CN115531558A - Method for marking animal liver lymphatic vasculature and three-dimensional atlas imaging - Google Patents
Method for marking animal liver lymphatic vasculature and three-dimensional atlas imaging Download PDFInfo
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- 241001465754 Metazoa Species 0.000 title claims abstract description 90
- 210000004185 liver Anatomy 0.000 title claims abstract description 88
- 210000004324 lymphatic system Anatomy 0.000 title claims abstract description 71
- 238000003384 imaging method Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 31
- 102100026849 Lymphatic vessel endothelial hyaluronic acid receptor 1 Human genes 0.000 claims abstract description 129
- 101710178181 Lymphatic vessel endothelial hyaluronic acid receptor 1 Proteins 0.000 claims abstract description 129
- 230000002440 hepatic effect Effects 0.000 claims abstract description 37
- 210000001365 lymphatic vessel Anatomy 0.000 claims abstract description 34
- 210000001953 common bile duct Anatomy 0.000 claims abstract description 33
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
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- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
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Abstract
The present disclosure provides a method for labeling and three-dimensional atlas imaging of an animal liver lymphatic vasculature, the imaging method comprising: injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids; injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; and (4) carrying out three-dimensional imaging on the marked animal liver lymphatic vessel system to obtain a three-dimensional map of the liver lymphatic vessel system. According to the spatial position difference of the liver blood vessels and lymphatic vessels, the invention provides a method for injecting different antibodies in a time-space sequence to specifically mark the lymphatic system of the complete liver of a mouse, for example, firstly injecting LYVE-1 antibody which is not coupled with a fluorophore, and then injecting LYVE-1 antibody which is coupled with the fluorophore to the common bile duct, so that the specific marking of the liver lymphatic system by a single fluorescent antibody is realized; during fluorescence microscopic imaging, only the liver lymphatic vessels have signals of fluorescent groups, so that a three-dimensional map of the animal liver lymphatic vessel system can be obtained.
Description
Technical Field
The disclosure relates to the technical field of lymphatic system imaging, in particular to a marking and three-dimensional atlas imaging method for an animal liver lymphatic system.
Background
The liver is the organ which generates most lymph, 25% -50% of lymph passing through the thoracic duct comes from the liver, the liver disease and the liver lymph are closely related, and if the three-dimensional map of the liver lymph system can be accurately determined, the research of the liver disease is facilitated.
In the textbook and the latest published research, no report on the precise and complete three-dimensional structural diagram of the hepatic lymphatic vessel system is found, and at present, only the schematic diagram of the three-dimensional structure of the hepatic lymphatic vessel system presumed based on the clinical image result or two-dimensional imaging is carried out after the liver section is marked with an antibody. The french anatomist Rouvi de re proposed the structure and distribution of hepatic lymphatic vessels from a macroscopic level based on anatomical and CT, MRI data, and in his proposed model, there were superficial and deep lymphatic systems in the liver, where superficial lymphatic vessels were located near the hepatic capsule and deep lymphatic systems comprised periportal and central perivenous lymphatic vessels. However, the anatomy, CT and MRI data have the disadvantages of insufficient specificity and low resolution. Thus, to confirm the three-dimensional map of the lymphatic vasculature of the liver, it must be specifically labeled and microscopically imaged at submicron resolution over the entire liver.
The existing mouse liver lymphatic vessel three-dimensional structure microscopic imaging report is not accurate at present, because the marking method is to directly use LYVE-1 antibody to soak a mouse liver sample and then carry out light slice imaging, thus liver blood sinuses cannot be excluded. In addition, the imaging results show vessels without the unidirectional dead-end structure of lymphatic vessels. Therefore, a method for marking and three-dimensional map imaging of animal liver lymphatic vasculature is needed.
Disclosure of Invention
The invention provides a method for marking and imaging a three-dimensional atlas of an animal hepatic lymphatic vasculature, which at least solves the technical problems of the prior art that the defects of a marking method exist and the three-dimensional atlas of the animal hepatic lymphatic vasculature is inaccurate.
In one aspect of the present disclosure, there is provided a method of labeling the lymphatic vasculature of an animal's liver, the method comprising:
injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids;
injecting a labeled LYVE-1 (lymphoreticular hyaluronan receptor 1) antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of a fluorophore-conjugated LYVE-1 antibody, a nuclide-labeled LYVE-1 antibody, a magnetic particle-labeled LYVE-1 antibody, and a contrast agent-labeled LYVE-1 antibody.
