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Effects of Laminarin on Maturation and Function of Human Monocyte Derived Dendritic Cells

Author: ChenLuYu
Tutor: ZhaoYiQiao
School: Guangzhou Medical College
Course: Internal Medicine
Keywords: Dendritic cells Oxidized low density lipoprotein Laminarin Atherosclerosis
CLC: R285.5
Type: Master's thesis
Year: 2010
Downloads: 16
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Abstract

ObjectiveLaminarin interfere in vitro culture of human monocyte derived dendritic cells after induced by oxidized low density lipoprotein. To investigat whether laminarin could influence human monocyte derived dendritic cells mature and function.To make a further discussion of immune regulation mechanism and resistance mechanism of atherosclerosis about laminarin.Materials and method1、Twenty healthy person were verification by medial examination. Everybody was extracted 20ml of peripheral blood. Peripheral blood mononuclear cell were isolated by density gradient centrifugation and culture with DCgro(containing rhGM-CSF 20ng/ml,rhIL-4 20ng/ml)at an atmosphere of 5%CO2 for 5 days. At the 5th day the above cultured cells were divided into 2 groups:the experimental group which treatment by OX-LDL(100ug/ml) and PBS group. DC were collectd at 7th day and the expression of CD1a、CD80、CD86、HLA-DR on the surface of DC were analyzed by flow cytometry. Culture changes in cell morphology were observed under an inverted microscope.2、Twenty four healthy person were verification by medial examination. Everybody was extracted 20ml of peripheral blood. Peripheral blood mononuclear cell were isolated by density gradient centrifugation and culture with DCgro(containing rhGM-CSF 20ng/ml,rhIL-4 20ng/ml)at an atmosphere of 5%CO2 for 5 days. At the 5th day all of the cell treatmented by OX-LDL(100ug/ml).At the 6th day the above cultured cells were divided into 4 groups:high-dose laminarin(5μg/ml)group,mediate -dose laminarin(2.5μg/ml)group,low-dose laminarin(0.5μg/ml)group and PBS group. DC were collectd at 7th day and the expression of CD1a、CD86、TLR-2 on the surface of DC were analyzed by flow cytometry. Collect cell culture supermatant of DC in each group,detecting IL-12 in accordance with the ELISE kit instructions. Culture changes in cell morphology were observed under an inverted microscope.Results1.1 0bservation on morphology of DC: After cultured 72 hours,adherent cells become scattered,similar to the cell cloning polymer,increasing bigger. When cells were cultured 5 days,cells float in culture fluid. When cultured 7 days,part of them aggregation in cluster,dendritic-like protrusionsim on cell surface in different guatity and shape . Difference between two groups is not obviously.1.2 DC influence of phenotype test results:collected the suspension cells and detected the molecules expressed on the surface by FACS analysis.The results showed that CD1a、CD80、CD86、HLA-DR positive rates were compared between the two groups had statistically singnificant differences(P<0.05),OX-LDL group higher than PBS group.2.1 Observation on morphology of DC: After cultured 48 hours, most of them adhesion to the bottom of the polystyrene bottle, smaller,and no significant difference shape. When cultured 6 days, part of them aggregation in cluster, dendritic-like protrusionsim on cell surface in different guatity and shape. When cultured 7 days, PBS group cells have the best group status, most of them aggregation cluster.can see more dendritic-like protrusionsim on cell surface; there is no singnificant differences between high-dose laminarin(5μg/ml)group,mediate -dose laminarin(2.5μg/ml)group and low-dose laminarin(0.5μg/ml)group after laminarin intervention . 2.2 DC influence of phenotype test results:collected the suspension cells and detected the molecules expressed on the surface by FACS analysis.The results showed that CD1a、CD86 positive rates of high-dose laminarin(5μg/ml)group,mediate -dose laminarin(2.5μg/ml)group and low-dose laminarin(0.5μg/ml)group were lower than PBS group(P<0.05).TLR-2 positive rates of high-dose laminarin(5μg/ml)group,mediate -dose laminarin(2.5μg/ml)group and low-dose laminarin(0.5μg/ml)group((33.06±0.91)%, (29.88±2.53) %,(27.94±1.40) %)was high than (P=0.000)((22.65±4.9) %). TLR-2 positive rates between high-dose laminarin(5μg/ml)group and low-dose laminarin(0.5μg/ml)group had statistically singnificant differences(P<0.05).2.3 Collect cell culture supermatant of DC in each group,detecting IL-12 in accordance with the ELISE kit instructions. The content of IL-12have singnificantly statistical difference in 4 groups of cells cultured supernatan(tP=0.000),compared the content of IL-12 in each two groups have singnificantly statistical difference(P=0.000). PBS group is (116.4117±13.14) pg/ml higher than high-dose laminarin(5μg/ml)group,mediate -dose laminarin(2.5μg/ml)group and low-dose laminarin(0.5μg/ml)group .Conclusions(1) The phenotype exspression of CD1a、CD80、CD86、HLA-DR in monocyte derived dendritic cells after Oxidized low density lipoprotein treatment are singnificantly higher ,DCs in a mature stste.(2)The phenotype exspression of CD1a、CD86 in monocyte derived dendritic cells after laminarin treatment are singnificantly lower ,as the same time, Toll-like receptors-2 are higher. (3)The levels IL-12 secreted in monocyte derived dendritic cells after laminarin treatment,are singnificantly lower then the control group.(4)Laminarin restrain the maturation and function of DCs,It may be one of the mechanisms of anti-atherosclerosis with laminarin.

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