The Role Of Exosomes Derived From PTPN11 Activating Mutant Bone Marrow Cells In Myeloproliferative Neoplasm

【Background】Myeloproliferative neoplasm is a type of hematological tumor caused by the aberrant differentiation of HSCs into one or more lines during the differentiation process.It has high mortality,recurrence,and metastasis.Traditional surgery can only refer to the removal of solid tumors.Hematological tumors can only be treated with chemotherapy drugs and hematopoietic stem cells transplantation.One of the biggest problems of MPN treatment is the extramedullary infiltration of cells,that is,the aggressive behavior of malignant tumors,and the tiny cell residues of cancer cells.These are the root causes of repeated attacks after chemotherapy drugs and hematopoietic stem cells transplantation.The occurrence of MPN is closely related to environmental physical inheritance and other factors,among which gene mutation is the main cause of MPN.Common gene mutations include JAK2,BCR-ABL1,RARS,SETBP1,PTPN11.This research group is mainly devoted to studying the role of PTPN11 activating mutations in the occurrence of MPN.SHP2 is a protein tyrosine phosphatase encoded by PTPN11,which plays a vital role in the growth and differentiation of hematopoietic cells.Mutations in PTPN11 have been reported to be highly correlated with the occurrence and development of many tumors,and hematological tumor MPN is one of them.The results of the study show that the mutation probability of PTPN11 in patients with Noonan syndrome is as high as 50%.In addition,other blood system tumors such as leukemia and multiple myeloma also have varying degrees of PTPN11 mutation.Exosomes are a kind of nano-scale vesicles secreted by cells with a particle size of less than 150nm.They have a typical lipid bilayer structure and contain a lot of rich lipids,nucleic acids,glycogen complexes and other substances.Extensive material exchange and information transmission between the two countries play an important role in the course of the disease.It is also used as a marker for disease diagnosis and patient prognosis.The results of previous studies have shown that PTPN11 activating mutations will cause the homeostasis of the bone marrow microenvironment,leading to the occurrence of MPN.Specifically,the secretion of inflammatory factors is increased,while the level of factors that maintain the function of hematopoietic stem cells is decreased,causing excessive activation and exhaustion of HSCs.The mice developed hepatosplenomegaly and myeloid cells increased significantly.At the same time,the sequencing results found that the expression capacity of exosome-related proteins were significantly enhanced in the mutant mice.In recent years,studies on exosomes have emerged one after another,but there have been no studies on bone marrow-derived exosomes after PTPN11activation mutations.See the report,combined with numerous reports on the function of exosomes,we suspected that whether exosomes derived from mutant bone marrow cells play a vital role in the occurrence of MPN.We have constructed a mouse model of PTPN11 activating mutation for the following research,hoping to provide a novel target for clinical research of MPN in the future.【Methods】1.Mice with PTPN11^E76K/+;Mx1-Cre and control mice PTPN11^+/+;Mx1-Cre were obtained through breeding and identification.2.Two months after PIPC injection,the experimental group and the control group mice were sacrificed to observe whether the PTPN11^E76K/+;Mx1-Cre activating mutation mice developed the MPN phenotype specifically as hepatosplenomegaly,myeloid cells increased significantly,decrease in the number of hematopoietic stem cells.3.After determining the MPN phenotype of the mutant mice,the supernatant of the bone marrow cells was separated to extract exosomes.4.Use TEM,NTA and WB to detect the purity of exosomes.5.Use magnetic beads to separate LSK cells from normal mouse bone marrow cells,and add exosomes derived from different bone marrow cells for co-cultivation.Cytometry was used to detect the changes of mutant exosomes on the myeloid differentiation ability of normal LSK cells.6.Separate the exosomes of the experimental group and the control group mice,and inject them into normal mice through the tail vein to observe whether the mice have MPN-like phenotypes.7.Use the kit to detect the difference between the exosomes derived from the mutant bone marrow cells and the normal exosomes.8.After PTPN11^E76K/+;Mx1-Cre activating mutation mice are treated with receptor antagonists,observe whether the symptoms of MPN are alleviated,and test the exosomes again.【Results】1.After PIPC induction,the mice showed symptoms of MPN,such as hepatosplenomegaly and myeloid cells increased.2.The purity of exosomes extracted by ultracentrifugation is higher,and the number of exosomes derived from mutant bone marrow cells is greater than that of the normal group.3.Exosomes derived from mutant bone marrow cells will accelerate the myeloid differentiation of normal LSK cells in vitro.4.Exosomes derived from mutant bone marrow cells can cause MPN-like phenotypes in normal mice.5.The test outcomes of the cytokine kit revealed that the level of inflammatory factors in exosomes derived from bone marrow cells in the mutant group was higher than that in the control group.6.After the use of the receptor antagonist,the MPN symptoms of the mice were alleviated,and the level of inflammatory factors in the exosomes was decreased.【Conclusion】After PTPN11^E76K/+;Mx1-Cre is used to induce activating mutations with PIPC,the homeostasis in the bone marrow microenvironment is unbalanced.The number of factors that maintain the steady state of the microenvironment is significantly reduced,however the inflammatory factors are significantly increased,and MPN occurs in mice.At the same time,the number of bone marrow-derived exosomes in mice has increased,and their functions have also changed.They will carry inflammatory factors to promote the myeloid differentiation of HSCs and lead to the MPN-like phenotype in mice.