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Application of FTY720-Phosphate in preparation of activating pharmacy of TREM2

A technology of myeloid cells and receptors, applied in the field of drug research and development, to achieve the effect of enhancing phagocytosis and promoting clearance

Pending Publication Date: 2020-03-17
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors newly discovered that FTY720 has the function of enhancing the phagocytosis of microglial cells, but whether it mediates the phagocytic function by activating microglial phagocytosis-related receptors, such as myeloid triggering receptor 2 (TREM2), has not been reported yet

Method used

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  • Application of FTY720-Phosphate in preparation of activating pharmacy of TREM2
  • Application of FTY720-Phosphate in preparation of activating pharmacy of TREM2
  • Application of FTY720-Phosphate in preparation of activating pharmacy of TREM2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Microthermal Surge Method Reveals the Relationship Between FTY720-P and TREM2

[0027] Using pET-24a(+) as a vector, construct TREM2 protein expression plasmids of various origins marked with a C-terminal His-tag. The sequences used for human-derived, rat-derived and mouse-derived TREM2 are shown in SEQ ID NO: 1. Shown in SEQ ID NO: 2, SEQ ID NO: 3.

[0028] Transform the recombinant plasmid into BL(DE3) Escherichia coli: Take 100 μL of freshly prepared competent BL(DE3) cells, add 10 μL of the recombinant plasmid, flick and mix well, place on ice for 30 minutes; heat shock at 42°C for 80 seconds, and quickly transfer to ice Cool in the bath for 5 minutes; add 400 μL of LB medium at 37°C, transfer to the incubator, and incubate at 37°C for 1 hour to recover the cells; take an appropriate amount of transformation products and spread them on LB plates containing kanamycin (agar 15g, Peptone 10g, yeast extract 5g, NaCl 10g, water 1000mL), 37 ℃ incubator upside-down cultur...

Embodiment 2

[0031] Experimental grouping and drug treatment

[0032] Rat primary neurons and microglia were extracted respectively, and the primary neurons were cultured in 24-well plates (200,000 cells / well) with Neurobasal medium + 2% B27 + 1% double antibody, and the medium was half changed every other day. Culture for one week until cell maturity; primary microglial cells were cultured with 10% Gibco FBS+DMEM+1% double antibody, and the medium was changed every three days, and culture for one week until cell maturity.

[0033]After the cells matured, microglia were added to neurons for co-culture (20,000 cells / well), co-cultured at a ratio of 1:10, and replaced with Neurobasal medium without B27: (10% Gibco FBS+DMEM+1% double antibody)=3:1 medium culture, divided into normal group, normal drug group, normal knockdown group, knockdown drug group, and each time point after oxygen glucose deprivation-reperfusion (OGD / R) injury These four groups of (3h, 5h, 7h, 9h, 12h). The specific op...

Embodiment 3

[0035] Immunofluorescence staining test

[0036] After the cells were fixed, they were washed 3 times with 0.01M PBS, discarded, and then blocked by adding blocking solution (containing 5% goat serum and 0.1% Triton X-100) at room temperature for 1 hour; adding the primary antibody dropwise (see Table 1 for antibody titer), 4 Incubate overnight at °C. After taking it out the next day, wash the primary antibody with 0.01MPBS, wash 3 times, 5min each time, add the corresponding fluorescent secondary antibody dropwise (see Table 2 for antibody titer), incubate at room temperature for 1h in the dark, and then wash 3 times with 0.01M PBS , 5 min each time; add 5 μg / mL Hoechst solution dropwise to react in the dark for 20 min, wash 3 times with 0.01M PBS, 5 min each time, and take pictures.

[0037] Table 1 Primary antibody for immunofluorescence staining

[0038]

[0039] Table 2 Secondary antibody for immunofluorescence staining

[0040]

[0041] The result is as image ...

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Abstract

The invention discloses application of FTY720-Phosphate in preparation of activating pharmacy of a TREM2, thereby realizing application of regulation of cell phagocytosis. The invention discloses thatFTY720-Phosphate is an agonist of TREM2 for the first time, discloses a fact that phosphorylated fingolimod enhances the phagocytic function of microglial cells by activating the TREM2, and illustrates a new mechanism of FTY720-Phosphate for regulating the phagocytic function of microglial cells.

Description

technical field [0001] The invention relates to the technical field of drug research and development, in particular to a drug for activating 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 inflam...

Claims

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Application Information

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IPC IPC(8): A61K31/661A61P29/00
CPCA61K31/661A61P29/00
Inventor 孙秀兰薛腾飞程虹郭若冰杨进季娟范益胡刚
Owner NANJING MEDICAL UNIV
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