Histone Methylation Of Peripheral White Blood Cells In Sepstic Patients

Sepsis is a systemic inflammatory response syndrome caused by infec-tion.Without control, it could cause multiple organ dysfunction. Sepsis with high in-cidence, has become the main cause of death of critically ill patients. Sepsis involves a variety of inflammatory cells, of which white blood cells (WBCs) mainly participate in excessive inflammatory response, but the specific regulatory mechanism is still unknown. Histone methylation is an important epigenetic modification. Its cellular functions involved in the regulation of gene expression, which is closely related to the embryonic development and tumorigenesis.Generally, histone H3lysine4(H3K4), H3K36and H3K79methylation regulate gene transcription activation, while methyla-tion of H3K9, H3K27and H4K20regulate gene transcription suppression. Recently cytology study also found that H3K4, H3K9, H3K27methylation were closely related to inflammation/infection. To explore the role of histone methylation in patients with clinical sepsis process, we extracted patient’s peripheral blood to obtain WBCs. Firstly we created and optimized immunofluorescence staining methodof WBCs, and thus we could observe differences of histone methylation among three kinds of WBCs. Then we chose trauma and postoperative esophageal cancer patients as study objects, and analyzed the changes of histone methylation of different patients due to sepsis.We aimed to obtain clinical evidence of histone methylation involved in the regulation of sepsis and seek epigenetic new indicators as early predictior of inflammation/infec-tion process.This study consists of two parts:Part I:Establishment and optimization of immunofluorescence staining method in human peripheral white blood cells. The number of leukocytes is the least in pe-ripheral blood, including neutrophils (Neu), lymphocytes (Lym), monocytes (Mon) and others, which have different counts and involve in infection, inflammation and immune responses. Short survival in vitro and suspended state makes it difficult to be fixed on slides, which limits the application of the immunofluorescence staining tech-nique. To address this difficult problem, we separated white blood cells as soon as possible.We explored the best cryopreservation and recovery conditions of WBCs to keep them survival as much as possible, and compared different suspensions and slides. Moreover we observed Eppendorf suspension method, blood smear method and drop method, looking for the best leukocyte fixed conditionsand optimizing im-munofluorescence staining techniques of WBCs.Part Ⅱ:Histone methylation of peripheral white blood cells in sepstic patients. We selected traumatic and postoperative esophageal cancer patients as study objects, Using the established immunofluorescence staining techniques of peripheral WBCs and293T cells fluorescence intensity as control, we analyzed three kinds of WBCs, Neu, Lym and Mon, relative quantitatively. Preliminary results suggested that:While sepsis could make H3K9dimethylation levels in three types of white blood cells in traumatic patients increased, but the changes compared with the pure trauma patients were not significant. While postoperative sepsis could reduce elevated H3K9di-methylation levels in three types of WBCs of postoperative patients with esophagus cancer. It was suggested that the impact of sepsis on trauma and postoperative esoph-ageal cancer patients was different. We speculated that cancer patients had been on the presence of abnormal histone methylation levels, which made it different with trauma patients. It was demonstrated that in patients with different underlying diseas-es, histone methylation related to infection/inflammation reaction process changed differently.With immunofluorescence analysis of histone methylation changes in pe-ripheral WBCs, We expected to construct a characteristic histone methylation spec-trum of white blood cells in different sepstic patients and explore new targets to per-sonalized diagnosis and treatment of sepsis.