Method for establishing a naked mole rat immunodeficient animal model

Abstract

The application relates to the field of animal model construction, in particular to a naked mole rat immunodeficiency animal model establishment method, which is established by regularly injecting a naked mole rat with a chloromethylene bisphosphonate-liposome, and the naked mole rat immunodeficiency animal model is established. The naked mole rat immunodeficiency animal model establishment method has the advantages of short cycle, simple operation, good reproducibility, remarkable immune intervention effect, and can be used for the research on the related functions of the naked mole rat macrophage action mechanism.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically, a method for establishing a naked mole-rat immunodeficiency animal model. Background Technology

[0002] Naked mole-rats (Heterocephalus glaber, NMR) are long-lived rodents that have long inhabited resource-scarce, damp, and harsh underground environments. However, they rarely contract viruses or parasites and almost never develop neurodegenerative diseases or cancer. Therefore, they provide favorable conditions for our research on anti-inflammatory and anti-cancer agents. In addition, naked mole-rats, as novel experimental animals, also possess unusual characteristics such as tolerance to low oxygen levels, pain tolerance, anti-cancer properties, and anti-aging effects.

[0003] The naked mole-rat's immune system has many unusual characteristics compared to mice and humans, especially its good anti-inflammatory and anti-cancer properties. Studies have found that the naked mole-rat's spleen is relatively small compared to that of mice, and the spleen tissue shows a lower cell density, smaller white medullary follicles, and fewer B and T cells.

[0004] Compared to bone marrow isolated from ICR mice, naked mole-rats exhibited higher phagocytic capacity. Furthermore, under LPS or polyI:C induction, naked mole-rats' macrophages showed greater resistance to apoptosis and higher expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interferon-β [IFN-β]). Macrophages are crucial for maintaining the body's defense and homeostasis. Macrophages possess a broad spectrum of PAMP, Fc-, and scavenger receptors on their surface. Under inflammatory conditions, macrophages secrete cytokines to fine-tune the immune response, aiding in pathogen clearance. On the other hand, the anti-inflammatory functions of macrophages (such as tissue remodeling and immunosuppression) are related to catabolism; macrophage function is not only closely related to cellular metabolism but also to tissue and systemic energy requirements. Gene expression analysis revealed that naked mole-rats possess systemic metabolic adaptations, such as lower mitochondrial respiratory chain performance, the use of fatty acids as a major source of OXPHOS, and elevated baseline levels of HIF1-α, indicating a metabolic response to environmental stress that differs significantly from most other mammals.

[0005] Given the prevalence and unique metabolism of myeloid cells in the naked mole-rat immune system, we anticipate that naked mole-rat macrophages may acquire some unique adaptive characteristics, which can be traced through transcriptomics and immunometabolic profiling. Therefore, this invention aims to construct an immune intervention model in naked mole-rats, laying the foundation for subsequent research on the mechanism of action of naked mole-rat macrophages. Summary of the Invention

[0006] The purpose of this invention is to provide a method for establishing an immune intervention model of naked mole rats that is short in duration, easy to operate, highly reproducible, and has significant effects on immunodeficiency.

[0007] To achieve the above objectives, the present invention provides a method for establishing a naked mole-rat immunodeficiency animal model, wherein the method involves periodically injecting naked mole-rats with chlorophosphonate-liposomes to establish the naked mole-rat immunodeficiency animal model.

[0008] Furthermore, the chlorophosphonate-liposome injection site is intraperitoneal injection in naked mole rats.

[0009] Furthermore, the chlorophosphonate-liposome injection method involves multiple repeated injections, repeated every 3 days, and continuous injection is required to maintain the animal model status.

[0010] Furthermore, the concentration of the chlorphosphoate-liposome injection is 0.05 mg / g.

[0011] Furthermore, the chlorphosphoate-liposome injection interval is 3 days.

