Application of antibody in preparation of marking preparation of femoral vascular system

Abstract

The invention belongs to the technical field of medical biomarkers, and particularly relates to application of an antibody to preparation of a marker preparation of a femoral vascular system, the marker preparation of the femoral vascular system is an anti-mouse CD31 antibody used for intravenous injection to a gene animal simultaneously containing Col10a1 gene and Rosa26-tdTomato reporter gene, the Rosa26-tdTomato reporter gene expresses tdTomato, and the Col10a1 gene and the Rosa26-tdTomato reporter gene are used for expressing the TdTomato. A CD31 antibody with a fluorophore is intravenously injected into a blood vessel, and the antibody reaches and marks blood vessels in a thighbone cortical bone, a bone trabecula and a marrow cavity along with blood circulation, so that the goal of three-dimensionally and simultaneously presenting thighbone hypertrophy cartilage cells, progeny cells thereof and a complete blood vessel system is achieved; the limitation that two-dimensional data cannot comprehensively reflect the real situation is broken through, and meanwhile compatibility of tdTomato signals and the transparency technology is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomarkers, and in particular to the application of antibodies in the preparation of femoral vascular system marker preparations. Background Art

[0002] Bone formation involves both endochondral ossification and intramembranous ossification. Early in skeletal development, the limbs and axial bones of vertebrates are generated from cartilage rudiments through endochondral ossification. Mesenchymal stem and progenitor cells condense and further differentiate into chondroblasts, or chondrocytes. These chondrocytes undergo rapid proliferation, depositing a type II collagen-rich extracellular matrix to form the initial immature cartilage rudiments. As cartilage growth proceeds, chondrocytes located closest to the center of each primordium exit the cell cycle and undergo maturation and hypertrophic differentiation. Hypertrophic chondrocytes secrete not only their specific type X collagen but also additional proteins, such as vascular endothelial growth factor α, platelet-derived growth factor β, and other factors, promoting vascular invasion and osteoblast differentiation of surrounding perichondrial progenitor cells. As blood vessels from the perichondrium invade the hypertrophic cartilage to establish the bone marrow cavity, newly formed osteoblasts and their progenitors are transported by these vessels to the bone marrow, where they secrete a type I collagen-rich matrix that is ultimately mineralized to replace the cartilage with bone. Bone formation, which includes the mineralization of plate-like bones, the formation of blood vessels and the bone marrow cavity, requires observation of intact bone tissue and acquisition of high-resolution three-dimensional structural data to study bone development.

[0003] In previous studies, bone tissue was fixed and decalcified before sectioning. Individual sections included paraffin sections and frozen sections. More recently, automated serial sectioning and associated imaging software analysis have been applied to intact bone analysis. While individual or serial sections are essential and valuable for studying bone development, even these methods still involve bone tissue damage. To this end, researchers have used optical techniques to study intact bone tissue. However, strong light scattering from tissue limits imaging depth, and bone's uniquely thick hard tissue structure is difficult for optical instruments to penetrate. With technological advancements, tissue optical scatter removal techniques have been applied to various tissues, using chemical reagents and tools to reduce scattering. Previously, FDISCO technology has been reported, which achieves both tissue preservation of fluorescent proteins and robust tissue scatter removal capabilities by regulating temperature and pH conditions. This technique has primarily been used in the central nervous system, but also encompasses internal organs, soft tissues, and even the skull, where calcified periosteal ossification occurs in hard tissue. However, it has not been tested in the femur, where both endochondral and intramembranous ossification occur simultaneously.

[0004] In summary, in the past, when chemical reagents were used to mark the vascular distribution of the femur, the vascular distribution of only a single slice or part of the femur in continuous slices was displayed, and the overall vascular distribution of the entire femur could not be displayed. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the use of antibodies in preparing femoral vascular system marker preparations.

[0006] Use of the antibody in preparing a femoral vasculature marker preparation, wherein the femoral vasculature marker preparation is an anti-mouse CD31 antibody for intravenous injection into genetic animals containing both the Col10a1 gene and the Rosa26-tdTomato reporter gene;

[0007] The Rosa26-tdTomato reporter gene expresses tdTomato.

[0008] Preferably, the anti-mouse CD31 antibody is diluted with a pharmaceutically acceptable solvent to a solution with a concentration of 1 μg / 10 μL to 2 μg / 10 μL, and then injected intravenously.

[0009] Preferably, the pharmaceutically acceptable solvent is sterile physiological saline or PBS buffer.