In a second aspect of the present disclosure, there is provided a method of three-dimensional atlas imaging of the lymphatic vasculature of an animal liver, the method comprising:
injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids;
injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of a fluorophore-conjugated LYVE-1 antibody, a nuclide-labeled LYVE-1 antibody, a magnetic particle-labeled LYVE-1 antibody, and a contrast agent-labeled LYVE-1 antibody;
and (4) carrying out three-dimensional imaging on the marked animal liver lymphatic vessel system to obtain a three-dimensional map of the liver lymphatic vessel system.
In one embodiment, the method for three-dimensional imaging of the lymphatic vasculature of a liver in a labeled animal to obtain a three-dimensional map of the lymphatic vasculature of the liver comprises:
if the LYVE-1 antibody coupled with the fluorophore is injected into the common bile duct of the animal, performing 3D fluorescence microscopic imaging on the labeled animal hepatic lymphatic system to obtain a three-dimensional map of the hepatic lymphatic system; or
If the LYVE-1 antibody marked by nuclide is injected into the common bile duct of the animal, performing PET imaging on the marked animal liver lymphatic system to obtain a three-dimensional map of the liver lymphatic system; or
If the LYVE-1 antibody marked by the magnetic particles is injected into the common bile duct of the animal, carrying out MRI imaging on the marked animal hepatic lymphatic system to obtain a three-dimensional map of the hepatic lymphatic system; or
If the LYVE-1 antibody marked by the contrast agent is injected into the common bile duct of the animal, CT imaging is carried out on the marked animal hepatic lymphatic system, and a three-dimensional map of the hepatic lymphatic system is obtained.
In one embodiment, an unlabeled LYVE-1 antibody is injected into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids, comprising:
mixing unlabeled LYVE-1 antibody with phosphate buffer solution, and injecting into the animal via tail vein; the mass of the unlabeled LYVE-1 antibody is 20-30 μ g, and the volume of the phosphate buffer solution is 140-160 μ L.
In one embodiment, the labeled LYVE-1 antibody is administered to the common bile duct of the animal 1-20min after administration of unlabeled LYVE-1 antibody to the tail vein of the animal.
In one embodiment, the animal is injected with a labeled LYVE-1 antibody into the common bile duct comprising:
mixing a labeled LYVE-1 antibody with a phosphate buffer solution to obtain a mixed solution;
the mixed solution is injected from the common bile duct in a bile retrograde direction.
In one embodiment, the mixed solution contains 5-30 μ g of labeled LYVE-1 antibody and 140-190 μ L of phosphate buffered saline.
In one embodiment, the injection rate of the mixed solution is not less than 40 μ L/s.
In yet another aspect of the present disclosure, there is provided a kit for labeling and three-dimensional imaging of lymphatic vasculature in an animal comprising reagents involved in the above method.
According to the method for labeling the lymphatic vasculature of the liver of the animal and imaging the three-dimensional map, unlabeled LYVE-1 antibody is injected through tail vein, the LYVE-1 antibody can be specifically bound to LYVE-1 antigen of endothelial cells of liver blood sinuses, and occupies LYVE-1 epitope of the endothelial cells of liver blood sinuses, for example, coupled fluorophore labeled LYVE-1 antibody is used, and during fluorescence microscopic imaging, no fluorescence signal is generated because the bound LYVE-1 antibody does not have a fluorescence molecule. And injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the lymphatic system of the liver of the animal, for example, by using the LYVE-1 antibody labeled by coupling fluorescent group, wherein the lymphatic system is specifically labeled by the LYVE-1 antibody coupled with the fluorescent group, and only the lymphatic system has the fluorescent signal of the fluorescent group during fluorescence microscopic imaging, so that the three-dimensional map of the lymphatic system of the liver of the animal can be obtained.
It should be understood that the statements in this section do not necessarily identify key or critical features of the embodiments of the present disclosure, nor do they limit the scope of the present disclosure. Other features of the present disclosure will become apparent from the following description.
Drawings
The above and other objects, features and advantages of exemplary embodiments of the present disclosure will become readily apparent from the following detailed description read in conjunction with the accompanying drawings. Several embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which:
in the drawings, the same or corresponding reference numerals indicate the same or corresponding parts.
