CN113229213B - Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe - Google Patents
Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe Download PDFInfo
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Abstract
The invention relates to a near-infrared fluorescent probe molecule capable of combining with thromboembolism and application thereof in pulmonary embolism modeling and noninvasive quantitative detection. The method comprises the steps of forming traceable thrombus emboli in vitro by rat plasma, thrombin, calcium chloride and near-infrared fluorescent probe molecules, shearing the thrombus emboli, appropriately grinding the thrombus emboli to a micrometer scale, and injecting the thrombus emboli into a mouse body through tail veins. The thromboembolus particle can be accumulated in a mouse lung blood vessel to further block the blood vessel to form embolism, so that a mouse pulmonary embolism model is constructed, noninvasive quantitative detection can be realized by the aid of a small animal living body fluorescence imaging system, and the thromboembolus particle can be applied to aspects such as thrombolysis effect evaluation of thrombolytics.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a mouse pulmonary embolism model constructed by using a thromboembolus particle marked by a near-infrared fluorescent probe and application thereof.
Background
Pulmonary Embolism (PE) is a cardiovascular emergency caused by the blockage of Pulmonary arteries or their branches by exogenous or endogenous emboli, and seriously threatens the life and health of humans. Right ventricular failure, circulatory shock and cardiac arrest secondary to acute PE are the causes of their high mortality. In recent years, the number of PE patients in China tends to increase year by year. If the early diagnosis and timely treatment can be realized, the fatality rate of patients with acute PE can be reduced from 30% to 8%. Treatment regimens for acute PE include anticoagulation, thrombolytic therapy, interventional therapy, surgical therapy, and the like. The thrombolytic therapy is that the medicine directly or indirectly converts plasma protein plasminogen in a non-activated state into activated plasmin, the plasmin acts on fibrin clot to cause the fibrin clot to be dissolved, and simultaneously, coagulation factors II, V and VIII are removed and inactivated to interfere coagulation, thereby playing an anticoagulation role. In recent years, numerous researchers have been working on the development of novel thrombolytic drugs, and several convincing findings have provided evidence-based medical evidence for acute PE thrombolytic therapy. Therefore, there is a need to develop a simple, effective and reproducible standardized pulmonary embolism model for further research of the molecular mechanism of pulmonary embolism and evaluation of the thrombolytic efficiency of different thrombolytic treatment regimens to reduce the bleeding complications of thrombolytic drugs and benefit patients.
At present, several mouse PE models are constructed for simulating pulmonary obstructive thromboembolism of human body, such as a blood coagulation factor-induced mouse PE model, a photochemical reaction-induced mouse PE model and an exogenous thrombus injection-induced mouse PE model. The mouse PE model induced by the blood coagulation factor has the advantages of simple and convenient operation, low price and the like, and is widely applied to detecting the in-vivo antithrombotic effect of the compound. However, coagulation factors often lead to fatal thromboembolism, and the pathophysiological processes that induce the formation of pulmonary embolism are different from PE in humans secondary to venous thrombosis. The mouse PE model induced by photochemical reaction utilizes rose bengal B to generate singlet oxygen free radicals under the irradiation of green light, so that vascular endothelium is damaged, blood platelets are adhered, the blood coagulation process is stimulated, and thrombus in blood vessels in an irradiation area is formed. The PE model established by the method is closer to the PE generated under human pathophysiological conditions, but the formed thrombus is mostly seen in pulmonary microvasculature and is rare in pulmonary macrovascular. The mouse PE model induced by exogenous thrombus injection is characterized in that blood clots prepared in vitro are injected into a mouse body through a tail vein or a jugular vein, microemboli or thrombus embolic particles are preferentially retained in a lung and are distributed uniformly, and the microemboli or thrombus embolic particles can be spontaneously and slowly dissolved under physiological conditions without lethality.
The rapid development of in vivo imaging technology for living animals has greatly promoted disease diagnosis and treatment and drug activity evaluation at the living level, and has also been used for evaluation of thrombolytic drugs. In vivo imaging techniques for living animals are mainly classified into optical imaging (optical imaging), nuclear-nuclear imaging (radio-nuclear imaging), Magnetic Resonance Imaging (MRI), ultrasonic imaging (ultrasound imaging), and Computed Tomography (CT). With the improvement of spatial and temporal resolution of multi-slice helical CT, CT pulmonary artery imaging (CTPA) has replaced angiographic examination and is the gold standard for clinical diagnosis of PE. The optical imaging has the unique advantages of simple and convenient operation, visual result, quick measurement, high sensitivity, low cost and the like, can be repeated for many times, monitors for a long time and the like, and has more advantages in drug research and screening.
