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Curtis Dobrowolski Department of Biology and Marine Science Jacksonville University Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus.

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Presentation on theme: "Curtis Dobrowolski Department of Biology and Marine Science Jacksonville University Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus."— Presentation transcript:

1 Curtis Dobrowolski Department of Biology and Marine Science Jacksonville University Rate of Phagocytosis in Macrophages Stimulated by Astragalus membranaceus

2 Introduction Macrophages are the first line of defense in the human immune system Phagocytose Important antigen presenting cells, required for adaptive immune response

3 Introduction Advantages of increased phagocytosis Removal of pathogen More T cell interaction, faster adaptive immune response More memory More Antibody production More cytokines released increased inflammation and response from other immune cells www.phototakeusa.com

4 Introduction Astragalus membranaceus Shrub native to east Asia, primarily China A. membranaceus is a common medicinal herb used in eastern medicine One of the 50 fundamental Chinese medicinal herbs Huáng qí- root tea Used primarily when symptoms of an infection first arise Shown to increase antibody production in mice (1) Increased cytokine production (2) http://nccam.nih.gov/health/astragalus/

5 50 Fundamental Chinese Medicinal Herbs 1Agastache rugosa26Gentiana loureiroi 2Alangium chininese27Gleditsia sinensis 3Anemone chininese28Glycyrrhiza uralensis 4Anisodus tanguticus29Hydnocarpus anthelmintica 5Aridisia j aponica30Ilex purpurea 6Aster tataricus31Leonurus japonicus 7Astragalus membranaceus32Ligusticum wallichii 8Camillia sinesis- Traditional Black Tea33Lobella chinensis 9Cannabis sativa- Marijuana34Phellodendron amurense 10Cathamus tinctorius35Platycladus orientalis 11Cinnamomum cassia- Cinnamon36Pseudolarix amabilis 12Cissampelous pareira37Psilopeganum sinense 13Coptis chinensis38Pueraria lobata 14Corydalis ambigua39Rauwolfia serpentina 15Croton tiglium40Rehmannia glutenosa 16Daphne genkwa41Rheum officinale 17Datura metel42Rhododendron tsinghaiense 18Datura tatula43Saussurea costus 19Dendrobium nobile -Orchid44Schisandrac hinensis 20Dichroa febrifuga45Scutellaria baicalensis 21Ephedra sinica46Stemona tuberosa 22Eucommia ulmoides47Stephania tetrandra 23Euphorbia pekinensis48Styphnolobium japonicum 24Flueggea suffruticosa49Trichosanthes kirilowii 25Forsythia suspensa50Wiksotroemia indica

6 Hypothesis In vitro phagocytosis in macrophages increases when subjected to A. membranaceus

7 Methods IC-21 macrophages were cultured in tissue culturing flasks Macrophages grown RPMI 1640 media supplemented with 10% fetal bovine serum and 1% pen/strep antibiotic Incubated at 37°C with 5% CO 2 until cells were fully confluent www.millan.com

8 Methods Macrophages were lifted with Ca 2+ depleted PBS Solution PBS= phosphate buffered saline PBS/macrophage solution was stained with trypan blue vital dye and counted using a hemocytometer Using (Q 1 +Q 2 +Q 3 +Q 4 )*10 4 =cells/ml equation concentrations were determined www.ruf.rice.edu

9 Methods Using the count from the hemocytometer we placed 300,000 cells in each micro-chamber Equal amount of RPMI 1640 media were added to each micro- chamber Incubated for minimum of 3 hours www.usascientific.com

10 Methods Media/PBS solution was removed Macrophages were incubated for a minimum of 24 hours to allow the medias to have its effects on the macrophages Media alone 0.3% EtOH Control 1% astragalus Control 1%A.M. EtOH Control Control 1%A.M. EtOH Control 1%A.M.

11 Methods Media was removed 1 μ diameter fluorescently tagged latex beads were layered on top of macrophages, at a concentration of 20 beads per cell Media alone, EtOH and astragalus media were then added to the respective wells

12 Methods Media and beads were removed and wells were washed 3 times To remove beads that have not been phagocytosed Wells were removed and slides were stained with protocol hema 3 stain Increasing phagocytosis More cells phagocytosing Subset of cells phagocytosing more

13 Methods Data collected Total number of cells (75-175 cells counted per experimental well) Number of cells that phagocytosed any beads Number of cells that phagocytosed >1 bead Rate of phagocytosis calculated

14 Results P Value 0.396169 P Value 0.000463 P Value 0.000538 N=9

15 Results P value 0.3867711 P value 0.445056 P value 0.0167912 N=9

16 Conclusions The rate of in vitro phagocytosis in macrophages increases when subjected to A. membranaceus Could be beneficial in helping immuno-compromised patients HIV/AIDS patients Genetic disorders More research is need to fully understand A. membranaceus’ immuno-stimulating properties and benefits

17 Acknowledgements I would like to thank Dr. Karen Jackson PhD, for all of her help in culturing macrophages in a less than ideal environment Also I would like to thank Sally and Judy in the Science and Math division office, for signing me out a lab key nearly everyday of the year

18 References 1. Zhao, K S, Mancini, C, et al., “Enhancement of the immune response in mice by Astragalus membranaceus extracts.”, Immunopharmacology, 1990 Nov-Dec; 20(2): 225-233 2. Bedir E, Pugh N, et al., “Immunostimulatory effects of cycloartane-type triterpene glycosides from astragalus species.”, Biol Pharm Bull, 2000 Jul; 23(7): 834-7


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