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Method for realizing pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus through near-infrared fluorescent probe

A fluorescent probe and near-infrared technology, applied in the field of biomedicine, can solve problems such as embolism and different pathophysiological processes, and achieve the effect of simple method and simplified experimental operation process.

Active Publication Date: 2021-08-10
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, coagulation factors often lead to fatal thromboembolism, and the pathophysiology of pulmonary embolism induced by them is different from PE secondary to venous thrombosis in humans

Method used

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  • Method for realizing pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus through near-infrared fluorescent probe
  • Method for realizing pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus through near-infrared fluorescent probe
  • Method for realizing pulmonary embolism modeling and non-invasive quantitative detection by labeling thrombus through near-infrared fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: ZnPc-(Lys) 5 Preparation and Characterization of Labeled Thromboembolic Microparticles

[0037] (1) ZnPc-(Lys) 5 Preparation of labeled thrombus microparticles: Blood was taken from the orbit of SD rats, anticoagulated with 3.2% sodium citrate (the volume ratio of anticoagulant to whole blood was 1:9), and the whole blood was separated by centrifugation at 1200 g for 10 min plasma. Take 500 μL plasma and add 2 μL ZnPc-(Lys) 5 (4.5 mM), 11.5 μL thrombin (10 U / mL) and 46.5 μL calcium chloride (240 mM), mix well and incubate in an oven at 37 °C for 2 h. ZnPc-(Lys) will be formed 5 The marked thrombus was placed in a mortar, cut into small pieces, resuspended in 1 mL Tyrodes-Hepes buffer (THB), and ground for 10 min to form thrombus particles.

[0038] (2) ZnPc-(Lys) 5 Characterization of labeled thrombus microparticles: randomly sample 100 μL of the prepared fluorescently labeled thrombus microparticles into a 96-well plate, and put them into a Spectra Ma...

Embodiment 2

[0040] Example 2: In vitro thrombolysis of unground thrombus and thrombus microparticles after grinding

[0041] (1) Thrombolysis of unground thrombus in vitro: blood was collected from the orbit of SD rats, anticoagulated with 3.2% sodium citrate (the volume ratio of anticoagulant to whole blood was 1:9), and 1200 g of whole blood Plasma was separated by centrifugation for 10 min. Add 30 μL of plasma to a 96-well plate, add 123.3 μL of Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and 6.7 μL of calcium chloride (240 mM) in sequence, mix well and store in an oven at 37 °C Incubate for 2 h. After thrombus formation, different concentrations of r-tPA were added for thrombolysis, and the absorption at 405 nm was recorded in a Spectra Max i3x microplate reader.

[0042] (2) Thrombolysis of the ground thrombus in vitro: Take out the unground thrombus formed above with microscopic tweezers, place it in a mortar, cut it into small pieces and add 0.2 mL Tyrodes-Hepes buffer (THB...

Embodiment 3

[0044] Example 3: Construction of mouse PE model and in vivo optical imaging

[0045] The prepared ZnPc-(Lys) 5 The labeled thrombus microparticles were injected into the ICR mice (200 μL / 20 g) through the tail vein. After the mice were anesthetized with isoflurane gas, the chest skin was depilated, and placed in a small animal in vivo imager (FMT2500 TM Real-time monitoring of mouse lung ZnPc-(Lys) in LX instrument, PerkinElmer) 5 fluorescent signal. The instrument uses a 680 nm laser diode to excite ZnPc-(Lys) 5 For molecules, mouse lungs were selected as Regions of interests (ROIs), and 50-60 source locations were scanned (the distance between adjacent scanning points was 3 mm). At the same time, the same concentration of free ZnPc-(Lys) 5 The solution was injected into mice as a control (free fluorescent probe set). For quantification of ZnPc-(Lys) 5 Concentration of 1 μM ZnPc-(Lys) 5 (dissolved in DMSO) was used as a standard to calibrate the FMT instrument. The ...

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Abstract

The invention relates to a near-infrared fluorescent probe molecule capable of being combined with thrombus emboli and application of the near-infrared fluorescent probe molecule in pulmonary embolism modeling and non-invasive quantitative detection. According to a method, rat plasma, thrombin, calcium chloride and near-infrared fluorescent probe molecules form traceable thrombus emboli in vitro, and the traceable thrombus emboli is cut into pieces, moderately ground to micron scale and then injected into a rat body through caudal vein. Thromboembolic particles can be accumulated in lung blood vessels of a rat to block the blood vessels to form embolism, construction of a rat pulmonary embolism model is achieved, non-invasive quantitative detection can be achieved with assistance of a small animal living body fluorescence imaging system, and the thromboembolic particles can be applied to evaluation of the thrombolysis effect of thrombolytic drugs and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a mouse pulmonary embolism model constructed by using near-infrared fluorescent probe-labeled thrombus particles and its application. Background technique [0002] Pulmonary embolism (PE) is a cardiovascular emergency caused by pulmonary circulation disorder caused by exogenous or endogenous emboli blocking the pulmonary artery or its branches, which seriously threatens human life and health. Right ventricular failure, circulatory shock, and cardiac arrest secondary to acute PE are responsible for its high mortality rate. In recent years, the number of PE patients in my country has been increasing year by year. If diagnosed early and treated in time, the fatality rate of acute PE patients can be reduced from 30% to 8%. The treatment options for acute PE include anticoagulant therapy, thrombolytic therapy, interventional therapy, and surgical therapy. Thrombolytic therapy is a drug t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02
CPCA01K67/02
Inventor 黄明东陈丹袁彩江龙光徐芃刘玉蓉
Owner FUZHOU UNIV
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