[0010] A method for three-dimensional atlas imaging of the femoral vascular system for non-diagnostic / therapeutic purposes comprises intravenously injecting the anti-mouse CD31 antibody into an animal containing both the Col10a1 gene and the Rosa26-tdTomato reporter gene to achieve the effect of labeling the femoral vascular system, and processing the labeled femoral tissue for three-dimensional atlas imaging.

[0011] Preferably, the femoral tissue is decalcified and transparentized before being subjected to three-dimensional atlas imaging;

[0012] Wherein, decalcification is carried out at 6°C to 8°C using an EDTA solution with a mass fraction of 20% and a pH of 8 to 10.

[0013] Preferably, the EDTA solution is used for decalcification for 6 to 8 days.

[0014] Preferably, clearing is performed using a tissue clearing kit.

[0015] Preferably, the three-dimensional atlas imaging is any one of light sheet microscopy imaging, 3D fluorescence microscopy imaging, PET imaging, MRI imaging or CT imaging.

[0016] Preferably, when imaging with the light sheet microscope, the numerical aperture of the 10× objective lens is 0.5 and the working distance is 5.5 mm.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Hypertrophic chondrocyte tracing marker gene mice were injected with antibodies into the tail vein to mark the femoral vascular system.tdT / + Experiments were conducted on transgenic mice. The Rosa26-tdTomato reporter gene activated the expression of tdTomato when the Col10a1 gene began to express. tdTomato labeled the hypertrophic chondrocytes of the mouse long bones, as well as the osteoblasts and other bone-related cells after the transdifferentiation of hypertrophic chondrocytes. Anti-mouse CD31 antibodies were used to label the femoral vascular system in vivo, thereby labeling the femoral hypertrophic chondrocytes, their progeny, and the entire femoral vascular system, and obtaining a three-dimensional atlas imaging of the femoral vascular system.

[0019] 2. Decalcify the mouse femurs at low temperatures using a high-concentration decalcifying solution and clearing them using FDISCO clearing technology. After deep anesthesia, the mice were perfused with saline and fixative and placed in the fixative overnight at 4°C. They were then decalcified at high concentrations and at 6°C-8°C for one week. Clearing was performed using FDISCO technology at 6°C-8°C and imaged using an optical microscope.

[0020] 3. Using Col10a1-Cre / R26 tdT / + Experiments were conducted on transgenic mice, in which tdTomato-labeled Col10a1-Cre cells and their later-stage progeny marked hypertrophic chondrocytes in the mouse bones, as well as osteoblasts and other bone-related cells that these hypertrophic chondrocytes transdifferentiate into. This invention, for the first time, utilized clearing technology in the mouse femur to visualize the distribution of femoral hypertrophic chondrocytes, their progeny, and the entire femoral vasculature. The imaging process required both preserving the tdTomato labeling signal and displaying a complete vascular image.

[0021] Cells labeled with tdTomato exhibit autofluorescence, as do antibodies injected into the tail vein. Therefore, decalcification requires both short-term effectiveness and fluorescence protection. Low temperatures are used to quench autofluorescence, while high-concentration alkaline decalcification solutions are used to achieve the desired decalcification effect without damaging fluorescence. While standard decalcification solutions require 21 days to decalcify the femur of a one-month-old mouse, high-concentration alkaline decalcification solutions can complete decalcification in just 7 days at low temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Gross three-dimensional imaging results of the femur before and after tissue clearing, where A is before clearing and B is after clearing;

[0023] Figure 2Three-dimensional images were obtained on an optical microscope platform after clearing. A is a fluorescent composite image of the mouse femur from the distal to mid-femur after clearing, showing tdTomato cells and their progeny and antibody-labeled tissue blood vessels. B is immunostained CD31-labeled tissue blood vessels in Figure A. C is autofluorescent tdTomato-labeled hypertrophic chondrocytes and their progeny in Figure A.

[0024] Figure 3 100um Z-axis fluorescence imaging was selected for the distal end of the mouse femur, and tdTomato cells and vascular configuration were clearly visible. A is a fluorescent composite image showing the distribution of autofluorescent tdTomato cells and the vascular configuration labeled with CD31 antibodies. B is an image of Figure A showing only the CD31-labeled vascular configuration. C is an enlarged image of the femoral growth plate in Figure A, where CD31-labeled blood vessels at the growth site are clearly visible extending from the growth plate into the medullary cavity, and autofluorescent tdTomato cells are distributed closely to the blood vessels. D is an enlarged image of the femoral growth plate in Figure B, where blood vessels are distributed in a dendritic manner, arranged neatly and passing through in an orderly manner. E is an enlarged image of the femoral cortical bone, endosteum and medullary cavity in Figure A. F is an enlarged image of the femoral cortical bone in Figure B, where blood vessels labeled at the endosteum extend closely to the endosteum, and there are blood vessels passing through the cortical bone. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0026] Table 1 Abbreviation comparison and information table

[0027]

[0028]

[0029] Rosa26-tdTomato mice were from the Jackson Laboratory in the United States; Col10a1-Cre mice were from Kathryn SECheah's laboratory at the University of Hong Kong, China.

