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miRNA regulation of platelet gelsolin and its application in screening antiplatelet drugs

An anti-platelet, drug candidate technology, applied in the direction of drug combination, microbial determination/inspection, medical preparations containing active ingredients, etc., can solve problems such as inability to express the same genus and prolonged bleeding time

Active Publication Date: 2021-04-27
XIYUAN HOSPITAL OF CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Promegakaryocytes can express gelsolin Gelsolin, but cannot express Adseverin and capping protein G (capG), which belong to the Gelsolin superfamily. With the expression of Gelsolin down, the bleeding time of Gelsolin protein knockout mice is longer than normal after injury, which indicates that Gelsolin may inhibit the process of promegakaryocyte differentiation into platelets

Method used

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  • miRNA regulation of platelet gelsolin and its application in screening antiplatelet drugs
  • miRNA regulation of platelet gelsolin and its application in screening antiplatelet drugs
  • miRNA regulation of platelet gelsolin and its application in screening antiplatelet drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Culture and Induced Maturation of Human MEG-01 Megakaryocytes

[0025] 1. Experimental grouping: 1.MEG-01 group: MEG-01 cells were cultured with RMPI-1640+10%FBS+4mmol / L L-glutamine culture medium; 2.MEG-01 / TPA group: MEG-01 cells were cultured with The culture solution of RMPI-1640+10%FBS+4mmol / L L-glutamine was kept at 37℃, 5%CO 2 Conditioned suspension culture, the initial concentration of each well of 6-well plate was 5×10 5 cell. The final concentration of TPA was 10nM to induce the differentiation of MEG-01 cells for 3 days. Cell images (200×) were collected with an inverted Olympus microscope.

[0026] 2. FACS surface staining

[0027] Aspirate 500 μL of MEG-01 cells induced for 3 days and the cells of the blank control group, first wash the two groups of cells with PBS (staining solution) containing 5% serum for 1-2 times, centrifuge at 500 g for 5 min, and adjust the cell concentration to 1×10 6 / mL. Add 2 μL anti-human Fc blocking antibody pe...

Embodiment 2

[0032] Example 2: Research on the expression levels of miRNA and Gelsolin protein in MEG-01 and MEG-01 / TPA cells

[0033] 1. Detection of miRNA expression level

[0034] Take 1×10 7 Quantities of MEGA-01 and MEGA-01 / TPA cells in the exponential growth phase were used to extract the RNA of these two cells by Trizol.

[0035] For the reverse transcription of miRNA, each miRNA needs to use its specific reverse transcription primer, as shown in Table 1. The reverse transcription method is as follows: Take a certain amount of RNA (0.1ng-5μg), add 1μL oligo miRNA-specific reverse primer to an RNase free tube, and add a certain amount of DEPC water to make the final volume 12μL. Place the tube in warm water at 65°C for 5 minutes. After the incubation period, quickly place the tubes on ice. Add 4μL 5×reaction buffer, 2μL 10mM dNTP mix, 1μL RNase inhibitor and 1μL reverse transcriptase to the tube, mix gently, place in a 42℃ water bath for 1 hour, then place in a 70℃ water bath in...

Embodiment 3

[0060] Example 3 Verification that miR-124 directly regulates the expression of Gelsolin

[0061] 1. Construction of 3'-UTR-containing vectors and mutant 3'-UTR-containing vectors

[0062] (1) Enzyme cut empty plasmid

[0063] 2 μg pmirGLO empty plasmid was double digested with PmeI and XbaI, the enzyme digestion system is as follows:

[0064] Table 4 enzyme digestion reaction system

[0065]

[0066]

[0067] Digest overnight at 37°C, then add 4 μL of 5M NaCl and 100 μL of ice-free ethanol to the EP tube after digestion, and mix well. Place in -80°C refrigerator for 30 minutes, centrifuge at 12,000 rpm for 15 minutes, quickly wash twice with 75% ice-free ethanol, and blow dry. Finally with 10 μL ddH 2 O dissolved.

[0068] (2) Synthesize the target DNA fragment

[0069] The following fragments were synthesized (sequences in bold in italics are miRNA binding sequences or binding mutation sequences, and the underlined sequences are NotI enzyme-cut recognition sequen...

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Abstract

The present invention relates to the application of miRNA in regulating the expression level of Gelsolin and its application in antiplatelet therapy, and the miRNA is miR‑124. It also relates to a method for screening potential anti-platelet therapeutic drugs by screening agents that regulate the above-mentioned miRNA in platelets. Screening experiments showed that ligustrazine, panax notoginseng saponins and aspirin could regulate the level of miR‑124 in platelet cells, thus playing a role in antiplatelet therapy.

Description

technical field [0001] The invention belongs to the field of platelet regulation, and specifically relates to the application of miRNA in regulating platelet gelsolin Gelsolin and screening drugs applied to platelet-related diseases, especially blood stasis syndrome of coronary heart disease. Background technique [0002] The occurrence and development of coronary heart disease are closely related to a variety of factors, and platelet activation plays a key role in it. Atherosclerotic plaque damages coronary arteries and activates platelets through various ways, leading to platelet adhesion and aggregation, forming platelet-rich plaques. Thrombosis, which completely or partially blocks a coronary artery, contributes to coronary heart disease. Blood stasis syndrome is the most common TCM syndrome of coronary heart disease. Previous studies have shown that it is closely related to various pathophysiological changes such as blood circulation disorder, high blood viscosity, plat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883A61K45/00A61P7/02
CPCA61K45/00C12Q1/6883C12Q2600/136C12Q2600/158C12Q2600/178
Inventor 蒋跃绒殷惠军刘玥贾敏缪宇杨琳
Owner XIYUAN HOSPITAL OF CHINA ACAD OF CHINESE MEDICAL SCI
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