FIG. 1 is a schematic flow chart diagram illustrating a method for labeling and three-dimensional atlas imaging of the lymphatic vasculature of an animal liver in accordance with an embodiment of the present disclosure;
FIG. 2 is a schematic flow chart of a method for labeling and three-dimensional atlas imaging of the lymphatic vasculature of a mouse liver according to an embodiment of the disclosure;
fig. 3 shows a three-dimensional map of the left lymphatic vasculature of the liver of a mouse provided by an embodiment of the present disclosure.
Detailed Description
In order to make the objects, features and advantages of the present disclosure more apparent and understandable, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present disclosure, and it is apparent that the described embodiments are only a part of the embodiments of the present disclosure, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.
Example 1
The invention provides a method for marking animal liver lymphatic vasculature, which comprises the following steps:
injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids;
injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of LYVE-1 antibody coupled with a fluorescent group, LYVE-1 antibody labeled with nuclides, LYVE-1 antibody labeled with magnetic particles, and LYVE-1 antibody labeled with contrast agent.
Example 2
Fig. 1 shows a schematic flow chart of a three-dimensional atlas imaging method for lymphatic vasculature of animal liver provided by the invention, which comprises the following steps:
step S101, injecting an unlabeled LYVE-1 antibody into the animal body to block LYVE-1 antigen on the endothelial cells of the liver sinusoids.
Mixing unlabeled LYVE-1 antibody and Phosphate Buffer Solution (PBS), and injecting into the animal body via tail vein, wherein the mass of unlabeled LYVE-1 antibody is 20-30 μ g, and the volume of Phosphate buffer solution is 140-160 μ L.
The injection of the unlabeled LYVE-1 antibody can be specifically bound to the LYVE-1 antigen of the liver blood sinus endothelial cells, and the LYVE-1 antigen epitope of the liver blood sinus endothelial cells is occupied, so that the LYVE-1 antigen on the liver blood sinus endothelial cells is sealed.
Step S102, injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of a fluorophore-conjugated LYVE-1 antibody, a nuclide-labeled LYVE-1 antibody, a magnetic particle-labeled LYVE-1 antibody, and a contrast agent-labeled LYVE-1 antibody.
After injecting unlabeled LYVE-1 antibody into the tail vein of the animal for 1-20min, the labeled LYVE-1 antibody is injected into the common bile duct of the animal, and because of the spatial position difference of the liver blood vessels and lymphatic vessels, different LYVE-1 antibodies (namely the unlabeled antibody and the labeled antibody) are injected at different time points, so that the lymphatic system of the whole liver of the mouse is specifically labeled.
In one example, an animal is injected with a labeled LYVE-1 antibody comprising:
mixing the labeled LYVE-1 antibody with a phosphate buffer solution to obtain a mixed solution;
the mixed solution is injected from the common bile duct in a bile retrograde direction.
For example, in this embodiment, using a fluorophore-conjugated LYVE-1 antibody, the mixed solution comprises: 5-30 μ g of LYVE-1 antibody conjugated to a fluorophore, 140-190 μ L of phosphate buffer, wherein the fluorophore is Alexa Fluur 488 fluorescein. The mixed solution is extracted by a syringe with the range of 300 mu L, and then 200 mu L of the mixed solution is injected within 5s, namely the injection speed is not less than 40 mu L/s.
And S103, carrying out three-dimensional imaging on the marked animal liver lymphatic vessel system to obtain an animal liver lymphatic vessel system three-dimensional map.
The imaging method differs depending on the labeled LYVE-1 antibody used, and the imaging method of the present invention includes 3D fluorescence microscopy, PET imaging, MRI imaging, CT imaging.
In one example, three-dimensional imaging of the lymphatic vasculature of a liver of a tagged animal to obtain a three-dimensional map of the lymphatic vasculature of the liver comprises:
if the LYVE-1 antibody coupled with the fluorophore is injected into the common bile duct of the animal, performing 3D fluorescence microscopic imaging on the labeled animal hepatic lymphatic system to obtain a three-dimensional map of the hepatic lymphatic system; or
If the LYVE-1 antibody marked by nuclide is injected into the common bile duct of the animal, performing PET imaging on the marked animal liver lymphatic system to obtain a three-dimensional map of the liver lymphatic system; or
If the LYVE-1 antibody marked by the magnetic particles is injected into the common bile duct of the animal, carrying out MRI imaging on the marked animal hepatic lymphatic system to obtain a three-dimensional map of the hepatic lymphatic system; or
If the LYVE-1 antibody marked by the contrast agent is injected into the common bile duct of the animal, CT imaging is carried out on the marked animal hepatic lymphatic system, and a three-dimensional map of the hepatic lymphatic system is obtained.
