Effects Of Oligomannuronaic Acid In Promoting Leucocyte Growth And Its Mechanism

Leukopeina is a common complication in the process of radiotherapy andchemotherapy of tumor, it refers that the peripheral white blood cell numbers below4×10~9/L. The main clinical symptoms is weakness, dizziness, limb weakness andcold. If not treatment in time, severe infection and complications may be occur.Therefore, regulating the body resistance to disease, especially the resistance ofneutropenia after cancer treatment, reduce the incidence of infection, is the key factorsto improve the quality of anticancer therapy.At present, the drugs to treatment leucopenia are vitamins, purine nucleotides,coenzymes and colony-stimulating factor. These drugs’ effects are not ideal, or theyare expensive, or patients are difficult to accept. Therefore, it is urgent to developmentsdrugs of elevated white blood cell, that have exact effect, low price, and no toxicand side effects.Alginate is a widely biological active substances. In previous research, wereasearched mannuronate by digestion with an alginate lyase, and found that theycould improve the white blood cell, and promote the recovery of hemopoietic systemof mice. Based on the above reasons, we studied the effects on leukocyte of mice ofdifferent molecular weight of mannuronate oligomers and guluronate oligomers, andmeasured serum granulocyte macrophage colony stimulating factors. According to theabove two indicators, mannuronate oligomers with molecular weight of5800was thebest effective fragment. We reasearched the effects of M5800on white blood cell, andreasearched its mechanism at the cellular level.This paper mainly carried out the following works:1. The contents of uronic acid in M5800were50.6%, which were measured by sulphuric acid and carbazole method. Using the HPGPC method to determine the average molecular weight and distribution of the molecular weight of M5800, and respectively was5.812KDa and1.080; the specific rotation and pH value of M5800was‐69.16°and6.51repectively.2. Used the moded of mice induced by cyclophosphamide, we studied theeffects on leukocyte of mice of different molecular weight of mannuronate oligomers and guluronate oligomers, and measured serum granulocyte macrophage colonystimuating factors.The results showed that, in addition to a guluronate oligomers wighmolecular weight of2000,the other5drugs could significantly increase the numberof white blood cells in mice, at the same time could greatly increased the GM-CSFlevels in mice serum,and mannuronate oligomers with molecular weight of5800had the strongest effect. Therefore, according to the above two indicators,mannuronate oligomers with molecular weight of5800could increase the numberof white blood cells in mice, and increase GM-CSF in serum.With the suppression of bone marrow induced by cyclophosphamide in micemodel for screening platform, we reasearched the effects of M5800on white bloodcell.The results showed that, M5800in the180mg/kg to540mg/kg range, can besignificantly increased the number of white blood cells and bone marrow nucleatedcells, compared with the model group, there was significant difference.3. We studied the mechanism of M5800at the cellular level:(1) We observed the effect of M5800on the production capability ofCFU-GM on normal mice and model mice by in vivo and in vitro, and by using agarculture method; and observed the effect on CFU-GM of supernatant of bone marrowstromal cells,spleen cells and thymocytes cells induced by M5800.The results showedthat:①Compared with normal group, M5800can promote the CFU-GM in normalmice, and in the range of3.125μg/mL to25μg/mL, as with the drug concentrationincrease, the effect has gradually increased, at the concentration of25μg/mL, itseffect reached the maximum.②M5800, in the dose of180mg/kg,360mg/kg and540mg/kg, could remarkably enhance the number of CFU-GM of cyclophosphamidemodel mice by in vivo, and compared with the model group, there was significantdifference.③The supernatant of bone marrow stromal cell induced by M5800couldobviously promote the formation of CFU-GM, and in6.25μg/mL to50μg/mL range,compared with the control group, there are significant differences. The supernatant ofspleen cells and thymocytes cells induced by M5800could also promote theformation of CFU-GM, compared with the control group, there are significantdifferences.The result indicated that the cell culture supernatant induced by M5800could increase content or activity of GM-CSF.(2) Using the method of MTT, the Proliefration of bone marrow cell of normalmice and cyclophosphamide inhibition model mice were detected by in vivo and in vitro.The results showed that:①M5800, from6.25μg/mL to50μg/mL concentrationrange, could significantly promote the proliferation of bone marrow stromal cells, andat25μg/mL, its action was the strongest.②M5800could promote the proliferationof bone marrow stromal cells of cyclophosphamide inhibition of mice, in which the50μg/mL group, compared with control group, there was significant difference.(3) Using the ELISA method, we researched the effect of M5800on GM-CSFand CSF, using MTT method, tested the secretion of IL-3of spleen lymphocyte. Theresults showed that,①M5800, at the range of12.5μg/mL to50μg/mL, canobviously promote the bone marrow stromal cells secreting GM-CSF, compared withcontrol group, has significant difference.②M5800at the rang of6.25μg/mL and50μg/mL, can obviously promote the bone marrow stromal cells to secret SCF, andcompared with control group, there was significant difference.③M5800canobviously improve the ability of spleen lymphocyte of secret IL-3, compared withcontrol group, there was significant difference.(4) M5800was reacted with fluorescein isothiocyanate (FITC), then thereducing terminal of M5800was labeled. We cultured bone marrow stromal cells, andthe fluorescent markers and bone marrow stromal cells were incubated together, usingconfocal laser scanning microscopy fluorescent to observe the location of fluorescentmarkers in cells.The results showed that M5800could act on the bone marrow stromalcells and into the cell plasma to play its role.