[0030] Example 1

[0031] A method for marking a femoral vascular system comprises the following steps:

[0032] (1) Model animal breeding

[0033] Col10a1-Cre model mice were obtained by mating with tdTomato fluorescent reporter transgenic mice Rosa26-tdTomato. After mating, Col10a1-Cre model mice and Rosa26-tdTomato mice were obtained to obtain tdTomato red-labeled hypertrophic chondrocyte-derived skeletal stem cells, namely Col10a1-Cre / R26 tdT / + Mice. Among them, skeletal stem cells derived from hypertrophic chondrocytes specifically express Col10a1. These transgenic mice were bred and maintained at the Experimental Animal Center of the Air Force Medical University. The experimental animals and conditions complied with the "Regulations on the Administration of Laboratory Animals" promulgated by the State Science and Technology Commission. All mice were housed in an SPF environment. Mice aged 6-8 weeks were used for mating to obtain offspring.

[0034] (2) Mouse gene identification

[0035] DNA extraction: age-appropriate Col10a1-Cre / R26 tdT / + The mice were marked and numbered, and a small amount of toe or tail tissue was cut for DNA extraction. The cut and numbered tissue was placed in an EP tube, and 30 μL of DNA Extraction Solution was added. The sample was placed in a centrifuge to completely mix the tissue and liquid. The sample was then transferred to a metal bath and incubated at 95°C for 20 minutes. The sample was then cooled to room temperature. Finally, 30 μL of stabilizing solution was added to the sample to terminate the reaction and stabilize the extracted DNA.

[0036] The DNA tissue samples obtained can be used for the next step of the experiment or stored at -20°C. For primer design and genotyping, the required primers are shown in Table 2, the reaction system is shown in Table 3, and the genotyping amplification procedure is shown in Table 4.

[0037] Table 2 List of primers for identification of model animal mouse

[0038]

[0039] Table 3 Reaction system

[0040] System solution Dosage Mix 12.5μL DNA 2μL ForwardPrimer 1 μL ReversedPrimer 1 μL ddH2O 8.5 μL total 25 μL

[0041] Table 4 PCR program

[0042]

[0043] Identification: For nucleic acid gel electrophoresis and sample loading, prepare a 2% nucleic acid gel. Weigh 2g of agarose powder using an electronic balance and add it to 100mL of 1×TAE buffer. Shake well and heat in a microwave oven until dissolved in the liquid. The liquid becomes transparent. Cool to room temperature, add an appropriate amount of nucleic acid developer, mix thoroughly, and pour into a nucleic acid electrophoresis mold. Once cooled and formed, it is ready for use. For electrophoresis loading, place the nucleic acid gel in an electrophoresis tank and add TAE electrophoresis buffer. Add the amplified PCR sample to the gel, along with the DNA ladder. Run the gel at a constant current of 120V-150V for 30-40 minutes. After completion, place the gel in a nucleic acid imaging instrument for analysis.

[0044] Through mouse gene identification, it is determined whether the mouse gene contains both the Col10a1 gene and the Rosa26-tdTomato reporter gene. If both the Col10a1 gene and the tdTomato reporter gene are present, the mouse is Col10a1-Cre / R26 tdT / + Genetic mice can be used as genetic mice for later experiments.

[0045] The Rosa26-tdTomato reporter gene initiates the expression of tdTomato when the Col10a1 gene begins to express.

[0046] (3) Antibody labeling: The genotype of Col10a1-Cre / R26 was obtained experimentally. tdT / + Three genetically modified mice were used. The time after birth of the genotyped mice was accurately calculated. The genetically modified mice were raised to one month of age for the experiment. In the experiment, anti-mouse CD31 antibodies were used for in vivo vascular labeling. The CD31 antibody is an autofluorescent Alexa Fluor 647 anti-mouse antibody. The antibody was diluted with sterile saline to 1μg / 10μL. The tail vein injection experiment was performed at 1μg / g according to the mouse body weight. The mouse was weighed and an appropriate amount of antibody injection was taken. At the same time, the mouse was fixed on the tail vein injection instrument, with the tail exposed, and the tail vein was injected to track blood vessels of different diameters. After injection, the mouse was placed in a cage with a certain degree of thermal insulation performance and raised in an SPF-level biological laboratory for 30 minutes before subsequent experiments.