Disclosure of Invention
The invention aims to establish a mouse PE model induced by exogenous fluorescence labeled thrombus, and the mouse PE model can be used for evaluating the thrombolytic effect of a medicament by tracing the fluorescence labeled thrombus by optical imaging.
Pulmonary Thromboembolism (PTE) is the most common type of acute PE, resulting from the occlusion of the pulmonary artery or its branches by thrombi from the venous system or right heart, with pulmonary circulation and respiratory dysfunction as the main pathophysiological features and clinical manifestations, accounting for the vast majority of acute PE, as well as the most severe clinical manifestations of Venous Thromboembolism (VTE), and in many cases secondary to Deep Venous Thrombosis (DVT).
In order to better simulate the natural occurrence and development process of PTE, the animal model is mainly established by an in vitro injection embolus particle method, in particular to an in vitro prepared blood clot embolus particle which is injected into a vein and embedded in a pulmonary artery through blood circulation to form a PE model. When the constructed PE model is used for evaluating the thrombolytic effect of the thrombolytic drug, in order to reduce the number of animals used and realize real-time monitoring of the thrombolytic process, the embolic particles prepared in vitro need to have traceable characteristics.
The invention is realized by the following technical scheme: a near-infrared fluorescent probe molecule able to bind thrombus is pentapolylysine beta-carbonyl zinc phthalocyanine (ZnPc- (Lys)5) The structure of the compound is shown in figure 1, wherein the penta-polylysine modification increases the hydrophilicity of the compound molecule, enables the compound molecule to have positive charge, and can be combined with negative charge components in thrombus embolus to target thrombus.
Further, the present invention provides a tracinable thromboembolic particle having a particle size of from about 1 to about 5 microns. The preparation method of the thrombus embolic particles comprises the following steps: the fluorescent probe penta-lysine beta-carbonyl zinc phthalocyanine is added into rat plasma, thrombin and calcium chloride are used as coagulants, the formed fluorescence labeled thrombus is cut into pieces, ground to a particle size by a mortar, and suspended in physiological saline. The traceable thrombus embolus particles formed by grinding mainly comprise highly crosslinked fibrin and platelets, and have compact embolus structures, so that the embolus cannot be easily destroyed by an animal fibrinolysis system in vivo to cause embolus autolysis.
Furthermore, the invention provides a novel mouse PE model construction method, which is characterized in that the fluorescent tracer thrombus particles formed in vitro are injected into a mouse body through tail veins, and accumulate in pulmonary arteries through blood circulation to cause pulmonary artery blockage. This process approaches the natural occurrence and development of PTE under pathophysiological conditions.
Furthermore, the invention provides a strategy for evaluating the thrombolytic effect of the thrombolytic drug, which is characterized in that signals of a near-infrared fluorescent probe in the lung of a mouse represent the accumulation and blockage levels of thrombus embolic particles in blood vessels in the lung of the mouse, so that the thrombolytic process can be represented and the thrombolytic drug effect can be evaluated by quantifying the concentration of the fluorescent probe in the lung of the mouse in real time.
Compared with the existing small animal PE model which can be used for evaluating and screening thrombolytic drugs, the invention has the innovation and special points that:
(1) the particle size of the thrombus embolic particle prepared in vitro is about 1-5 microns, the surface is charged negatively and can be charged positively ZnPc- (Lys)5The target label can generate stronger fluorescent signals under the excitation of a 680 nm light source. The mouse modeling method is simple and convenient, is close to the generation and development process of PTE in vivo disease physiological state, and can carry out noninvasive real-time quantitative monitoring subsequently through FMT.
(2) After tail vein injection, the thrombus particles can be uniformly distributed in the pulmonary artery of a mouse, are not easily dissolved by the fibrinolytic system of the mouse, can be stably accumulated in the pulmonary artery for more than 6 h, and provide a long enough time window for subsequent evaluation of the thrombolytic drug effect.
(3) The mouse PE model constructed by the invention can be used for in vivo evaluation and screening of novel thrombolytic agents, and has the advantages of high efficiency, reliability, low price and the like.
In conclusion, the invention develops a method for constructing the mouse PTE model by injecting the thrombus embolus particles marked by the near-infrared fluorescent probe into the tail vein, greatly simplifies the experimental operation process of mouse PTE modeling, and has small variability of animal experimental data and no death. In addition, the application of live 3D real-time quantitative imaging greatly reduces the number of small animals required to evaluate thrombolytic effects, and may facilitate current research on PE intervention.
