HVOD. At present, the clinical effect of prevention and treatment of HVOD is not satisfactory. Our previous study showed that endothelial progenitor cells (EPCs) promoted the repair of damaged HSECs and thus alleviated symptom of HVOD. Unfortunately, it is hard to obtain large amounts of EPCs; In addition, the implanted EPCs have impaired function and activity with low survival rate. Therefore, starting with exploration of pathogenesis, new approaches are required to be considered which might result in a favorable outcome in preventing HVOD. Thymosin beta-4 (Tβ4) was shown to enhance the activity, inhibit apoptosis and senescence of EPCs, and might be potentially effective in enhancement of the anti-HVOD effect of EPCs. Therefore, in this project, Tβ4-pretreated EPCs combined with Tβ4 were administrated during murine HSCT to observe their anti-HVOD effects, repair of damaged HSECs as well as anti-inflammatory roles; furthermore, Tβ4 gene was silenced in EPC cells to further explore the key role and mechanism of Tβ4 gene in the anti-HVOD process of EPC. Our study may provide new strategies for management of HVOD.

肝静脉闭塞病(HVOD)是造血干细胞移植（HSCT）重要并发症之一。肝窦内皮细胞(HSECs)受损是HVOD发生的关键，其不仅促使炎性因子释放，加重自身损伤，还致肝窦毛细血管化，引起HVOD。目前HVOD防治效果尚不理想。我们前期研究发现EPC可促进受损HSECs修复，减轻HVOD。但获得大量EPC较难; 此外植入的EPC功能及活性易受损害、存活率低。因此，有必要进一步从探索发病机制入手，优化HVOD防治策略。研究发现胸腺肽β4（Tβ4）可增强EPC活性，抑制EPC凋亡和衰老，应是增强EPC抗HVOD效应的潜在有效策略。本研究拟在HSCT动物实验中予Tβ4预处理的EPC联合Tβ4应用，观察其抗HVOD作用及修复HSECs和抗炎效应；并沉默细胞Tβ4基因，探讨Tβ4基因在EPC抗HVOD中发挥的作用及机制。如获成功可为HVOD防治开辟新的方向。

肝窦阻塞综合征（HSOS）是造血干细胞移植（HSCT）后危及生命的并发症之一，临床常用的化疗方案如白消安/环磷酰胺及高剂量全身照射与之密切相关。预处理毒性及移植后免疫排斥等因素导致的肝窦内皮细胞（LSECs）损伤是 HSOS 的始动因素，早期逆转LSECs损伤及控制肝纤维化是HSOS治疗的关键。目前临床上仍缺乏有效的HSOS治疗方案。胸腺肽β4（Tβ4）是一种活性多肽，在多种病理和生理状态下发挥作用，如抗氧化应激、调节炎症、抗纤维化等。我们前期研究发现在辐照损伤的内皮细胞中Tβ4的mRNA表达显著降低，因此我们预想Tβ4在HSOS发生发展中可能发挥作用。本研究探讨了Tβ4对LSECs存活和功能的作用，并在γ射线辐照诱导的LSECs损伤模型中探讨了Tβ4抗凋亡、抗炎及抗氧化的作用及机制，并建立骨髓移植诱导的HSCT-HSOS小鼠模型，评估Tβ4抗小鼠HSOS作用。通过BrdU、划痕修复实验、内皮细胞成管实验、western blot、RT-qPCR、ELISA等方法，我们发现 Tβ4外源性给药可显著促进LSECs增殖、迁移和成管能力，而Tβ4 siRNA转染后对细胞增殖，迁移和成管起到了抑制作用，其机制可能与AKT，ERK1/2通路的激活相关；Tβ4通过上调抗凋亡蛋白Bcl-2和Bcl-xL，显著抑制了辐照诱导的LSECs凋亡，降低caspase3蛋白的活化。此外，Tβ4在体外负调控辐照后LSECs NF-κB和MAPK p38通路，降低了促炎细胞因子IL-6, IL-1β 和 TNF-α的释放以及黏附分子ICAM-1和VCAM-1的蛋白水平。另一方面，Tβ4减少了辐照后LSECs活性氧(ROS)水平，并增加了还原性谷胱甘肽（GSH）和超氧化物歧化酶（SOD）。在体内，我们发现Tβ4腹腔注射显著改善了HSOS小鼠的肝功能指标，减少了血浆促炎因子IL-6, IL-1β 和 TNF-α的水平，并减轻肝脏水肿及炎性细胞浸润；更重要的是，Tβ4腹腔注射减少了HSEC损伤指标VWF，PAI-1和TM，肝脏免疫组化还显示其显著减少了LSEC脱落。总之，Tβ4 具有增强LSECs存活和功能的作用。它还在辐射引起的肝损伤过程中表现出抗凋亡和抗炎特性。因此我们认为Tβ4 的使用可能是预防或治疗HSOS中肝损伤的潜在治疗方法，为临床HSOS的防治提供了新方法。