In the scheme of the invention, because the endothelial cells of the liver blood sinuses express LYVE-1 antigen, and the spatial positions of blood vessels and lymphatic vessels are different, the scheme injects different LYVE-1 antibodies at different time points according to the spatial position difference of the liver blood vessels and the lymphatic vessels, and provides a method for injecting the antibodies in a spatiotemporal sequence to specifically mark the lymphatic system of the whole liver of a mouse. The LYVE-1 antibody is injected into tail veins of animals to seal LYVE-1 antigen of liver blood sinus endothelial cells, liver lymphatic vessels enter the lymphatic vessels from a distance gap (perisinus gap) to a Mall gap (a space between a portal interval matrix and liver cells on the outermost layer of liver lobules) and then enter the lymphatic vessels, and the tail ends of bile ducts exist in the Mall gap, so that the labeled LYVE-1 antibody is injected from the common bile duct along the retrograde direction of bile, and a mixed solution injected at high pressure breaks through the tail ends of the bile ducts to enter the Mall gap so as to quickly enter the lymphatic vessels. While the mixed solution injected at high pressure also breaks down the liver's sinuses into the hepatic vasculature during this procedure, the previously tail vein injected unlabeled LYVE-1 antibody has blocked the LYVE-1 antigen on the liver's sinuses. Therefore, the labeled LYVE-1 antibody injected by common bile duct high pressure does not combine with endothelial cells of hepatic sinusoids, can specifically label the hepatic lymphatic vessel system, and combines with an imaging technology, such as 3D fluorescence microscopy, PET imaging, MRI imaging or CT imaging, to finely image the lymphatic vessel system of the liver after the complete hepatic lymphatic vessel system is specifically labeled, so as to obtain a three-dimensional map.
Example 3
The invention also provides a kit for labeling and three-dimensional imaging of lymphatic vasculature of an animal comprising reagents involved in the labeling methods and imaging methods of the invention.
The kit comprises: unlabeled LYVE-1 antibody;
labeled LYVE-1 (fluorophore-conjugated LYVE-1 antibody, nuclide-labeled LYVE-1 antibody, magnetic particle-labeled LYVE-1 antibody, contrast agent-labeled LYVE-1 antibody);
phosphate buffer.
Example 4
Taking a mouse as an example, fig. 2 shows a flow chart of a method for imaging a three-dimensional atlas of a hepatic lymphatic vasculature of the mouse, which comprises the following steps:
step S201, taking 25 mu g of LYVE-1 antibody which is not coupled with a fluorescent group, adding 150 mu L of Phosphate Buffer Solution (PBS), injecting tail vein into a mouse body, and sealing LYVE-1 antigen on mouse liver blood sinus endothelial cells;
after the step S202 and the step 10min, 20 mu g of LYVE-1 antibody coupled with a fluorescent group is taken, 175 mu L of phosphate buffer solution is added, wherein the fluorescent group is Alexa flow 488 fluorescein, and the mixed solution is marked as LYVE-1-Alexa flow 488; injecting LYVE-1 antibody coupled with fluorophore in 5s from common bile duct along bile retrograde direction to mark mouse hepatic lymphatic system;
the LYVE-1-Alexa flow 488 solution injected under high pressure breaks through the end of the bile duct and enters into the gaps of the Mall, so as to rapidly enter into the lymphatic vessels, although the LYVE-1-Alexa flow 488 injected under high pressure also breaks through the liver blood sinuses and enters into the liver vascular system, the LYVE-1 antibody injected by the caudal vein in the step S201 already seals the LYVE-1 antigen on the liver blood sinuses, therefore, the LYVE-1-Alexa flow 488 antibody injected under high pressure in the common bile duct cannot be combined with the endothelial cells of the liver blood sinuses.
And S203, performing 3D fluorescence microscopic imaging on the marked mouse hepatic lymphatic vasculature to obtain a three-dimensional map of the mouse hepatic lymphatic vasculature.