Drawings
FIG. 1 shows ZnPc- (Lys)5The chemical structure of (a);
FIG. 2 shows ZnPc- (Lys)5Preparing and characterizing labeled thrombus embolic particles; wherein:
(A) in vitro formed ZnPc- (Lys)5A picture of labeled thrombi;
（B）ZnPc-(Lys)5the particle size distribution of the labeled thromboembolic microparticles;
（C）ZnPc-(Lys)5the surface zeta potential of the labeled thrombo-embolic particles;
（D）ZnPc-(Lys)5an ultraviolet-visible absorption spectrum of the labeled thromboembolic microparticles;
（E）ZnPc-(Lys)5a fluorescence spectrum of the labeled thromboembolic microparticles;
FIG. 3 is an in vitro thrombolysis of thromboembolism and embolic particles; wherein:
(A) thrombolysis results of unground thrombus emboli treated by r-tPA with different concentrations;
(B) the average particle size change of the main particles of the grinded thrombus particles after being treated by 200 nM r-tPA;
FIG. 4 is the construction of mouse PE model and FMT characterization; wherein:
(A) free fluorescent probe group ZnPc- (Lys)5Accumulation in mouse lung, distribution of 3D imaging results;
(B) PE model group ZnPc- (Lys)5Accumulation in mouse lung, distribution of 3D imaging results;
(C) quantitative free fluorescent probe group and ZnPc- (Lys) in PE model group5Concentration in the mouse lung;
FIG. 5 is an evaluation of the thrombolytic effect of r-tPA; wherein:
(A) physiological saline treatment group and r-tPA treatment group ZnPc- (Lys)5Accumulation in mouse lung, distribution of 3D imaging results;
(B) quantitative physiological saline treatment group and r-tPA treatment group ZnPc- (Lys)5Concentration in mouse lung (. times.P < 0.0001）;
FIG. 6 shows the 2D imaging results of isolated lung tissues of mice of different experimental groups;
FIG. 7 shows H & E staining results of ex vivo lung tissue sections of mice of different experimental groups (magnification 10X, scale bar: 300 μm).
Detailed Description
Example 1: ZnPc- (Lys)5Preparation and characterization of labeled thromboembolic microparticles
（1）ZnPc-(Lys)5Preparation of labeled thromboembolic microparticles: SD rat orbital blood is collected, anticoagulated with 3.2% sodium citrate (volume ratio of anticoagulant to whole blood is 1: 9), whole blood is centrifuged at 1200 g for 10 min, and plasma is separated. Taking 500. mu.L of plasma, adding 2. mu.L of ZnPc- (Lys)5(4.5 mM), 11.5. mu.L thrombin (10U/mL) and 46.5. mu.L calcium chloride (240 mM) were mixed and incubated in an oven at 37 ℃ for 2 h. ZnPc- (Lys) to be formed5The labeled thromboembolism was placed in a mortar, cut into small pieces and resuspended by adding 1 mL of Tyrodes-Hepes buffer (THB), and ground for 10 min to form thromboembolic microparticles.
（2）ZnPc-(Lys)5Characterization of labeled thromboembolic microparticles: 100 mu L of prepared fluorescence labeled thrombus particles are randomly sampled and placed in a 96-well plate, and the plate is placed in a Spectra Max i3x enzyme-linked analyzer to scan the ultraviolet-visible absorption spectrum (400-. Additional random sampling of fluorescently labeled blood1 mL of embolic particles was centrifuged at 13000 rpm for 10 min and resuspended in deionized water, and the particle size distribution and surface zeta potential were measured by dynamic light scattering (Zetasizer Nano-ZS, Malvern, PA, USA) with a fixed scattering angle of 90 degrees and an equilibration time of 90 s and the measurement was repeated 3 times.
The results are shown in FIG. 2, ZnPc- (Lys)5The particle size distribution of the labeled thromboembolus particles showed 3 peaks, in which 80% of the particles had an average particle size of 1.2. mu.m, 16% of the particles had an average particle size of 5 μm, and 4% of the particles had an average particle size of 100 nm. The surface of the unlabeled thrombus embolus particle is charged with strong negative electricity and positive electricity ZnPc- (Lys)5The negative charge of the surface portion is neutralized after marking. ZnPc- (Lys)5The labeled thrombo-embolic particles show a characteristic absorption at 680 nm and a strong fluorescence signal in the near infrared region. However, ZnPc- (Lys) dissolved in THB buffer5Due to aggregation, its maximum absorption is at 630 nm and no fluorescence in the near infrared region.
