ATF4基因在噬血细胞性淋巴组织细胞增生症发病机制中作用的研究

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome with a high lethality caused by over-activation of lymphocytes and macrophages, and one of the key processes of pathogenesis is the cytokine storm. Our preliminary studies showed activating transcription factor 4 (ATF4) was related with inflammatory response and cytokine secretion, and its expression was increased in HLH patients. Besides, it was reported that ATF4 could improve the secretion of many kinds of cytokines, which had been demonstrated to be important in the HLH cytokine storm. Therefore, a question is raised that how ATF4 plays a role in the pathogenic process of HLH. To answer this question, we are intent to choose several downstream molecules of ATF4 using ATF4 knockout mice. Then, we will measure the expression of these downstream molecules and cytokines after knocking down (or over expressing) ATF4, and explore whether ATF4 directly binds to the promoter of these downstream molecules by chromatin immunoprecipitation and luciferase assay. Lastly, in vivo experiments will be performed to verify the function of ATF4 in HLH patients and murine models. This study might reveal molecular pathways of ATF4 in HLH and bring a new idea for its clinical therapy.

噬血细胞性淋巴组织细胞增生症(HLH)是一种临床上严重威胁患儿生命的危急重症，炎症因子风暴的产生是发病的关键环节之一。申请人在前期工作中发现，转录激活因子4（ATF4）与炎症反应和细胞因子分泌密切相关，且在HLH患儿中表达量明显升高，另有文献显示ATF4促进多种HLH相关炎症因子分泌。在此基础上我们提出科学问题：ATF4在HLH发病中如何发挥作用。为解答该问题，我们拟利用ATF4敲除小鼠细胞选取2-3个与炎症因子分泌相关的ATF4可能的下游分子；继而探索ATF4敲低（或过表达）对上述分子及炎症因子表达的影响，并利用染色质免疫共沉淀和双荧光素酶实验研究ATF4能否直接结合于下游分子启动子区；最后行体内功能学验证，测定HLH患儿中上述分子表达量，并探索敲除ATF4是否减轻HLH模型小鼠的发病情况。阐明ATF4在HLH炎症因子风暴发生中的作用机制，有助于找到新的治疗靶点，为临床应用提供理论依据。

噬血细胞性淋巴组织细胞增生症（hemophagocytic lymphohistiocytosis，HLH）又称噬血细胞综合征，是一组由于巨噬细胞和细胞毒性T细胞等细胞的过度活化导致的致命性临床综合征，严重威胁患儿生命，其发病过程与巨噬细胞和细胞毒性T淋巴细胞的持续过度活化从而引发的细胞因子大量分泌密切相关，但其具体机制尚未完全清楚。转录激活因子4（activating transcription factor 4，ATF4）是活化转录因子家族成员之一，广泛表达于多种组织细胞中，发挥造血发育、骨骼形成、能量代谢、细胞增殖分化等多种生物学功能。在前期的研究中，我们发现ATF4在炎症反应和细胞因子分泌过程中发挥着重要作用，并发现ATF4在HLH患儿外周血单个核细胞中的表达量有所升高。因此，我们推测ATF4可能在HLH发病机制中炎症因子风暴的产生过程发挥重要作用。本研究通过临床标本检测、体内及体外动物学实验等方法验证了ATF4在细胞因子产生过程中的重要作用。结果提示：1.HLH患儿外周血单个核细胞中ATF4的表达量较正常对照组有所增加，尤其是在EB病毒感染相关性HLH患儿中的表达量增加明显；2.利用ATF4敲除小鼠胎肝的单个核细胞在转录水平上进行细胞因子分泌相关的分子进行表达量测定，继而利用siRNA敲低的方式对ATF4调控的下游分子进行筛选，发现无翅型MMTV整合位点家族成员5A（wingless-type MMTV integration site family member 5A，Wnt5A）的表达量减低，推测ATF4可能通过Wnt5A调控细胞因子分泌；3.最后，通过在人急性单核细胞白血病细胞系（THP-1）中敲低ATF4发现ATF4敲低后IL-2和IL-6的表达量减低，验证ATF4对细胞因子分泌的调控作用。本研究的意义在于更加深入全面地了解并丰富了炎症因子风暴的产生机制，阐明ATF4对细胞因子分泌的调控机制，有利于临床上研制新的抑制炎症因子分泌及治疗HLH的靶向药物，为将来的临床应用提供理论依据。