[0012] In a more preferred embodiment of the present invention, the method specifically includes the following steps:

[0013] Healthy adult naked mole-rats, weighing 25-40g, were kept in a housing with a temperature of 30℃ and humidity of 40-70%. Before the experiment, the animals were fasted but allowed to drink water for 12 hours. The naked mole-rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg / kg, 2.5%, 0.2ml / 100g body weight). After anesthesia, the naked mole-rats were restrained, and clophosphonate-liposomes were slowly injected intraperitoneally using a syringe. Before injection, the clophosphonate-liposomes were brought to room temperature and mixed thoroughly by inverting the syringe. The injection dose was 0.05mg / g. The injection interval was 3 days.

[0014] The advantages and beneficial effects of this invention are as follows:

[0015] 1. The method for establishing a naked mole-rat immunodeficiency animal model according to the present invention can establish a naked mole-rat immunodeficiency animal model with a short cycle, simple operation, good reproducibility, and significant immune intervention effect.

[0016] 2. The animal model established in this invention can be used to study the relevant functions of the naked mole rat macrophage mechanism. Attached Figure Description

[0017] Figure 1 Serum ALT / AST levels in naked mole rats.

[0018] Figure 2 Changes in liver macrophage markers in naked mole rats.

[0019] Figure 3 Changes in macrophages in the liver after macrophage clearance.

[0020] Figure 4 Pathological changes in the spleen, liver, and lungs of naked mole-rats. Detailed Implementation

[0021] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0022] Example 1: Establishment of an immunodeficient animal model of chlorphosphoside-liposome clearance of naked mole rat macrophages

[0023] (1) Materials and reagents

[0024] The chlorphosphophosphate-liposome reagent was purchased from LIPOSOMA in the Netherlands (catalog number CP-005-005).

[0025] (2) Animals and Grouping

[0026] Twenty-four healthy adult naked mole-rats, weighing 25-40g, were raised and preserved in our laboratory. Based on their weight, they were randomly divided into a control group and a model group, with 12 animals in each group. Each naked mole-rats was housed individually in a cage, fed in a normal environment and with normal feed. The temperature in the rearing room was controlled at 30℃, and the humidity at 40-70%. The cages and experimental environment were regularly disinfected. The experiment was conducted after one week of acclimatization.

[0027] (3) Establishment Method

[0028] Before the experiment, the animals were fasted for 12 hours but allowed free access to water. All naked mole-rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg, 2.5%, 0.2 ml / 100 g body weight). After anesthesia, the naked mole-rats were restrained by hand, and clophosphonate-liposomes were slowly injected intraperitoneally using a syringe (clophosphonate-liposomes were brought to room temperature and mixed thoroughly by inverting before injection; the injection dose was 0.05 mg / g). The animals were positioned with their abdomens facing upwards during injection. The injection interval was 3 days. The control group naked mole-rats were anesthetized in the same way and then injected with PBS liposomes (injection dose of 0.05 mg / g) using the same injection method.

[0029] After modeling, the naked mole-rats were observed daily to monitor their survival status, including their mental state, behavior, skin and fur, nasal and oral secretions, and defecation and urination. The naked mole-rats were weighed twice a week after modeling.

[0030] Example 2: Detection of an immunodeficient animal model of naked mole rat macrophages cleared by clophosphate-liposomes

[0031] (1) Animal weight changes

[0032] After the macrophages were cleared, the naked mole rats maintained normal daily activities, and their weight did not change significantly compared to the control group.

[0033] Table 1. Weight changes in naked mole rats

[0034]

[0035]

[0036] Note: *P>0.05 compared with the control group.

[0037] (2) Detection of organ indices

[0038] Organ coefficient refers to the ratio of the weight of internal organs to body weight (kg or 100g), and water content (g) refers to the difference between the wet weight and dry weight of the organ. After rinsing slightly with physiological saline and drying the surface water of the organs, they were immediately weighed on a balance with a sensitivity of 1 / 100 gram. The weight obtained was divided by the body weight to obtain the organ coefficient. The heart, liver, spleen, lungs, and kidneys were separated, and the mass of the heart, liver, spleen, lungs, and kidneys was measured using an analytical balance. The indices of the heart, liver, spleen, lungs, and kidneys in each group of naked mole rats were calculated: for example, spleen index = spleen mass (mg) / body mass (g). The results showed that there was no significant difference in body weight between the clophosphonate liposome clearance group and the control group. In terms of organ coefficient results, there was no significant difference between the model group and the control group (Table 3).

