Method for detecting platelet α7nachr activity and its application

A technology of platelets and preplatelets, applied in the field of medical biology, can solve the problems of platelet adhesion and aggregation functions that have not been reported, affect platelets, etc., and achieve the effect of reducing platelet aggregation

Inactive Publication Date: 2018-09-25
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 α7nAChR function of platelets, platelet activation, platelet adhesion and aggregation functions has not been reported so far

Method used

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  • Method for detecting platelet α7nachr activity and its application
  • Method for detecting platelet α7nachr activity and its application
  • Method for detecting platelet α7nachr activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Human and mouse platelets express α7nAChR

[0024]

[0025] ICR mice (10 males and 10 females each), weighing 25-30 grams, were purchased from the Experimental Animal Center of Jiangsu Province. Use a 3.8% citrate anticoagulant tube to extract 2 mL of blood from the human cubital vein or the inner canthus of the mouse (the volume ratio of blood:anticoagulant is 1:9), mix well and place it at room temperature for 8 minutes at low speed and centrifuge at 800r / min After that, 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).

[0026]

[0027] Western Blot detection. Specimen preparation for WB test: Add RIPA protein lysate (mainly including phosphatase inhibitors, protease inhibitors, PMSF and other components) to plasma, homogenate by ultrasonic, let stand in ice for 30 minutes, ...

Embodiment 2

[0030] Example 2 Ca of platelet α7nAChR 2+ inflow.

[0031]

[0032] With embodiment 1.

[0033] Selective α7nAChR agonist acetylcholine (acetylcholine, ACh), α7nAChR antagonist α-BGT (α-bungarotoxin), MLA (methyl-lycaconitine) were purchased from Sigma, selective α7nAChR agonist 3-(2,4-dimethoxybenzylidene)- Anabaseine (DMXB, also known as GTS-21) is provided by Jokho Pharmaceutical Company of Japan.

[0034]

[0035] platelet free Ca 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 Fluo-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 spe...

Embodiment 3

[0041] Example 3 α7nAChR activation can promote platelet adhesion and aggregation.

[0042]

[0043] With embodiment 1 and embodiment 2.

[0044]

[0045] Platelet adhesion test method : Suck out 1.5mL of anticoagulant blood and pour it into a long-necked glass bottle to the end, then take blood from it to calculate the number of platelets before rotation, calculate the average number of two or three times, and use it 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. Normal value: 20-40%.

[0046] Platelet ag...

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Abstract

The invention discloses a method for detecting the activity of alpha7nAChR of blood platelets and application of alpha7nAChR. The Ca2+ internal flow of a blood platelet alpha7nAChR positive ion channel is detected, and the adhesion and aggregation effects of alpha7nAChR on blood platelets are detected. Compared with alpha7nAChR in a non-activated state, selectively-activated alpha7nAChR can cause increase of the Ca2+ internal flow of blood platelets and induce increase of the adhesion rate and aggregation rate of blood platelets, and thus the activity of alpha7nAChR of blood platelets can be evaluated.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and in particular relates to a method for detecting platelet α7 cholinergic receptor (α7nAChR) 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. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/49G01N33/50
CPCG01N21/6428G01N33/49G01N33/50
Inventor 陈玲张宝峰张婷婷曹馨元缪冶炼
Owner NANJING MEDICAL UNIV
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