Method for tracing macrophages by targeting liver tissues
By using Dil probe to label macrophages and combining intraperitoneal injection and live imaging technology, the problems of poor light stability and high background noise of macrophage tracing in the liver site in the prior art are solved, and high precision and long-term trace of liver macrophages are achieved.
Patent Information
- Application Number
- CN202510325005.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-06-20
AI Technical Summary
The existing macrophage tracing technology has problems such as poor light stability and high background noise in the liver area, which is difficult to meet the traceability needs of targeted liver tissue.
Macrophages were labeled with Dil probes and combined with in vivo imaging technology by intraperitoneal injection, labeling parameters were optimized to improve the clarity of fluorescence signals and long-term traceability.
High-precision traceability of liver macrophages is achieved, non-specific signal interference is reduced, signal-to-noise ratio is improved, and the purpose of long-term traceability is achieved.
Smart Images
Figure CN120168668A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of in vivo imaging, and particularly relates to a method for targeting liver tissue to trace macrophages. Background Art
[0002] Macrophages are a type of important immune cells and play a key role in the body's defense mechanism. The tracing technology can help researchers observe the migration path and distribution of macrophages in vivo. This is crucial for understanding how macrophages respond to inflammation, infection, or other stimuli in biological immune functions. In some diseases (such as cancer, atherosclerosis, etc.), the behavior and quantity of macrophages will change accordingly. The tracing technology can be used to monitor these changes, so as to evaluate the progress of the disease and the effect of treatment. Tracing and inducing macrophages has important scientific and clinical significance and helps to promote the development of immunology and related medical fields.
[0003] Traditional macrophage tracing methods mostly rely on immunohistochemistry or staining techniques, which are complex to operate and difficult to observe in real time. Moreover, existing macrophage tracing technologies have problems such as poor photostability (such as CFSE) and high background noise (such as fluorescence protein transfection), and it is difficult to meet the requirements of macrophage tracing in the liver. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a method for targeting liver tissue to trace macrophages, select a Dil probe and optimize the labeling parameters of the Dil probe, and combine intraperitoneal injection to achieve good macrophage tracing accuracy in the liver.
[0005] The present invention provides a method for targeting liver tissue to trace macrophages, comprising the following steps:
[0006] Mix macrophages with a cell membrane staining working solution containing a Dil probe and incubate for 5 - 20 min, remove impurities, and obtain Dil probe-labeled macrophages;
[0007] The Dil probe-labeled macrophages are injected into a living animal through intraperitoneal injection, and the fluorescence signal is detected by in vivo imaging of the animal. The migration path and distribution of macrophages are obtained according to the distribution of the fluorescence signal.
[0008] Preferably, the incubation density of the macrophages is 1×10 6 cells / ml - 2×10 6 cells / ml.
[0009] Preferably, the working volume concentration of the Dil probe in the cell membrane staining working solution containing the Dil probe is 0.25%.
[0010] Preferably, the cell membrane staining working solution containing Dil probe further includes a staining enhancer with a volume concentration of 0.25% and a staining buffer solution of 99.5%.
[0011] Preferably, the incubation temperature is 36 - 38 °C.
[0012] Preferably, the incubation is carried out in the dark.
[0013] Preferably, the method for removing impurities includes removing the liquid phase in the incubation system and washing with cell culture medium.
[0014] Preferably, the number of times of removing impurities is 2 - 3 times.
[0015] Preferably, the injection dose of Dil probe-labeled macrophages is 1×10 6 cells / live animal - 5×10 6 cells / live animal.
[0016] Preferably, the live animals include at least one of the following: mice, rats, and hamsters.
