CN113229213A - 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
- Publication number
- CN113229213A CN113229213A CN202110524861.XA CN202110524861A CN113229213A CN 113229213 A CN113229213 A CN 113229213A CN 202110524861 A CN202110524861 A CN 202110524861A CN 113229213 A CN113229213 A CN 113229213A
- Authority
- CN
- China
- Prior art keywords
- thrombus
- mouse
- fluorescent probe
- pulmonary embolism
- thrombolytic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 53
- 208000010378 Pulmonary Embolism Diseases 0.000 title claims abstract description 45
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 title claims abstract description 6
- 239000002245 particle Substances 0.000 claims abstract description 62
- 210000004072 lung Anatomy 0.000 claims abstract description 36
- 208000005189 Embolism Diseases 0.000 claims abstract description 34
- 230000002537 thrombolytic effect Effects 0.000 claims abstract description 30
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- 210000003462 vein Anatomy 0.000 claims abstract description 10
- 210000004204 blood vessel Anatomy 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000001110 calcium chloride Substances 0.000 claims abstract description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 5
- 108090000190 Thrombin Proteins 0.000 claims abstract description 4
- 229960004072 thrombin Drugs 0.000 claims abstract description 4
- 230000009424 thromboembolic effect Effects 0.000 claims description 16
- 239000003527 fibrinolytic agent Substances 0.000 claims description 14
- 238000003384 imaging method Methods 0.000 claims description 14
- 239000011859 microparticle Substances 0.000 claims description 9
- 239000003146 anticoagulant agent Substances 0.000 claims description 8
- 238000009825 accumulation Methods 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- 239000004570 mortar (masonry) Substances 0.000 claims description 4
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000011701 zinc Substances 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- IXPHOHNWDLRFJH-UHFFFAOYSA-N 6-amino-2-[[6-amino-2-[[6-amino-2-[[6-amino-2-(2,6-diaminohexanoylamino)hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid Chemical compound NCCCCC(N)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(O)=O IXPHOHNWDLRFJH-UHFFFAOYSA-N 0.000 claims description 2
- 239000000701 coagulant Substances 0.000 claims description 2
- 108010091617 pentalysine Proteins 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 238000003325 tomography Methods 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 11
- 208000001435 Thromboembolism Diseases 0.000 abstract description 8
- 238000011156 evaluation Methods 0.000 abstract description 8
- 229960000103 thrombolytic agent Drugs 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000000227 grinding Methods 0.000 abstract description 3
- 238000000799 fluorescence microscopy Methods 0.000 abstract 1
- 238000010008 shearing Methods 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 238000011282 treatment Methods 0.000 description 16
- 101001095260 Mus musculus Prolyl endopeptidase Proteins 0.000 description 15
- 210000001147 pulmonary artery Anatomy 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 230000001154 acute effect Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 230000003073 embolic effect Effects 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 238000012634 optical imaging Methods 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 4
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 4
- 239000003114 blood coagulation factor Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000002591 computed tomography Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 3
- 108010073385 Fibrin Proteins 0.000 description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 3
- 206010047249 Venous thrombosis Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 229950003499 fibrin Drugs 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 206010051055 Deep vein thrombosis Diseases 0.000 description 2
- 206010014522 Embolism venous Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 229940019700 blood coagulation factors Drugs 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000012633 nuclear imaging Methods 0.000 description 2
- 230000004963 pathophysiological condition Effects 0.000 description 2
- 238000006552 photochemical reaction Methods 0.000 description 2
- 229940012957 plasmin Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 208000004043 venous thromboembolism Diseases 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000010496 Heart Arrest Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 206010039163 Right ventricular failure Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000005796 circulatory shock Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- VQHHOXOLUXRQFQ-UHFFFAOYSA-L dipotassium;4,5,6,7-tetrachloro-2',4',5',7'-tetraiodo-3-oxospiro[2-benzofuran-1,9'-xanthene]-3',6'-diolate Chemical compound [K+].[K+].O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC(I)=C([O-])C(I)=C1OC1=C(I)C([O-])=C(I)C=C21 VQHHOXOLUXRQFQ-UHFFFAOYSA-L 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 238000011155 quantitative monitoring Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000001454 recorded image Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012285 ultrasound imaging Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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, it is urgently needed to develop a simple, effective and reproducible standardized pulmonary embolism model for deeply researching the pathogenesis molecular mechanism of pulmonary embolism and evaluating the thrombolysis efficiency of different thrombolysis treatment schemes so as to reduce the bleeding complications of thrombolysis 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 the signal of the near-infrared fluorescent probe in the lung of the mouse represents the accumulation and blockage level of thrombus particles in the blood vessel of 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 diameter of the thrombus embolus particle prepared in vitro is about 1-5 microns, the surface is charged with negative electricity and can be positively charged 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 marked thrombus embolus 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 the H & E staining results of the isolated lung tissue sections of mice from 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-. Additionally, 1 mL of fluorescently labeled thrombo-embolic particles were randomly sampled, centrifuged at 13000 rpm for 10 min, resuspended in deionized water, and their particle size distribution and surface zeta potential were measured by dynamic light scattering (Zetasizer Nano-ZS, Malvern, Pa., USA) at a fixed scattering angle of 90 degrees for 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 hours. 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 selection of mouse Lung as Regions of interests (ROIs)Scanning 50-60 source positions (adjacent scanning points are 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)5Labeled thrombus particles are rapidly gathered in the lung, and the highest concentration is reached in the lung after 1 h of injection, and then the concentration of the labeled thrombus particles 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 (6)
2. an exogenous traceable thrombus embolus particle, which is characterized in that the thrombus embolus is marked by the near-infrared fluorescent probe molecule of claim 1, and the particle size of the thrombus embolus particle is 1-5 microns.
