培门冬酶通过干扰mTOR信号通路治疗EB病毒相关噬血细胞综合征的机制研究

Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a rare life-threatening disease. In our center, we have used the PEG-aspargase of principal combination regimen to treat EBV-HLH patients who have not achieved response on the HLH-94 regimen and achieve encouraging effect, and EBV-DNA was reduced even to 0 copy/ml after PEG-aspargase regimen. But the role of PEG-aspargase therapy for EBV-HLH is unclear.The apoptotic effects of PEG-Asp on EBV positive cell line and PBMC extracted from EBV-HLH patients were observed in a couple of in vitro experiments. It has been reported that asparagine depletion may inhibit the mTOR pathway. Therefore, we hypothesize that the potential mechanism of PEG-aspargase in the treatment of EBV-HLH is that the mTOR pathway was inhibited and the EBV-DNA replication was disrupted by L-asparagine depletion. To test this hypothesis, this proposed study will evaluate that the replication of EBV positive cells is through mTOR pathway, and that the PEG-aspargase may inhibit this pathway, and evaluate the effects of asparagine blocks the enzyme, growth factors on mTOR pathway activation and mTOR inhibitors on related markers and EBV-DNA through CyTOF flow cytometry. The above study is designed to verify the role of PEG-aspargase therapy for Epstein–Barr virus-associated hemophagocytic lymphohistiocytosis, to improve the treatment status of this severe disease, and to provide a theoretical basis for improving survival.

EBV相关噬血细胞综合征（EBV-HLH）是一种进展迅速、高死亡率的危重疾病。我们前期发现培门冬酶可显著降低EBV-HLH患者的EBV-DNA，但机制不清。前期发现培门冬酶促进EBV(+)细胞株和EBV-HLH患者PBMC的凋亡。据报道培门冬酶耗竭门冬酰胺可能抑制mTOR通路。据此提出假说：培门冬酶治疗EBV-HLH的可能机制为培门冬酶耗竭EBV感染细胞的门冬酰胺，抑制mTOR通路，促进细胞凋亡，降低EBV-DNA。本研究拟应用质谱流式技术分析1、培门冬酶对EBV(+)细胞株和EBV-HLH患者PBMC的mTOR信号通路、细胞凋亡和EBV-DNA的作用。2、门冬酰胺阻断培门冬酶的作用。3、生长因子活化mTOR通路及mTOR抑制剂对上述标记物和EBV-DNA的影响。旨在探索EBV-HLH的发病机制，培门冬酶治疗EBV-HLH的可能机制，为改善这一危重症的生存提供理论依据。

EBV相关噬血细胞综合征（EBV-HLH）是一种进展迅速、高死亡率的危重疾病。我们前期发现培门冬酶可显著降低EBV-HLH患者的EBV-DNA，但机制不清。本研究通过体外实验结果验证培门冬酶促进EBV(+)细胞株的凋亡，且发现培门冬酶对EBV-HLH患者PBMC的体外促凋亡作用。. 此外，本研究主要通过质谱流式技术检测EBV-HLH患者培门冬酶治疗前、后淋巴细胞亚群及细胞表面标记变化。质谱流式聚类得到42个亚群，其中配对t检验后，P＜0.05的亚群仅有C29亚群（经典单核细胞），治疗后该亚群比例升高，并且对C29上的marker进行治疗前后的变化分析发现CD272在治疗后的样本中表达显著降低，该marker为共抑制受体。配对t检验后，0.1＞P＞0.05的亚群有3个，均为记忆T细胞，在治疗后记忆T细胞升高。. 培门冬酶治疗前后细胞表面标记Foxp3有明显统计学差异。LMP1与EBNA1双表达的单核细胞、NK细胞和CD8+T细胞在培门冬酶治疗后CD8+T淋巴细胞和NK细胞LMP1表达均下降。NK细胞mTOR在治疗后明显下降。CD8+T细胞亚群在治疗后caspaes3升高。初步说明培门冬酶治疗后EBV受累的NK细胞、CD8+T淋巴细胞和单核细胞LMP1表达较前下降，mTOR下降，促进细胞凋亡，提示了培门冬酶治疗EBV-HLH的可能机制，为改善这一危重症的生存提供理论依据。.