Multi-cycle Treatment Of Leukopenia With Recombinant Ganoderma Lucidum Immunoregulatory Protein And Identification Of Its Binding Receptors On Hematopoietic Stem Cells

Purpose:In our previous studies,it was found that recombinant ganoderma immunoregulatory protein acts on hematopoietic stem cells in the mouse bone marrow and promotes their self-renewal and differentiation into granulocyte precursor cells such as myeloid progenitor cells and granulocyte-macrophage progenitor cells.At the same time,it promotes release of mature neutrophils from bone marrow into peripheral circulation by regulating the CXC chemokine receptor type 4(CXCR4)-Stromal cell derived factor-1(SDF1)axis to treat neutropenia caused by cyclophosphamide in a mouse model.To further study the therapeutic effect and mechanism of rLZ-8 in this disease,considering the use of granulocyte colony-stimulating factor(G-CSF)and Peg G-CSF as the main drug in clinical treatment,it is still needed to evaluate the satisfactory sustainability and stability of rLZ-8 treatment effect under multiple treatment cycles.Besides,as a first-stage clinical test substance,it will be of great significance to evaluate the effect of this protein on peripheral blood in the treatment of leukopenia in non-human primate models.It has been determined that mouse bone marrow-derived hematopoietic stem cells are the main effector cells of rLZ-8,and screening out the receptors on cell surface is crucial for understanding the protein function and regulation.Method:First,the fermentation and purification process of rLZ-8 were optimized to obtain high-purity active protein for downstream experiments.Subsequently,a stable mouse and cynomolgus monkey leukopenia model were established.In the mouse model,Peg G-CSF and G-CSF were used as positive control drugs to test the effectiveness and stability of rLZ-8’s effect in multiple treatment cycles.In the cynomolgus monkey model,the effect of rLZ-8 by subcutaneous administration or tail vein administration on the steady state of peripheral blood cells was examined,to evaluate the therapeutic effect of rLZ-8 in non-human primate models.Finally,based on TriCeps-living cell surface receptor recognition screening technology,the receptors bound to rLZ-8 on the surface of hematopoietic stem cells were screened and verified to supplement the treatment mechanism of leukopenia caused by cyclophosphamide.Results:In the first part of the study,the p GAPZα A-rLZ-8 expression strain was designed and constructed,and then the strain was fermented and cultivated at the pilot level.Subsequently,through the fine purification technology based on cation exchange chromatography and gel chromatography,high-purity recombinant ganoderma immunomodulating protein was obtained,which provided stable and high-quality recombinant protein samples for downstream experiments.In the second part of the study,we first constructed a stable mouse and cynomolgus monkey leukopenia model.In the mouse model,we tested the effect of rLZ-8 on peripheral blood cells during multiple cycles of treatment and confirmed that neutrophil is the main effector cell.Compared with the Peg G-CSF and G-CSF treatment groups,the therapeutic effect of rLZ-8 is stable and smooth,with obvious dose correlation,and can maintain the peripheral circulation in a long-term stable state.Subsequently,it was confirmed that in non-human primate leukopenia model,subcutaneous injection of rLZ-8 also has the effect of increasing peripheral circulation neutrophils.In the third part of the study,we first confirmed that rLZ-8 can be coupled with TriCEPS,and optimized the coupling conditions.Subsequently,an immunomagnetic column was used to isolate the hematopoietic stem cells with higher purity.Then,the coupled TriCEPs rLZ-8 were used to capture and enrich potential proteins on the cell surface that could bind to rLZ-8,and were identified and relatively quantified by proteomics.Comparing with the control group,we obtained the candidates for binding to rLZ-8.Finally,based on the immunofluorescence technology,CSF1 R was regarded as one of the receptors of rLZ-8.Conclusion:The study proves that CSF1 R is the receptor that rLZ-8 binds to the surface of murine hematopoietic stem cells.During multiple treatment cycles,rLZ-8 has good sustainability and stability in the treatment of murine leukopenia model.Neutrophils are the main effector cells and have a certain restorative effect on peripheral circulating platelets.Once again it proves that rLZ-8 acts on more primitive cells than G-CSF cytokines.During treatment,the bone marrow cell reserves are more abundant and can bear the bone marrow suppression caused by multiple cyclophosphamide administration.At the same time,subcutaneous rLZ-8 administration in the cynomolgus leukopenia model has the effect of promoting neutrophil production.At present,there is no report of a protein that has both anti-tumor effect and maintenance of blood cell homeostasis.RLZ-8 may become neutral in the treatment / prevention of tumor therapy in the treatment of chemotherapy-induced leukocyte / neutropenia Therapeutic drugs or supplements for neutropenia,and potential adjuvants for stem cell mobilization and expansion in vitro.