[0047] Example 2

[0048] A method for three-dimensional atlas imaging of the femoral vascular system comprises the following steps:

[0049] (1) Col10a1-Cre / R26 tdT / + Excised femoral tissue from transgenic mice was fixed overnight in pre-chilled 4% paraformaldehyde. Because the tissue exhibits autofluorescence, the tissue container was wrapped in tin foil.

[0050] (2) Femoral decalcification: After overnight fixation, the femoral tissue was washed with PBS for 5 min each time. After washing three times, 20% EDTA solution with a pH of 8-10 was added and decalcified at 6°C to 8°C for 7 days. During this period, the decalcification solution was changed 3 to 4 times until the tissue became soft and bendable, and decalcification was completed.

[0051] (3) Tissue clearing: This study used the FDISCO tissue clearing kit, a three-dimensional imaging technology for organ clearing with superior fluorescence preservation capabilities. The clearing procedure was performed in full accordance with the kit instructions at 6°C to 8°C with gentle shaking.

[0052] (4) Optical imaging: After the tissue was cleared, it was imaged using a light sheet microscope and scanned using a 10× objective lens. The thin light sheet was illuminated from all four sides of the sample, and the merged image was saved. To acquire the image, the cleared sample was manually connected to the sample holder adapter. Subsequently, the sample was immersed in the imaging reagent in the 3D-printed sample chamber and excited with light sheets of different wavelengths. The information of light sheet microscope imaging is LiToneXL, Light Innovation Technology, China, NA of the 10× objective lens is 0.5, and the working distance WD is 5.5 mm.

[0053] Imaging results are shown in Figures 1 to 3 ,The gross three-dimensional imaging results of the femur before and after tissue clearing. Before clearing, the femur was fixed and decalcified and was a milky white rod-like object, while after clearing, the femur became transparent; Figure 2 The three-dimensional images were obtained on an optical microscope platform after clearing. A is a fluorescent composite image of the mouse femur from the distal to the mid-femur after clearing. B is the distribution of blood vessels in the tissue marked by immunostaining CD31 in green, and C is the distribution of chondrocytes and their progeny in the hypertrophic zone marked by autofluorescence tdTomato in red. Figure 3 100 μm Z-axis was selected for imaging, where tdTomato cells and vascular structures were clearly visible.

[0054] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0055] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0056] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of an antibody in the preparation of a femoral vascular system marker preparation, characterized in that: The femoral vasculature marker preparation is an anti-mouse CD31 antibody for intravenous injection into animals containing both the Col10a1 gene and the Rosa26-tdTomato reporter gene; The Rosa26-tdTomato reporter gene expresses tdTomato.

2. The use according to claim 1, characterized in that: The anti-mouse CD31 antibody is diluted with a pharmaceutically acceptable solvent to a solution with a concentration of 1 μg / 10 μL to 2 μg / 10 μL, and then injected intravenously.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable solvent is sterile physiological saline or PBS buffer.

4. A method for three-dimensional atlas imaging of the femoral vascular system for non-diagnostic / therapeutic purposes, characterized in that: The anti-mouse CD31 antibody of claim 1 is intravenously injected into an animal containing both the Col10a1 gene and the Rosa26-tdTomato reporter gene to achieve the effect of marking the femoral vascular system, and the marked femoral tissue of the femur is processed for three-dimensional atlas imaging.

5. The three-dimensional atlas imaging method according to claim 4, characterized in that: Decalcifying and transparentizing the femoral tissue and then performing three-dimensional atlas imaging; Wherein, EDTA solution with a mass fraction of 20% and a pH of 8 to 10 is used for decalcification at 6°C to 8°C.

6. The three-dimensional atlas imaging method according to claim 5, characterized in that: The EDTA solution was used for decalcification for 6 to 8 days.

7. The three-dimensional atlas imaging method according to claim 5, characterized in that: Clearing was performed using a tissue clearing kit.

8. The three-dimensional atlas imaging method according to claim 4, characterized in that: The three-dimensional atlas imaging is any one of light sheet microscopy imaging, 3D fluorescence microscopy imaging, PET imaging, MRI imaging or CT imaging.

9. The three-dimensional atlas imaging method according to claim 8, characterized in that: When imaging with the light sheet microscope, the numerical aperture of the 10× objective lens is 0.5, and the working distance is 5.5 mm.

Images