Ganoderma Recombinant Immunomodulatory Protein (rlz-8) Expression In Eukaryotic Expression System And Pharmacodynamics

Leukopenia is a fairly common clinical disease. Many reasons causing the disease, when the WBC count remains below 4.0×109/L, most accompanied by dizziness, fatigue, loss of appetite, heart palpitations, tinnitus and other non-specific symptoms, severe infections due to neutropenia bleeding and death. The most common cause is chemotherapy-induced neutropenia and immune dysfunction. In recent years, the incidence of cancer increased with increasing trend, except for surgical treatment of tumors, the use of more or radiotherapy and chemotherapy, but radiotherapy and chemotherapy side effects, such as hematopoietic suppression induced neutropenia, immune function and other reasons, was forced to suspend treatment or treatment failure, which is an important factor affecting its efficacy. Cancer is a kind of serious threat to human life and quality of life of the major diseases are global for this purpose the number of patients receiving chemotherapy and more than 1000 million, of which more than 80% occurred in patients with mild chemotherapy leukopenia. So, for the neutral treatment of neutropenia in cancer treatment is a very important aspect. The most common chemotherapy toxicity of bone marrow suppression, can be expressed as the proliferation of hematopoietic stem cells. Bone marrow suppression limits the amount of chemotherapy drugs and chemotherapy coherence, thus affecting the efficacy of chemotherapy, some patients often delay or interruption of treatment thus, the effect of greatly reduced, even to patients with severe infection and death.This study Ganoderma immunomodulatory protein family, treating leukopenia pharmacodynamic and its possible mechanism was studied and discussed. In 1989, the fungus immunomodulatory protein from Ganoderma lucidum by Kino purified fruiting bodies named as the Lz-8, by amino acid sequencing and analysis of immunological activity, indicating that the LZ-8 and 110 amino acids, N terminal serine acetylation, molecular weight To 12.4kD, isoelectric point of 4.4, the LZ-8 is known immune activity to promote proliferation of peripheral blood lymphocytes, macrophages and induced stimulation of lymphocytes secrete cytokines (such as interleukin, tumor cells necrosis factor and interferon, etc.). This study, the Pichia expression system recombinant PichiaPinkTM the Lz-8, and optimize the purification process.Through animal experiments in mice, rats, dogs proven model of leukocyte poor, rlz-8 with the prevention and treatment of leukopenia pharmacodynamic function, the results of the study in the international community realized the first time to clinical fungal immunomodulatory protein drug research and development of new ways. In in vitro experiments, we found rLz-8 can promote the mice with bone marrow mononuclear cell proliferation, and promote bone marrow cell cycle G0/G1 to S phase proliferation. Through the hematopoietic progenitor cell colony culture proved, rlz-8 on mouse bone marrow cells to promote proliferation, can increase bone marrow CD45+ cells in bone marrow hematopoietic tissue structure and no effect. Using ELISA assay rlz-8 can promote bone marrow cells in serum GM-CSF, IL-2, IL-4, y-IFN levels and other factors increased.Using gene chip data analysis combined with bioinformatics rLz-8 on mouse spleen cells and signaling pathways related to the major gene expression, preliminary analysis showed that, rLZ-8 molecules through CD28-mediated T cell activation and cytokine production to increase effects of lymphatic enable the activation of T cells from TCR/CD3 lymphokine secretion at low levels to higher forms of self-secreted lymphokine paracrine secretion of the level of activation of PI3K, Akt occurs leading to the plasma membrane from the cytoplasmic translocation and activation the further activation of Akt in turn activates its downstream factor NF-kB signaling pathway to stimulate T cell proliferation, increased white blood cell count and immune regulation. rLZ-8 by CD 19, CD21 molecule activation, activation of PI3K/Akt signaling pathway, activation of the IkB kinase complex, IkB phosphorylation, NF-kB nuclear localization site of exposure. Free rapid NF-kB translocation to the nucleus, binding with specific IkB sequence to stimulate B cell proliferation, increased the number of white blood cells, regulating proliferation of immune cell activation, rlz-8 through the stimulation of IL-2, IL-3 and IL-6, activation of cytokine receptors, the role of JAK-STAT signaling pathway; the other hand, JAK role in SHP 2 factor, activation of Ras-MAPK pathway to regulate the c-Myc, Cyc D factor, stimulate cell proliferation, increased number of neutrophils; regulation BclX, Spred factors, play against apoptosis, stimulate cell proliferation.More research on the rlz-8 in the treatment of leukopenia and mechanism of the pharmacodynamics were analyzed. First time defined the rLz-8 treated rats, mice, dogs leukopenia pharmacodynamic effects; and to explore the rlz-8 treatment of the possible mechanism of leukopenia. rLz-8 Leukopenia prevention and treatment revealed a new mechanism in the international field for the first time similar research for future clinical application to lay a solid foundation.