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Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro

A red cinnamon lectin, dendritic cell technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of peripheral blood dendritic cell maturation and proliferation that have not been reported, and achieve functional Strong, low cost, easy to operate effect

Inactive Publication Date: 2012-09-26
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After searching, there is no report on the induction of maturation and proliferation of human peripheral blood dendritic cells by laurel wood agglutinin

Method used

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  • Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro
  • Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro
  • Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Obtaining of laurel wood lectin

[0028] All operations for separation and purification of laurel wood lectin were carried out at 4°C. Firstly, take 30g of red osmanthus seeds, crush and grind them, add 0.01mol / L phosphate buffer solution containing 0.1mol / L NaCl, pH7.2 150mL at a ratio of 1:5, stir with a magnetic stirrer for 2 hours, soak and extract for 24 hours. The coagulation activity of the extract is at 2 9 -2 11 between. The extract was filtered, the filtrate was centrifuged at 3000rpm for 15min, and ammonium sulfate was added to the supernatant to reach 30% saturation. Centrifuge at 3000rpm for 20min, and discard the precipitate. Ammonium sulfate was added to the supernatant to 60% saturation in the same way as above, and left for 12 hours. Centrifuge at 3000rpm for 20min. Discard the supernatant, and dissolve the precipitate with a small amount of 0.01mol / L phosphate buffer solution containing 0.1mol / L NaCl, pH7.2-7.4. Dialyze the crude extra...

Embodiment 2

[0029] Example 2 Induction of mature dendritic cells

[0030] 1. Isolation of peripheral blood mononuclear cells (PBMC): Take 50ml of venous blood from a healthy person, inject it into a sterile blood collection tube containing heparin sodium solution and shake slowly, then add an equal amount of sterile PBS buffer and mix well. Take 5ml of lymphocyte separation solution and add it to a 15ml centrifuge tube, and slowly superimpose the blood dilution solution on the separation solution along the tube wall to form a clear interface. The appropriate volume ratio of the blood diluent to the separating liquid is 2:1-3:1. After standing for 10 minutes, centrifuge at 2000 rpm for 20 minutes at room temperature. Use a dropper to draw the mononuclear cell layer and place it in another centrifuge tube, add 2-3 times the volume of red blood cell lysate, mix well, let stand on ice for 3 minutes, add 10 times the volume of PBS buffer, and mix well , 1500rpm, centrifuge at 4°C for 5min. A...

Embodiment 3

[0034] Example 3 Identification of dendritic cells

[0035] 1. Observation of cell morphology

[0036] Such as figure 1 As shown, after adding different concentrations of ALL and culturing for 2 days, the cells in each group grew well and were in an irregular state, most of which were suspended or semi-adhered to the wall. The cell colonies are typical dendritic cell morphology.

[0037] 2. Detection of DC surface markers by flow cytometry

[0038] Count the cells and adjust the DC concentration to 1 x 10 6 / mL, add to Ep tube, wash once with PBS, centrifuge at 1500rpm for 5min; discard the supernatant, leave 100μL, mix the cells, add monoclonal antibodies CD1a, CD83, CD86, CD40 and HLA-DR respectively; react in the dark at 4°C for 30min, Wash once with PBS, add 500 μL PBS to resuspend cells, and detect cell surface markers by flow cytometry.

[0039] The DCs of the blank control group and the TNFα control group were subjected to the same steps to detect the surface marke...

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Abstract

The invention discloses Artocarpus lingnanensis lectin capable of inducing dentritic cells (DC) to mature and proliferate in vitro, a special culture medium for inducing DC to mature and proliferate, and a preparation method for the DC. The Artocarpus lingnanensis lectin is prepared from red cassia tree seeds through separation and purification by the conventional separation method. According to the special culture medium, inducing factors and the Artocarpus lingnanensis lectin are added into a DC culture medium. The preparation method comprises the following steps: adding precursor cells of the DC and the inducing factors into the DC culture medium, and adding the Artocarpus lingnanensis lectin when the cells are cultured in the sixth day; and continuously culturing to obtain mature DC, wherein the precursor cells are peripheral blood mononuclear cells; and the inducing factors are granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). A stable culture technical system for inducing the DC to mature and proliferate is established, and is easy to operate, convenient and practical; the required cell factors are a few, and the cost is low; and the dentritic cells (DC) have high maturity and strong functions. The Artocarpus lingnanensis lectin provides a novel effective way for further developing vaccine enhancers.

Description

technical field [0001] The invention relates to a method for inducing the maturation and proliferation of dendritic cells (DC) with plant lectin, in particular to a medium for inducing the maturation and proliferation of dendritic cells with laurel wood lectin. Background technique [0002] Plant lectins are a class of proteins with highly specific sugar-binding activity. Their greatest feature is the recognition of glycoproteins and glycolipids, especially the sugar chains with complex structures in the cell membrane: that is, the surface determinant of the cell membrane. Plant lectins can specifically interact with animal, plant, and microbial cells, causing or enhancing endocytosis or intercellular transport, and inducing certain physiological effects. It can store substances; resist the invasion of pathogens such as bacteria, fungi, and viruses; and also act as a mitogenic factor to regulate cell division and growth. Because lectins can selectively recognize different s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
Inventor 曾麒燕王婧娉李璐
Owner GUANGXI MEDICAL UNIVERSITY
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