Example 2: non-ground thrombus embolus and ground thrombus embolus particle external thrombolysis
(1) In vitro thrombolysis of unmilled thromboemboli: SD rat orbital blood is collected, anticoagulated with 3.2% sodium citrate (volume ratio of anticoagulant to whole blood is 1: 9), whole blood is centrifuged at 1200 g for 10 min, and plasma is separated. mu.L of plasma was added to a 96-well plate, 123.3. mu.L of Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and 6.7. mu.L of calcium chloride (240 mM) were added in this order, mixed well and incubated in an oven at 37 ℃ for 2 h. After the thrombus embolus is formed, r-tPA with different concentrations is added for thrombolysis, and the thrombolysis is put into a Spectra Max i3x microplate reader to record the absorption at 405 nm.
(2) In vitro thrombolysis of ground thromboemboli: the unground thromboembolism formed above was removed with a pair of tweezers and placed in a mortar, cut into small pieces and resuspended in 0.2 mL of Tyrodes-Hepes buffer (THB), ground for 10 min to form thromboembolus microparticles. The particle size distribution was determined by dynamic light scattering (Zetasizer Nano-ZS, Malvern, PA, USA). Adding r-tPA with the final concentration of 200 nM for thrombolysis and detecting the change of the particle size of the thrombus particles at different time points.
The results are shown in FIG. 3, where the absorbance at 405 nm of the unground thromboembolism remained unchanged without r-tPA treatment. The thrombolysis of the r-tPA is dose-dependent, the 10 nM r-tPA and the thrombus embolus can only partially dissolve the thrombus embolus after being incubated for 2 h, the 50 nM r-tPA and the thrombus embolus can completely dissolve after being incubated for 100 min, the 100 nM r-tPA and the thrombus embolus can completely dissolve after being incubated for 60 min, and the 200 nM r-tPA and the thrombus embolus can completely dissolve after being incubated for 50 min. For the milled thromboembolic particles, 80% of which had an average particle size of 1.2 microns, incubated with r-tPA at a final concentration of 200 nM, the average particle size of the thromboembolic particles was gradually reduced over time to about 700 nM after 6 h of incubation. The results show that the thrombus embolus particles formed by grinding have compact structures and are not easy to be completely dissolved.
Example 3: mouse PE model construction and living body optical imaging
Preparing ZnPc- (Lys)5Labeled thromboembolus particles were injected via tail vein into ICR mice (200. mu.L/20 g) which were subjected to chest skin dehairing after isoflurane gas anesthesia and placed in a small animal in vivo imager (FMT 2500)TMLX Instrument, Perkinelmer) for real-time monitoring of ZnPc- (Lys) in mouse lung5A fluorescent signal. The instrument adopts a 680 nm laser diode to excite ZnPc- (Lys)5Molecular, mouse lungs were selected as Regions of interests (ROIs) and 50-60 source locations were scanned (adjacent scan points 3 mm apart). At the same time, the same concentration of free ZnPc- (Lys)5The solution was injected into mice as a control (free fluorescent probe set). To quantify ZnPc- (Lys)5At a concentration of 1. mu.M ZnPc- (Lys)5FMT instruments were calibrated (dissolved in DMSO) as standards. And (3) carrying out three-dimensional reconstruction on the recorded image through TrueQuant v3.0 software, and obtaining a quantitative result.
The results are shown in FIG. 4, which shows the formation of free ZnPc- (Lys)5And ZnPc- (Lys)5The marked thrombus embolus particles are respectively injected into mice through tail veins (respectively a free fluorescence probe group and a PE model group), and the FMT imaging result shows that ZnPc- (Lys)5Labelled bloodThe embolus particles are rapidly gathered in the lung, the maximum concentration is reached in the lung after 1 h of injection, and then the concentration in the lung is gradually reduced; and free ZnPc- (Lys) as a control5Only a small amount is ingested into the lung tissue, free ZnPc- (Lys) at each time point5The concentration in the lung is obviously lower than that of ZnPc- (Lys) in the PE model group5Concentration of (A), (B) toP <0.001), indicating that the fluorescence signals of the lungs displayed by the PE model group through FMT imaging are mainly caused by aggregation of thrombo-embolic particles in the pulmonary arteries.