[0039] Table 2 Weights of various organs in naked mole rats

[0040]

[0041] Note: *P>0.05 compared with the control group.

[0042] Table 3. Organ coefficients of naked mole rats

[0043]

[0044] Note: *P>0.05 compared with the control group.

[0045] (3) Serum ALT / AST levels in naked mole rats were related to liver injury.

[0046] ALT and AST are important indicators for evaluating liver injury. When hepatocytes are damaged, these two enzymes are released from hepatocytes, leading to a significant increase in their levels in serum. This study measured serum ALT and AST levels, which are related to liver tissue injury. Blood samples were collected from naked mole rats and incubated at 37°C for 1 hour. After centrifugation at 1000g, the serum was collected and stored at -80°C. 2 μL of serum was used for ALT / AST analysis.

[0047] The ALT / AST liver injury experiment results showed no significant difference in ALT / AST levels between the Cl-Lip 0.05 mg / g group and the PBS-lip group in naked mole rat livers, demonstrating that neither the PBS-lip group nor the Cl-Lip 0.05 mg / g group caused liver damage. Figure 1 ).

[0048] (4) Changes in the macrophage marker CD68

[0049] Macrophages are typically identified using methods such as real-time quantitative PCR and immunofluorescence assays. Detection is generally performed using macrophage markers, including CD68, CD80, CD86, and CD32. Primers were designed based on the naked mole-rat genome (XM_004857437), specifically for CD68. The primer design is as follows:

[0050]

[0051] Real-time quantitative PCR results showed that, compared with the PBS-lip group, the transcriptional level of CD68, a macrophage marker in the liver of naked mole rats, was significantly decreased in the Cl-Lip 0.05 mg / g group, demonstrating that the Cl-Lip 0.05 mg / g group had a significant effect on clearing macrophages. Figure 2 ).

[0052] (5) Changes in macrophage markers in the liver and spleen after macrophage clearance

[0053] Naked mole-rat tissues and organs were dissected and fixed in a neutral, universal tissue fixative. Pathological sections of major organs were prepared and immunohistochemically stained. One section of each tissue was prepared and examined under a light microscope to observe the pathological damage. Remaining tissues were placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for later use. Naked mole-rat liver sections were stained with a monoclonal antibody (CD11b). Normal livers were positive for Kupffer cells, while the naked mole-rat model group showed depletion of Kupffer cells in the liver.

[0054] Immunohistochemical results of naked mole-rat liver macrophages showed that the number of macrophages in the model group was significantly reduced, and the macrophage clearance effect was significant. Figure 3 ).

[0055] (6) Pathological changes in the liver, lungs and spleen

[0056] Organs (liver, spleen, and lungs) from naked mole-rats were dissected and fixed in a neutral general-purpose tissue fixative. Pathological sections were prepared and stained with hematoxylin and eosin. Three sections of each tissue were prepared and examined under a light microscope to observe the pathological damage. No obvious lesions were found in the naked mole-rats' organs (liver, spleen, and lungs). Figure 4).

[0057] The above results demonstrate that the present invention can effectively clear macrophages from naked mole rats, with no pathological damage observed in the spleen, lungs, or liver. The symptoms reported are consistent with those of other mice or rats after macrophage clearance, indicating that chlorphosphodiesterate-liposomes can successfully clear macrophages from naked mole rats, successfully constructing a naked mole rat immune intervention model. This model is characterized by its short cycle, good reproducibility, simple operation, and significant immune intervention effect.

[0058] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for establishing a naked mole-rat immunodeficiency animal model, characterized in that, Includes the following steps: Healthy adult naked mole-rats, weighing 25-40g, were used. The temperature in the enclosure was controlled at 30℃ and the humidity at 40-70%. Before the experiment, the animals were fasted but allowed to drink water for 12 hours. The naked mole-rats were anesthetized by intraperitoneal injection of sodium pentobarbital. After the animals were anesthetized, they were restrained and clophosphonate-liposomes were slowly injected intraperitoneally using a syringe. Before injection, the clophosphonate-liposomes were brought to room temperature and mixed thoroughly by inverting the syringe. The injection dose was 0.05mg / g. The injection interval was 3 days.

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