3. A method of preparing the exogenously traceable thromboembolic microparticle of claim 2, comprising the steps of: 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.
4. A mouse pulmonary embolism model, wherein the exogenous traceable thromboembolic particles of claim 2 are injected into a mouse body via the tail vein, so that the particles accumulate in blood vessels of the mouse lung and block the blood vessels, thereby causing pulmonary embolism.
5. A method for non-invasive quantitative detection of pulmonary embolism model of mouse as claimed in claim 4, wherein the small animal living body is imaged by fluorescent molecular tomography, a 680 nm laser diode is used to excite the near infrared fluorescent probe molecule, and the quantitative result of the probe molecule is obtained by three-dimensional reconstruction.
6. The application of the method for non-invasive quantitative detection of pulmonary embolism model of mouse as claimed in claim 5 in evaluating the thrombolytic effect of thrombolytic drug, wherein the accumulation of fluorescent probe in mouse lung is monitored and quantified in real time by a small animal living body imaging system after administration of thrombolytic drug, the concentration of fluorescent probe in lung reflects the dissolution degree of thrombolytic particles, and the lower the concentration of fluorescent probe, the better the thrombolytic effect of thrombolytic drug is.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110524861.XA CN113229213B (en) | 2021-05-14 | 2021-05-14 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110524861.XA CN113229213B (en) | 2021-05-14 | 2021-05-14 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113229213A true CN113229213A (en) | 2021-08-10 |
CN113229213B CN113229213B (en) | 2022-06-14 |
Family
ID=77134240
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110524861.XA Active CN113229213B (en) | 2021-05-14 | 2021-05-14 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113229213B (en) |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030190653A1 (en) * | 2002-01-11 | 2003-10-09 | Mehrdad Shamloo | Regulated gene in the pathophysiology of ischemic stroke |
US20050120392A1 (en) * | 2002-02-28 | 2005-06-02 | Rubinstein Amy L. | Transgenic zebrafish models for thrombosis |
US20100268070A1 (en) * | 2008-11-26 | 2010-10-21 | Visen Medical, Inc. | Methods and Compositions for Identifying Subjects at Risk of Developing Stent Thrombosis |
CN104013977A (en) * | 2014-06-06 | 2014-09-03 | 复旦大学附属中山医院 | Fibrous protein targeted multi-modal nano particles for micro-thrombus detection and application thereof |
CN105308184A (en) * | 2013-04-16 | 2016-02-03 | 瑞泽恩制药公司 | Targeted modification of rat genome |
CN106798934A (en) * | 2017-03-03 | 2017-06-06 | 南开大学 | A kind of PDT assisted surgery treats the test method of mouse mastopathy cell |
WO2017212298A1 (en) * | 2016-06-10 | 2017-12-14 | Edinburgh Molecular Imaging Limited | Imaging agents and methods of use |
CN107760660A (en) * | 2017-12-13 | 2018-03-06 | 福州大学 | A kind of tissue-type plasminogen activator mutant and its application |
CN110628751A (en) * | 2019-10-31 | 2019-12-31 | 福州大学 | Mutant design and application of reteplase |
CN111657861A (en) * | 2020-06-04 | 2020-09-15 | 浙江大学 | Thrombolytic drug effect evaluation method based on two-photon microscope technology |
CN112274510A (en) * | 2020-11-23 | 2021-01-29 | 福州大学 | Application of montelukast in preparation of medicines for preventing and treating thrombotic diseases |
-
2021
- 2021-05-14 CN CN202110524861.XA patent/CN113229213B/en active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030190653A1 (en) * | 2002-01-11 | 2003-10-09 | Mehrdad Shamloo | Regulated gene in the pathophysiology of ischemic stroke |
US20050120392A1 (en) * | 2002-02-28 | 2005-06-02 | Rubinstein Amy L. | Transgenic zebrafish models for thrombosis |
US20100268070A1 (en) * | 2008-11-26 | 2010-10-21 | Visen Medical, Inc. | Methods and Compositions for Identifying Subjects at Risk of Developing Stent Thrombosis |
CN105308184A (en) * | 2013-04-16 | 2016-02-03 | 瑞泽恩制药公司 | Targeted modification of rat genome |