Example 4: application of mouse PE model in evaluation of r-tPA thrombolytic effect
Injecting the intravenous ZnPc- (Lys)5The PE model mice constructed by the marked thrombus plug particles are randomly divided into two groups (12 mice in each group), after 10 min of thrombus plug particle injection, physiological saline or r-tPA (10 mg/kg) is injected into the vein, and the lung ZnPc- (Lys) of the mice is monitored at 30 min, 1 h, 3 h and 6 h respectively5Fluorescent signal, expressed as ZnPc- (Lys)5The concentration of (2) represents accumulation and blockage conditions of the thrombus embolic particles in the lung, and the thrombolytic effect of r-tPA is evaluated.
The results are shown in fig. 5, and FMT imaging results show that lung accumulation is significantly reduced at 1 h, 3 h, 6 h in r-tPA-treated mice compared to saline controls (P <0.001), indicating that r-tPA produced a thrombolytic effect.
Example 5: ex vivo tissue 2D imaging and tissue section staining verification mouse PE model applicability to thrombolytic agent screening
At each time point (30 min, 1 h, 3 h and 6 h), 3 mice in each group (free fluorescence probe group, normal saline treatment group and r-tPA treatment group) were each subjected to cervical dislocation and sacrificed, lung tissue specimens were taken out after dissection, washed with normal saline, drained and placed in an FMT instrument for 2D imaging. Then, lung tissue was fixed in 4% paraformaldehyde, and after continuous dehydration and paraffin embedding, the sections were sectioned, dewaxed, rehydrated, and stained with hematoxylin and eosin.
The result is shown in fig. 6, and 2D imaging results of lung tissues of mice in each group show that the fluorescence of the lung of the mice with free probe group is weak at each time point, the fluorescence of the lung of the mice with PE model group is strong, especially the fluorescence intensity of the lung of the mice with PE model group is obviously stronger than that of the mice with r-tPA treatment group after 1 h and 3 h of molding, and the fluorescence intensity of the lung of the mice with r-tPA treatment group is rapidly weakened after 1 h.
The staining result of the lung tissue section is shown in figure 7, obvious thromboembolism can be seen in lung blood vessels of mice of a normal saline treatment group and a r-tPA treatment group within 1 hour, emboli in the lung blood vessels of the mice of the r-tPA treatment group can be dissolved within 3 hours, and the emboli in the lung blood vessels of the mice of the r-tPA treatment group within 6 hours are obviously reduced compared with the control mice of the normal saline control group. Further verifies that the PE model constructed by the invention and the evaluation platform based on optical imaging are suitable for developing and screening novel thrombolytic agents.
The above-mentioned embodiments are further detailed descriptions of the objects, technical solutions and advantageous effects of the present invention. It should be understood that the above-mentioned embodiments are only exemplary of the present invention, and are not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. An exogenously traceable thromboembolic microparticle, characterized by: labeling thrombus embolus by utilizing near-infrared fluorescent probe molecules, wherein the particle size of thrombus embolus particles is 1-5 microns; the near-infrared fluorescent probe molecule is penta-polylysine beta-carbonyl zinc phthalocyanine, and the structural formula is as follows:
the preparation method of the thrombus embolic particles comprises the following steps: a fluorescence probe, namely pentalysine beta-carbonyl zinc phthalocyanine is added into rat plasma, thrombin and calcium chloride are used as coagulants, formed fluorescence-labeled thrombus is cut into pieces, ground by a mortar and suspended in physiological saline.
2. A noninvasive quantitative detection method of a mouse pulmonary embolism model is characterized by comprising the following steps: carrying out living body imaging of the small animals by fluorescent molecule tomography, exciting near-infrared fluorescent probe molecules by using a laser diode with the wavelength of 680 nm, and obtaining a quantitative result of the probe molecules by three-dimensional reconstruction; the mouse pulmonary embolism model is characterized in that exogenous traceable thromboembolism particles are injected into a mouse body through tail veins to accumulate and block blood vessels in the mouse lung to cause pulmonary embolism.
3. The application of the method for non-invasive quantitative detection of the mouse pulmonary embolism model according to claim 2 in evaluating the thrombolytic effect of a thrombolytic drug, wherein the method comprises the following steps: after the thrombolytic drug is given, the accumulation condition of the fluorescent probe in the lung of the mouse is monitored in real time through a small animal living body imaging system and quantified, the concentration of the fluorescent probe in the lung reflects the dissolution degree of the thrombolytic particles, and the lower the concentration of the fluorescent probe is, the better the thrombolytic effect of the thrombolytic drug is.
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