Method for detecting activity of NMDA (N-methyl-D-aspartate) receptors of blood platelets and application of method

A technology of platelets and platelet aggregation, applied in the field of medical biology, can solve the problems of affecting the functional activity of platelets, platelet adhesion and aggregation functions have not been reported, and achieve the effect of reducing platelet aggregation

Inactive Publication Date: 2016-07-27
NANJING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aβ 1-42/1-40 Whether it affects the NMDA receptor functional activity of platelets, p...

Method used

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  • Method for detecting activity of NMDA (N-methyl-D-aspartate) receptors of blood platelets and application of method
  • Method for detecting activity of NMDA (N-methyl-D-aspartate) receptors of blood platelets and application of method
  • Method for detecting activity of NMDA (N-methyl-D-aspartate) receptors of blood platelets and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Platelets of Example 1 Human and Mouse Express NMDA Receptors

[0026]

[0027] ICR mice (10 males and 10 females each), weighing 25-30 grams, were purchased from the Experimental Animal Center of Jiangsu Province. Use a 3.8wt.% citrate anticoagulant tube to extract 2 mL of blood from the human cubital vein or the inner canthus of mice (the volume ratio of blood:anticoagulant is 1:9), mix it and place it at room temperature for low-speed centrifugation at 800 r / min After 8 minutes, carefully remove the upper layer of plasma, which is platelet-rich plasma (PRP). The remaining blood sample was then centrifuged at 3000r / min for 10 minutes, and the clear liquid on the upper layer was platelet poor plasma (PPP).

[0028]

[0029] Five subunits of NMDA receptors, NMDA receptor 1 and NMDA receptor 2 (A-D), have been cloned so far. Among them, NMDA receptor 1 can form functional pure oligomer NMDA receptor alone, but NMDA receptor 2 subunit does not have this function. T...

Embodiment 2

[0033] Ca of embodiment 2 platelet NMDA receptor 2+ inflow.

[0034]

[0035] With embodiment 1.

[0036] Selective NMDA receptor agonists N-methyl-D-aspartic acid (NMDA) and glutamate (glutamate), selective NMDA receptor antagonists MK-801 and memantine were purchased from Sigma.

[0037]

[0038] Free Ca in platelets 2+ ([Ca 2+ ] i) Concentration determination: Ca 2+ For fluorescent probe loading, the platelet-rich plasma suspension was centrifuged at 1000 r / min, 10 minutes each time x 3 times, the supernatant was aspirated and mixed by pipetting. Take 50 μL of cell suspension, add 17 μL of Fura-2 calcium probe, and incubate in a water bath at 36.5°C for 40 minutes. Fluorescence dual-wavelength spectrophotometer determination of [Ca 2+ ] i, The fluorescence intensity of Fura-2 was measured with a Hitachi F-3000 fluorescence dual-wavelength spectrophotometer. The measurement conditions are: the excitation light grating is 5nm, the emission light grating is 10nm, a...

Embodiment 3

[0045] Example 3 NMDA receptor activation can promote platelet adhesion.

[0046]

[0047] With embodiment 1 and embodiment 2.

[0048]

[0049] Platelet adhesion test method: suck out 1.5mL of anticoagulant blood, pour it into a long-necked glass bottle and insert it to the bottom, then take blood from it to calculate the number of platelets before rotation, and calculate the average number of two or three times as the number of platelets before rotation. After the flask was inserted into the platelet adhesion analyzer, it was rotated for 15 minutes at a rate of 3 revolutions per minute, and then blood was taken from the flask. The platelets were counted two or three times according to the conventional method, and the average value was calculated as the number of platelets after rotation. (number of platelets before rotation-number of platelets after rotation) / number of platelets before rotation=platelet adhesion rate.

[0050] Add NMDA receptor agonist NMDA (0-100 μM) o...

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Abstract

The invention provides a method for detecting activity of NMDA (N-methyl-D-aspartate) receptors of blood platelets and an application of the method. The activity of the NMDA receptors of the blood platelets is reflected through regulation of Ca<2+> influx of NMDA receptor channels of the blood platelets and detection for an acceleration function of the NMDA receptors on blood platelet adhesion and aggregation. The method comprises specific steps as follows: (1), the blood platelets are processed by a selective NMDA receptor stimulant NMDA, glutamate or glycine, and the NMDA receptors are activated, so that the Ca<2+> influx of the blood platelets can be increased and the adhesion rate and the aggregation rate of the blood platelets can be increased; (2), the blood platelets are processed by a selective NMDA receptor antagonist and memantine, and the NMDA receptors are blocked, thus, the Ca<2+> influx of the blood platelets can be decreased and the adhesion rate and the aggregation rate of the blood platelets can be decreased; therefore, the activity of the NMDA receptors of the blood platelets is reflected.

Description

technical field [0001] The invention belongs to the technical field of medical biology, and in particular relates to a method for detecting platelet N-methyl-D-aspartic acid (NMDA) receptor activity and an application thereof. Background technique [0002] Platelet (blood platelet) is one of the formed components in mammalian blood, and it is a small piece of cytoplasm that is shed from the cytoplasm of bone marrow mature megakaryocytes. Platelets have a specific morphological structure and biochemical composition, and have a relatively constant number in blood (the number of human platelets is 100,000 to 300,000 per cubic millimeter). Platelets are only found in mammalian blood. No nuclear structure, ie no chromosomes. The life span of platelets is about 7-14 days, and about 1 / 10-20 of the total amount is renewed every day. Most of the aging platelets are cleared in the spleen. The main function of platelets is to participate in the process of coagulation and hemostasis....

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6458G01N21/6486
Inventor 陈玲华旭狄婷婷王玲缪冶炼
Owner NANJING MEDICAL UNIV
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