When fluorescence microscopic imaging is carried out, because the LYVE-1 antibody combined on the endothelial cells of the liver blood sinuses is not coupled with a fluorescent group, and the LYVE-1 antibody combined on the endothelial cells of the lymphatic vessels is coupled with the fluorescent group, namely coupled with Alexa Flour488 fluorescein, the specific labeling of the liver lymphatic vessel system by the single antibody is realized. As shown in fig. 3 (a), the three-dimensional imaging result of the left lobe lymphatic vessel of the liver of the mouse is obtained according to the imaging method of example 2; fig. 3 (b) is an enlarged view of a rectangular frame in (a).
It should be understood that various forms of the flows shown above may be used, with steps reordered, added, or deleted. For example, the steps described in the present disclosure may be executed in parallel, sequentially, or in different orders, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved, and the present disclosure is not limited herein.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present disclosure, "a plurality" means two or more unless specifically limited otherwise.
The above description is only for the specific embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure, and all the changes or substitutions should be covered within the scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.
Claims (9)
1. A method of labeling the lymphatic vasculature of an animal's liver, comprising:
injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids;
injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of a fluorophore-conjugated LYVE-1 antibody, a nuclide-labeled LYVE-1 antibody, a magnetic particle-labeled LYVE-1 antibody, and a contrast agent-labeled LYVE-1 antibody.
2. A method of three-dimensional atlas imaging of the lymphatic vasculature of an animal's liver, the method comprising:
injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids;
injecting a labeled LYVE-1 antibody into the common bile duct of the animal to label the hepatic lymphatic system of the animal; the labeled LYVE-1 antibody comprises one of a LYVE-1 antibody coupled with a fluorescent group, a nuclide labeled LYVE-1 antibody, a magnetic particle labeled LYVE-1 antibody and a contrast agent labeled LYVE-1 antibody;
and (4) carrying out three-dimensional imaging on the marked animal liver lymphatic vessel system to obtain a three-dimensional map of the liver lymphatic vessel system.
3. The method of claim 2, wherein the three-dimensional imaging of the lymphatic vasculature of the liver of the labeled animal to obtain a three-dimensional map of the lymphatic vasculature of the liver comprises:
if the LYVE-1 antibody coupled with the fluorophore is injected into the common bile duct of the animal, performing 3D fluorescence microscopic imaging on the labeled animal hepatic lymphatic system to obtain a three-dimensional map of the hepatic lymphatic system; or
If the LYVE-1 antibody marked by nuclide is injected into the common bile duct of the animal, performing PET imaging on the marked animal liver lymphatic system to obtain a three-dimensional map of the liver lymphatic system; or
If the LYVE-1 antibody marked by the magnetic particles is injected into the common bile duct of the animal, carrying out MRI imaging on the marked animal liver lymphatic vessel system to obtain a three-dimensional map of the liver lymphatic vessel system; or
If the LYVE-1 antibody marked by the contrast agent is injected into the common bile duct of the animal, CT imaging is carried out on the marked animal hepatic lymphatic system, and a three-dimensional map of the hepatic lymphatic system is obtained.
4. The method of claim 2, wherein injecting an unlabeled LYVE-1 antibody into the animal to block LYVE-1 antigen on endothelial cells of the liver sinusoids comprises:
mixing unlabeled LYVE-1 antibody with phosphate buffer solution, and injecting into the animal via tail vein; the mass of the unlabeled LYVE-1 antibody is 20-30 μ g, and the volume of the phosphate buffer solution is 140-160 μ L.
5. The method of claim 4, wherein the labeled LYVE-1 antibody is administered to the common bile duct of said animal 1-20min after administration of unlabeled LYVE-1 antibody to the tail vein of said animal.
6. The method of claim 2, wherein injecting into the common bile duct of the animal a labeled LYVE-1 antibody comprises:
mixing the labeled LYVE-1 antibody with a phosphate buffer solution to obtain a mixed solution;
the mixed solution is injected from the common bile duct in a bile retrograde direction.
7. The method of claim 6, wherein the mixed solution comprises 5-30 μ g of labeled LYVE-1 antibody and 140-190 μ L of phosphate buffered saline.
8. The method according to claim 7, wherein the injection speed of the mixed solution is not less than 40 μ L/s.
9. A kit for labeling and three-dimensional imaging of lymphatic vasculature of an animal comprising the reagents of the method of claims 1-8.
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| CN202211131799.9A CN115531558B (en) | 2022-09-16 | Marking and three-dimensional map imaging method for animal liver lymphatic system |
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| CN202211131799.9A CN115531558B (en) | 2022-09-16 | Marking and three-dimensional map imaging method for animal liver lymphatic system |
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