CN104013977A (en) * | 2014-06-06 | 2014-09-03 | 复旦大学附属中山医院 | Fibrous protein targeted multi-modal nano particles for micro-thrombus detection and application thereof |
WO2017212298A1 (en) * | 2016-06-10 | 2017-12-14 | Edinburgh Molecular Imaging Limited | Imaging agents and methods of use |
CN106798934A (en) * | 2017-03-03 | 2017-06-06 | 南开大学 | A kind of PDT assisted surgery treats the test method of mouse mastopathy cell |
CN107760660A (en) * | 2017-12-13 | 2018-03-06 | 福州大学 | A kind of tissue-type plasminogen activator mutant and its application |
CN110628751A (en) * | 2019-10-31 | 2019-12-31 | 福州大学 | Mutant design and application of reteplase |
CN111657861A (en) * | 2020-06-04 | 2020-09-15 | 浙江大学 | Thrombolytic drug effect evaluation method based on two-photon microscope technology |
CN112274510A (en) * | 2020-11-23 | 2021-01-29 | 福州大学 | Application of montelukast in preparation of medicines for preventing and treating thrombotic diseases |
Non-Patent Citations (4)
Title |
---|
曹丰等: "分子影像在动脉血栓诊治的研究进展", 《中华保健医学杂志》 * |
李春艳等: "高效液相色谱-荧光检测法测定血浆中五聚赖氨酸-β-羰基酞菁锌", 《中国新药杂志》 * |
杨永帅等: "酞菁光敏剂抗小鼠宫颈癌的光动力学实验", 《福州大学学报(自然科学版)》 * |
王也飞等: "《临床血液学检验》", 31 July 2005, 北京科学技术文献出版社 * |
Also Published As
Publication number | Publication date |
---|---|
CN113229213B (en) | 2022-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Willmann et al. | US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice | |
JP6382208B2 (en) | System and computer program product | |
AU2020254985B2 (en) | Platelet membrane self-assembly nanobubble, and preparation method and application thereof | |
Bai et al. | Multimodal and multifunctional nanoparticles with platelet targeting ability and phase transition efficiency for the molecular imaging and thrombolysis of coronary microthrombi | |
Zhao et al. | Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin | |
CN109316608B (en) | Low-intensity focused ultrasound response type phase-change thrombolytic nanoparticles, application and preparation method thereof | |
Prikhozhdenko et al. | Target delivery of drug carriers in mice kidney glomeruli via renal artery. Balance between efficiency and safety | |
Spivak et al. | Low-dose molecular ultrasound imaging with E-selectin-targeted PBCA microbubbles | |
Fu et al. | Magnetic iron sulfide nanoparticles as thrombolytic agents for magnetocaloric therapy and photothermal therapy of thrombosis | |
Cao et al. | Thrombus-targeted nano-agents for NIR-II diagnostic fluorescence imaging-guided flap thromboembolism multi-model therapy | |
CN113229213B (en) | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe | |
Gusliakova et al. | Renal Artery Catheterization for Microcapsules’ Targeted Delivery to the Mouse Kidney | |
JP2003509125A (en) | Tumor imaging method | |
JP2002515887A (en) | Use of hollow microcapsules in diagnosis and therapy | |
Chen et al. | A Clot‐Homing Near‐Infrared Probe for In Vivo Imaging of Murine Thromboembolic Models | |
Li et al. | A facile theragnostic nano-platform for the effective treatment and real-time imaging of acute liver injury | |
Wang et al. | Exploring and Analyzing the Systemic Delivery Barriers for Nanoparticles | |
CN114831770A (en) | Pulmonary hypertension non-human primate model and construction method thereof | |
Lin et al. | Molecular photoacoustic imaging for early diagnosis and treatment monitoring of rheumatoid arthritis in a mouse model | |
Nam et al. | Ultrasound and photoacoustic imaging to monitor mesenchymal stem cells labeled with gold nanoparticles | |
US6986740B2 (en) | Ultrasound contrast using halogenated xanthenes | |
Setia et al. | Nanoparticles for Thrombus Diagnosis and Therapy: Emerging Trends in Thrombus-theranostics | |
Zhang et al. | Cyclic RGD functionalized PLGA nanoparticles loaded with noncovalent complex of indocyanine green with urokinase for synergistic thrombolysis | |
Liu | Visualized Medicine: Emerging Techniques and Developing Frontiers | |
KR101474063B1 (en) | CT contrast agent comprising glycol chitosan-gold nano particle conjugated fibrin target peptide sequence |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |