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Endogenous ligand of myelocyte trigger receptor 2 and application thereof

An endogenous, myeloid cell-based technology, applied to medical preparations containing active ingredients, pharmaceutical formulas, organic active ingredients, etc.

Active Publication Date: 2019-11-29
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

S1P can enhance the phagocytosis of macrophages, but whether it promotes phagocytosis by acting on TREM2 in microglia has not been reported

Method used

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  • Endogenous ligand of myelocyte trigger receptor 2 and application thereof
  • Endogenous ligand of myelocyte trigger receptor 2 and application thereof
  • Endogenous ligand of myelocyte trigger receptor 2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction of stable transfection strain

[0025] Construct the CD8 leader sequence-TREM2-DAP12 chimeric protein plasmid, load the CD8 leader sequence, the chimeric mRNA sequence of the extracellular and transmembrane regions of TREM2 and the intracellular region of DAP12 into the pGEFP-N1 vector to obtain a plasmid, And amplified, the plasmid sequence is shown in Table 1. Lipofectamine 2000 was used to transfect CHO cells. After the transfection was completed, the culture medium containing G418 was selected and cultured for 2 days, digested and serially diluted, and seeded into 96-well plates to ensure that there were only single cells in most of the wells. After the cells grow into clusters, they are digested and planted in a 24-well plate. After the cells form a cluster in the 24-well plate, they are digested and planted in a 6-well plate. Repeated amplification in this way, the whole process is selectively screened with a medium containing G418. Per...

Embodiment 2

[0029] Example 2: Phagocytosis experiment of stably transformed strains

[0030] The stable cells and common CHO cells were seeded into 24-well plates at a density of 100,000 / well. After the cells adhered to the wall, they were stained with 5 mM live cell cytoplasmic dye Cell tracker, and then 3 μl / 100 μl of red fluorescent-labeled yeast-conjugated pHrodo phagocytosis dye was treated with 20 μM S1P or 10 μg / ml LPS (Pink ginseng), or S1P was treated with 2 μM cytoD to block phagocytosis (Pink ginseng), and photographed at 2 h or 4 h. Such as figure 1 As shown, there is basically no phagocytosis in untransfected cells, which is consistent with the characteristics of CHO cells that do not have phagocytic function; after the transfected cells are given S1P or LPS, compared with the non-administered transfected cells, 2h There was a certain increase in phagocytosis, the difference was statistically significant, and the phagocytosis enhancement was more obvious at 4 hours; after th...

Embodiment 3

[0031] Embodiment 3: liquid mass spectrometry

[0032]Spread 2 large dishes for the stable transfected strains, give 20μM S1P or normal culture medium to culture respectively after overgrown, wash 3 times with PBS after acting for 2 hours, add 400μl homogenization buffer to each dish for lysis after discarding, homogenization buffer formula See Table 2. Scrape the cells with a spatula and collect them in a 1.5ml EP tube, put them in a -80°C ultra-low temperature refrigerator for freezing, take them out and thaw after the freezing is complete, and repeat freezing and thawing 5 times to promote cell lysis. After freezing and thawing, grind with glass beads, grind for 40 times, and then stand on ice for 5 minutes, repeating this 4 times. Centrifuge at 12000 rpm for 15 min at 4°C, take the supernatant, add 3 μl of TREM2 antibody for immunoprecipitation and incubate overnight, then add 100 μl proteinA+G beads / ml cell lysate supernatant, rotate overnight at 4°C to capture the immun...

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Abstract

The invention discloses an endogenous ligand of myelocyte trigger receptor 2 and application thereof, belonging to the technical field of drug research and development. The endogenous ligand of the myelocyte trigger receptor 2 is sphingosine-1-phosphate. On the premise that S1P does not depend on an S1PRs receptor, the phagocytic function of microglial cells is enhanced by activating TREM2, the S1P as the endogenous ligand of the TREM2 is disclosed for the first time, and a new mechanism that the S1P regulates the phagocytic function of the microglial cells is preliminarily prompted.

Description

technical field [0001] The invention relates to the technical field of drug research and development, in particular to an endogenous ligand of myeloid cell triggering receptor 2 and its application. Background technique [0002] Microglia are intrinsic immune effector cells in the central nervous system. In the normal development of the nervous system, microglia play a role in the rapid removal of apoptotic neurons, the pruning of neuronal synapses, and the secretion of cytokines to regulate the survival of neurons; in pathological conditions, microglia cells are among the first immune cells to respond. In the event of brain injury such as ischemic stroke, a large number of neurons die, and the dead neurons release many dangerous molecules such as nucleic acids, proteins, and lipids, which in turn lead to an inflammatory response. If these neurotoxic cell fragments are not obtained Effective removal will weaken the plasticity of neurons after injury, trigger secondary infl...

Claims

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Application Information

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IPC IPC(8): C07F9/113A61P35/00A61P29/00A61K31/661
CPCC07F9/113A61P35/00A61P29/00A61K31/661
Inventor 孙秀兰薛腾飞杨进胡刚王富强季娟
Owner NANJING MEDICAL UNIV
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