[0017] The present invention provides a method for tracing macrophages targeting liver tissue. Macrophages are mixed and incubated with a cell membrane staining working solution containing Dil probe for 5 - 20 min, and then impurities are removed to obtain Dil probe-labeled macrophages. The Dil probe-labeled macrophages are intraperitoneally injected into live animals, and fluorescence signals are detected by in vivo animal imaging. According to the distribution of the fluorescence signals, the migration path and distribution of macrophages are obtained. The present invention selects Dil probe to label primary mononuclear macrophages. Compared with traditional dyes (such as CFSE), it has stronger photostability, can be traced for a long time, and at the same time avoids the overlap of macrophage fluorescence signals and liver fluorescence signals. The fluorescence imaging is clear, reduces non-specific signal interference, improves the signal-to-noise ratio of the detection results, and achieves the purpose of tracing macrophages for a long time. The present invention also optimizes the incubation time during labeling to further improve the clarity of fluorescence imaging. The preparation method of the present invention provides basic materials for the study of macrophage biological immune functions, the occurrence and development of diseases, and the evaluation of drug efficacy. The present invention uses Dil probe-labeled macrophages by intraperitoneal injection, improves the liver targeting of Dil probe-labeled macrophages, avoids the dispersion of macrophages throughout the body, combines in vivo animal imaging technology to observe the fluorescence signals of Dil probe, and can observe the migration and distribution of macrophages in real time, providing a reliable tool for the study of inflammation mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 It is the HE staining result of an inflammatory mouse model; the observation magnification is 20×;
[0019] Figure 2In vivo imaging results after injecting Dil probe-labeled macrophages prepared by incubating for 5 min into an inflammatory mouse model;
[0020] Figure 3 In vivo imaging results after injecting Dil probe-labeled macrophages prepared by incubating for 10 min into an inflammatory mouse model;
[0021] Figure 4 In vivo imaging results after injecting Dil probe-labeled macrophages prepared by incubating for 20 min into an inflammatory mouse model;
[0022] Figure 5 Comparison chart of the tracing results of CFSE-labeled macrophages and Dil probe-labeled macrophages. Detailed implementation method
[0023] The present invention provides a method for tracing macrophages targeting liver tissue, comprising the following steps:
[0024] Mix macrophages with a cell membrane staining working solution containing a Dil probe and incubate for 5 - 20 min, then remove impurities to obtain Dil probe-labeled macrophages;
[0025] The Dil probe-labeled macrophages are intraperitoneally injected into a living animal, and the fluorescence signal is detected by in vivo animal imaging, and the migration path and distribution of the macrophages are obtained according to the distribution of the fluorescence signal.
[0026] In the present invention, the DiI is DiIC18(3), with the full English name of 1,1'-dioctadecyl-3,3,3',3'-tetr-amethylindocarbocyanine perchlorate, presenting orange-red fluorescence. The DiI is a lipophilic membrane dye, which can laterally diffuse into the cell membrane and gradually stain the entire cell membrane. In the present invention, the DiI is purchased from Beyotime Institute of Biotechnology, with the product number C1991S.
[0027] In the present invention, the working volume concentration of the Dil probe in the cell membrane staining working solution containing the Dil probe is preferably 0.25%. The cell membrane staining working solution containing the Dil probe preferably further includes a staining enhancement solution with a volume concentration of 0.25% and a staining buffer solution of 99.5%. The staining enhancement solution and the staining buffer solution are purchased from Beyotime Institute of Biotechnology, with the product number C1991S.
[0028] The present invention has no special limitation on the source of the macrophages, and the well-known sources of macrophages in the art can be adopted. The macrophages are divided into autologous macrophages or allogeneic macrophages according to the individuals for subsequent research. The autologous macrophages refer to individuals with the same source for separation and injection. The allogeneic macrophages refer to individuals with different sources for separation and injection. The separation method of the macrophages preferably separates the macrophages from the abdominal cavity or ex vivo tissues of animals by Percoll centrifugation method. The incubation density of the macrophages is preferably 1×10 6 cells / ml to 2×10 6 cells / ml, and can be 1.5×10 6 cells / ml.
[0029] In the present invention, the incubation temperature is preferably 36 - 38 °C, and can be 37 °C. The incubation is preferably carried out in the dark. The incubation time is preferably 8 - 18 min, and can be 10 - 15 min. Experiments of the present invention show that with the extension of the incubation time, the labeling efficiency of the Dil probe for macrophages increases, and the fluorescence signal is stronger during in vivo imaging.
[0030] In the present invention, the impurity removal method preferably includes removing the liquid phase in the incubation system and washing with cell culture medium. The number of impurity removal times is preferably 2 - 3 times. The method of removing the liquid phase in the incubation system preferably includes centrifugation. The rotation speed of the centrifugation is preferably 1500 - 3000 rpm, and can be 1500 - 2000 rpm. The centrifugation time is preferably 5 - 10 min, and can be 7 - 8 min.
[0031] In the present invention, the macrophages labeled with the Dil probe are resuspended in physiological saline to prepare an injection solution.
[0032] In the present invention, the injection dose of the macrophages labeled with the Dil probe is preferably 1×10 6 cells / mouse to 5×10 6 cells / mouse, and can be 2×10 6 cells / mouse to 4×10 6 cells / mouse, and can also be 3×10 6 cells / mouse.
[0033] In the present invention, after the live animal is intraperitoneally injected with the Dil probe-labeled macrophages, in order to trace the distribution and migration path of macrophages in the inflammation model, it is preferably further included to construct an inflammation model for the live animal. The construction method of the inflammation model preferably uses lipopolysaccharide (LPS) to induce liver inflammation. The administration dose of the lipopolysaccharide is preferably 5-20 mg / kg body weight of mice, and the optimal is 10 mg / kg body weight of mice. In the embodiment of the present invention, after intraperitoneal injection of Dil probe-labeled macrophages, after in vivo imaging detection of the inflammation model, the fluorescence signal aggregates in the liver, and a strong fluorescence signal can still be observed 72 hours after injection. It shows that the method of the present invention can not only achieve the targeted tracing of macrophages in the liver tissue, but also has a long-term tracing effect.
