Study Of β-glucan For Its Effects In Mice Monocytes-Macrophages Cell Line RAW264.7

Background and PurposeIn recent years,with broad-spectrum antibiotics,antineoplastic agents,immuno suppressants,and the long-term application of radiation therapy,especially in patients with AIDS increased,the incidence of fungal infections increased year by year.At the same time,clinical use of large quantities of antifungal drugs,fungidrug resistance is rapidly increasing,a fungal infection treatment of a difficult problem.β-glucan exist extensively in various types of fungi cell walls.Thereby clear pathogenic mechanism of glucan,eliminating these proinflammatory cytokines have become new ideas of the treatment of fungal infections.The purpose of our study is to explore mice monocytes -macrophage cell line RAW264.7 proliferation effects byβ-glucan,as well as the expression of TNF-αin the cell line RAW264.7.To investigate ERK1/2 signal transduction pathway in pathogenic process,provide experimental basis for the drug treatment of fungal infectionsMethodsPreparation 5μg/ml solution ofβ-glucan,4℃preservation.The macrophage cell line RAW264.7 cells from BALB/c mice,cultured in 37℃,5%CO2 and saturated humidity conditions,with RPMI1640 contain of 10%fetal bovine serum.Observation of cell morphology in microscope,counting after trypsin digestion and adjustment with cell culture medium for different density.Prepare for the following experiments: MTT to observe impacts on the RAW264.7 cells;ELISA,RT-PCR to detect mRNA and protein expression of TNF-α;Western blot detected t-ERK and p-ERK protein expression;observation the concentration change of[Ca²⁺]i and cAMP before and after the stimulation byβ-glucanResultsMTT results showed that cell proliferation rate related with final concentration and stimulating time ofβ-glucan; TNF-αsecretion selectivity related with final density ofβ-glucan as well as the response time;Western Blot results showed that p-ERK1 protein expression in the RAW264.7 cells stimulating byβ-glucan in final concentration of 100μg/ml;[Ca²⁺]i and cAMP concentration changed before and after theβ-glucan stimulate;ConclusionAppropriate concentration ofβ-glucan can stimulate the proliferation of RAW264.7 cells;β-glucan can dose-dependently induce the expression of TNF-α;β-glucan can activated ERK1/2-MAPK signal transduction pathway;β-glucan stimulate the concentration change of[Ca²⁺]i and cAMP in RAW264.7 cells, the rate of change related with theβ-glucan concentration.