肥大细胞通过FoxO3a调节中性粒细胞凋亡延迟在肝移植后急性肺损伤中的作用研究

The acute phase of acute lung injury (ALI) following orthotopic liver transplantation (OLT) is characterized by massive polymorphonuclear leukocytes (PMNs) infiltration in alveoli. Delayed apoptosis of PMN would lead to the persistence and exacerbation of ALI. However, the mechanisms remain largely unclear. Studies demonstrated that the activation of mast cells was associated with PMN infiltration in alveoli and ALI. At the same time, the expression levels of tumor necrosis factor (TNF)-α and tryptase released by mast cells in lung tissue were consistent with the severities of pathological changes in ALI following OLT. Activation of mast cell would decrease the apoptosis rates of PMN in cell co-culture system in our preliminary experiment. Studies showed that TNF-α and tryptase could active the upstream signal enzyme of FoxO3a, a nuclear transcription factor. FoxO3a mediates cell apoptosis through regulating the expression of Bcl-2 family composed of proapoptotic and anti-apoptotic proteins. Therefore, it is reasonable to hypothesize that TNF-α and tryptase released by activated mast cells may play important role in ALI by inducing the delayed apoptosis of PMN through mediating transcription of FoxO3a. We will use liver transplantation model in rats, isolation and co-culture of mast cells and PMNs, as well as the intervention of TNF-α, tryptase and mast cells to explore the role of mast cells and the underlying mechanisms of delayed apoptosis of PMNs in ALI. This study will shed light on the molecular mechanisms of ALI and provide new insight to the treatment of ALI following OLT.

肝移植术后急性肺损伤（ALI）的急性期以肺泡内大量中性粒细胞（PMN）浸润为主，PMN的凋亡延迟会致ALI持续存在与恶化，但其凋亡延迟机制尚不明确。肥大细胞激活与以及其释放的TNF-α和类胰蛋白酶的肺部表达与ALI肺部损伤和PMN浸润相一致；预实验细胞共培养中，激活肥大细胞会导致PMN凋亡率降低。TNF-α和类胰蛋白酶可激活核转录因子FoxO3a的上游信号酶，进而调节Bcl-2 家族成员的表达调节细胞凋亡。因此，我们提出“肥大细胞激活释放TNF-α和类胰蛋白酶改变FoxO3a转录活性，诱导PMN凋亡延迟参与肝移植后ALI”。本课题拟用大鼠肝移植模型、肥大细胞和PMN共培养等，结合对肥大细胞、类胰蛋白酶及TNF-α的干预，检测大鼠肝移植肺损伤、肥大细胞激活、PMN凋亡率及FoxO3a细胞内定位等，阐明肥大细胞参与PMN凋亡延迟的机制及其在肝移植后ALI中的作用，为防治肝移植后ALI提供依据。

肝移植术后急性肺损伤（ALI）的急性期以肺泡内大量中性粒细胞（PMN）浸润为主，PMN的凋亡延迟会致ALI持续存在与恶化，但其凋亡延迟机制尚不明确。本研究发现肥大细胞激活与以及其释放的TNF-α和类胰蛋白酶的肺部表达以及相应炎症因子明显升高，升高趋势与肺组织损伤一致，在8h达到高峰。而 PMN的凋亡在16h达到峰值，明显滞后于肺部损伤，与肺部急性炎症消退一致。肥大细胞抑制剂，类胰蛋白酶和TNF-α 抑制剂可以增加PMN细胞凋亡，减轻肺部损伤。OLT术后肺组织PMN中总的FoxO3a水平降低，且磷酸化FoxO3a水平升高，提示OLT术后肺组织PMN中FoxO3a失去活性。通过对大鼠进行病毒注射进行FoxO3a敲除，发现在FoxO3a敲除后肺部损伤明显加重，逆转类胰蛋白酶抑制剂和TNF-α抑制剂的肺保护作用，抑制PMN凋亡。实验结果表明肝移植术后ALI中，肥大细胞激活释放的类胰蛋白酶和TNF-α可能通过FoxO3a导致PMN延迟凋亡参与了ALI的进程。