[0034] The following is a detailed description of a method for tracing macrophages targeting the liver tissue provided by the present invention in conjunction with the embodiments, but they cannot be understood as a limitation to the protection scope of the present invention.
[0035] Example 1
[0036] A method for tracing macrophages targeting the liver tissue based on Dil probe
[0037] 1. Dil probe labeling method
[0038] 1) Prepare the cell membrane staining working solution: When the total volume of the cell membrane staining working solution is 10 ml, add 25 μl of DiI (400×) and the staining enhancer respectively, and add 9.95 ml of the staining buffer.
[0039] 2) Add the cell membrane staining working solution to the separated and prepared macrophages to make its density 1×10 6 / ml - 2×10 6 / ml.
[0040] 3) Incubate the cells in the dark at 37°C for 5 min, 10 min and 20 min respectively.
[0041] 4) Centrifuge at 1000×g at room temperature for 5 min.
[0042] 5) Aspirate the supernatant, and slowly add the pre-warmed cell culture medium at 37°C to resuspend the cells again.
[0043] 6) Repeat steps 3) - 4) twice to obtain Dil probe-labeled macrophages.
[0044] 2. Intraperitoneal injection
[0045] Inject the Dil probe-labeled macrophages into C57BL / 6 mice by intraperitoneal injection at an injection volume of 2×10 6 cells / mouse.
[0046] 3. Establishment of Inflammatory Mouse Model
[0047] Inject LPS (lipopolysaccharide) into the above mice at a dose of 10 mg / kg to induce liver inflammation. After 24 h, perform HE staining on the liver tissues of the modeled mice. The staining results are shown in Figure 1 , where the left figure is the HE staining result of the mouse liver tissue before modeling, and the right figure is the staining result of the liver tissue of the mice injected with LPS. The results show that obvious inflammatory reactions occur in the liver tissue, indicating that the inflammatory mouse model is successfully constructed.
[0048] 4. Liver Imaging:
[0049] Use a live fluorescence imaging system (excitation wavelength 549 nm, emission wavelength 565 nm) to collect liver fluorescence signals at 24 h, 48 h, and 72 h after injection respectively.
[0050] The liver imaging results are shown in Figures 2 to 4 . When the Dil probe labels macrophages, at 5 min of incubation, significant fluorescence signals can still be detected in liver imaging at 72 h, and the fluorescence signals are weak (signal-to-noise ratio 1.5). When the Dil probe labels macrophages, at 10 min of incubation, significant fluorescence signals can still be detected at 72 h, the signals are enhanced (signal-to-noise ratio 2.8), and the background interference is reduced by 30%. When the Dil probe labels macrophages, at 20 min of incubation, significant fluorescence signals can still be detected at 72 h, the signals are stable (signal-to-noise ratio 4.8), which is suitable for long-term tracing.
[0051] Comparative Example 1
[0052] A method for labeling and tracing macrophages based on CFSE (carboxyfluorescein diacetate succinimidyl ester)
[0053] 1. Cell Preparation: Isolate macrophages from the animal peritoneal cavity.
[0054] 2. CFSE Labeling: Resuspend macrophages in serum-free PBS at a cell density of 2×10 6 cells / mL to obtain a macrophage suspension. Add CFSE dye to the macrophage suspension at a final concentration of 5 μM, mix gently, and incubate at 37 °C for 15 min. Add 5 volumes of pre-cooled medium to the system to quench the excess dye, and incubate on ice for 5 min. Centrifuge to remove the supernatant, and wash the labeled macrophages with medium containing 10% fetal bovine serum.
[0055] 3. Cell Treatment and In Vivo Injection:
[0056] Resuspend the labeled macrophages in physiological saline to 4×10 6 cells / mL. Inject the labeled macrophages at 2×10 6The dose per mouse was injected into the mice via the tail vein.
[0057] 4. In vivo imaging:
[0058] The animals were subjected to in vivo imaging 72 h after injection.
[0059] The results are shown in Figure 5 , with CFSE labeling on the left side of the figure and Dil probe labeling on the right side. The CFSE signal decays rapidly (signal-to-noise ratio < 1.0 at 72 h), while the Dil probe-labeled macrophages (prepared under the condition of incubation for 20 min) can maintain a tracer signal for a longer time in live animals.
[0060] Comparative Example 2
[0061] For the method of tracing macrophages targeting the liver tissue based on DiR probe labeling, the labeling experiment was carried out using the method of Example 1, with the only difference being that macrophages were labeled with DiR probe instead of Dil probe, and other methods such as intraperitoneal injection, inflammatory mouse model, and in vivo imaging were the same.
[0062] The results showed that due to the overlap of the excitation / emission wavelength of the DiR probe with the autofluorescence of the liver tissue, during in vivo imaging, the fluorescence signal of the DiR probe overlapped with the fluorescence signal of the liver tissue, resulting in a decrease in the signal-to-noise ratio, and it was not possible to well observe the migration path and distribution of macrophages, and the ideal tracer effect of macrophages could not be achieved.
[0063] Comparative Example 3
[0064] For the method of tracing macrophages targeting the liver tissue based on DiO probe labeling, the labeling experiment was carried out using the method of Example 1, with the only difference being that macrophages were labeled with DiO probe instead of Dil probe, and other methods such as intraperitoneal injection, inflammatory mouse model, and in vivo imaging were the same.
[0065] The results showed that due to the overlap of the excitation wavelength and emission wavelength of the DiO probe with the autofluorescence of the liver tissue, during in vivo imaging, the fluorescence signal of the DiO probe overlapped with the fluorescence signal of the liver tissue, resulting in a decrease in the signal-to-noise ratio, and it was not possible to well observe the migration path and distribution of macrophages, and the ideal tracer effect of macrophages could not be achieved.
[0066] Comparative Example 4
[0067] The macrophages labeled with the Dil probe prepared by incubation for 20 min in Example 1 were injected into the inflammatory mouse model by intravenous injection. The injection dose was the same as that recorded in Example 1. After 72 h of injection, the distribution of fluorescence signals in the mice was observed by in vivo imaging.
[0068] The results showed that intravenous injection dispersed Dil probe-labeled macrophages throughout the body of the inflammatory mouse model, unable to target only the liver region, reducing the liver targeting of macrophages.
[0069] Comparative Example 5
[0070] Using the Dil probe-labeled macrophages prepared by incubating for 20 min in Example 1, when intraperitoneally injecting mice, the injection dose was 2×10 5 cells / mouse, and other steps were the same. After 72 h of injection, the fluorescence signal distribution in the mice was observed by in vivo imaging.
[0071] Compared with the Dil probe-labeled macrophages prepared by incubating for 20 min and injected in Example 1, the fluorescence signal in the mice in this experiment could not be detected, probably because the injection dose of macrophages was less and could not be observed.
[0072] Comparative Example 6
[0073] Using the Dil probe-labeled macrophages prepared by incubating for 20 min in Example 1, when intraperitoneally injecting mice, the injection dose was 2×10 7 cells / mouse, and other steps were the same. A total of 10 mice were injected. After 72 h of injection, 4 mice died after 72 h of injection, which might be caused by the burden of injecting high-dose Dil probe-labeled macrophages. The surviving mice were subjected to in vivo imaging to observe the fluorescence signal distribution in the mice.
[0074] The results showed that in addition to the liver tissue, there was also fluorescence signal accumulation in other tissues of the surviving mice, greatly increasing the background noise. This indicated that injecting a large dose of macrophages might not effectively target the liver.
[0075] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A method for targeting liver tissue to trace macrophages, characterized in that: The following steps are involved: The macrophages are mixed with the cell membrane staining working solution containing the Dil probe and incubated for 5 to 20 minutes, and impurities are removed to obtain macrophages labeled with the Dil probe; The Dil probe-labeled macrophages are intraperitoneally injected into a living animal, and the fluorescent signal is detected by in vivo imaging of the animal, and the migration path and distribution of the macrophages are obtained according to the distribution of the fluorescent signal.
2. The method according to claim 1, characterized in that: The incubation density of the macrophages was 1×10 6 / ml~2×10 6 Pieces / ml.
3. The method according to claim 1, characterized in that: The working volume concentration of the Dil probe in the cell membrane staining working solution containing the Dil probe is 0.25%.
4. The method according to claim 3, characterized in that: The cell membrane staining working solution containing the Dil probe also includes a staining enhancement solution with a volume concentration of 0.25% and a staining buffer solution with a volume concentration of 99.5%.
5. The method according to claim 1, characterized in that: The incubation temperature is 36-38°C.
6. The method according to claim 1, characterized in that: The incubation is carried out in the dark.
7. The preparation method according to any one of claims 1 to 6, characterized in that: The impurity removal method includes removing the liquid phase in the incubation system and washing with the cell culture fluid.
8. The preparation method according to claim 7, characterized in that: The number of times of removing impurities includes 2 to 3 times.
9. The method according to claim 1, characterized in that: The injection dose of the Dil probe-labeled macrophages was 1×10 6 Live animals~5×10 6 Live animals.
10. The method according to claim 1, characterized in that: The living animal comprises at least one of the following: a mouse, a rat and a hamster.