Mouse hepatic capillary nematode infection model and construction method and application thereof

Abstract

The invention relates to the field of biomedicine, in particular to a mouse hepatic capillary nematode infection model and a construction method and application thereof, and the construction method of the model comprises the following steps: separating and collecting hepatic capillary nematode eggs from naturally infected mouse livers through an artificial digestion method; placing the obtained hepatic capillary nematode eggs in a filter paper culture system containing 1% formaldehyde, and culturing the eggs at a constant temperature of 30 DEG C until the eggs in an infection period; the eggs in the infection period are inoculated into experimental animal bodies through an intragastric administration way, and the inoculation dosage is 500-1500 eggs per animal. The model provided by the invention can be used for researching the pathogenesis of hepatic capillary nematode and evaluating the curative effects of anti-parasitic drugs and immunomodulators, and has important scientific research and clinical application values. And the model has the advantages of simplicity and convenience in operation, good repeatability, reliable experimental result and the like, and provides an important experimental tool for researching the pathological mechanism of hepatic capillary nematode infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a mouse hepatic capillariasis infection model, a construction method thereof, and an application thereof. Background Art

[0002] The theory of reproductive cost mediated by parasites suggests that the allocation trade-off between reproduction and immunity is at the core of regulating the trade-off between reproduction and survival, because increasing the resource allocation to reproduction will inhibit the resource allocation to immune defense. Although the evidence for this trade-off is extensive, the mechanism behind it remains poorly understood. Although there are many cases showing the impact of parasites at the individual level, relatively few studies have been conducted on the impact of parasites at the population level, and the existing research results also have limitations. Field studies often involve field observations of wild animals and sample collection for analysis, while model predictions use ecological models to predict the impact of parasites on host populations. Although these studies have revealed the regulatory role of parasites on host populations in some cases, there are also inconsistent and contradictory research results.

[0003] Currently, the existing mouse models infected with hepatic capillaria are generally constructed by the following methods: selection of experimental animals, obtaining hepatic parasite metacercariae through natural infection, oral infection, and obtaining a mouse model infected with hepatic capillaria. The functions of this type of mouse model infected with hepatic capillaria have great limitations. For example, it cannot show the impact of hepatic capillaria on the number of mice and is used for the study of the regulatory mechanisms of reproduction and the immune system. Summary of the Invention

[0004] To solve the above problems, the present invention provides a mouse hepatic capillariasis infection model, a construction method thereof, and an application thereof. The constructed model can be used to further study the pathogenesis mechanism of hepatic capillaria and the impact of hepatic capillaria infection on the mouse population, especially the regulatory mechanisms of its reproduction and immune system.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A construction method of a mouse hepatic capillariasis infection model includes the following steps:

[0007] S1. Separating and collecting hepatic capillaria eggs from the liver of naturally infected mice by an artificial digestion method; specifically:

[0008] a. Tissue pretreatment stage: finely cutting the infected liver tissue in physiological saline at 4°C, and mixing it with artificial gastric juice containing 1% (w / v) pepsin, 0.7% (v / v) hydrochloric acid, and 0.9% (w / v) sodium chloride at a ratio of 1:10 (w / v);

[0009] b. Enzymatic hydrolysis and separation stage: Digest at 120 rpm in a constant temperature shaker at 42°C for 4 - 6 hours;

[0010] c. Purification stage: Filter through a 100 - mesh sieve and then centrifuge and wash until clear to obtain a high - purity egg suspension.

[0011] S2. Place the obtained Capillaria hepatica eggs in a filter paper culture system containing 1% formaldehyde and culture at a constant temperature of 30°C until infective eggs are obtained;

[0012] S3. Inoculate the infective eggs into the experimental animals through gavage, with the inoculation dose being 500 - 1500 eggs per animal. Specifically, gradient infection is used: 500 eggs / animal in the low - infection group, 1000 eggs / animal in the medium - infection group, and 1500 eggs / animal in the high - infection group;

[0013] Furthermore, the screening criteria for the experimental animals are: healthy male mice, 6 - 8 weeks old, weighing 30 ± 5 g, and adaptively raised for 1 week.

[0014] The present invention also provides a Capillaria hepatica infection model in mice, which is constructed by the above - mentioned method for constructing a Capillaria hepatica parasite infection model in mice and can be used to study the mechanism of the impact of parasite infection on the host immune - reproduction trade - off.

[0015] Specifically, the Capillaria hepatica infection model in mice of the present invention can be used to detect the changes in the levels of host immunoglobulin IgG and inflammatory factors IL - 6 and IL - 10 after infection.

[0016] The Capillaria hepatica infection model in mice of the present invention can be used to analyze the mechanism of secretion disorders of hormones GnRH, FSH, LH, and testosterone in the hypothalamic - pituitary - testicular axis (HPG axis).

[0017] The Capillaria hepatica infection model in mice constructed by the present invention can be used to study the pathogenic mechanism of Capillaria hepatica, evaluate the efficacy of anti - parasite drugs and immunomodulators, and has important scientific research and clinical application values. And through this model, the regulatory mechanism of its reproduction and immune system can be mainly studied. This model has the advantages of simple operation, good repeatability, and reliable experimental results, and provides an important experimental tool for the research on the pathological mechanism of Capillaria hepatica infection. Description of the Drawings

[0018] Figure 1 It is a flowchart for establishing a Capillaria hepatica infection model in mice;

[0019] Figure 2Comparison chart of the concentrations of immunoglobulin G (IgG), interleukin 6 (IL-6), and interleukin 10 (IL-10) between the mouse model infected with Capillaria hepatica and normal mice;

[0020] Figure 3 Liver pathological section diagram of the mouse infected with Capillaria hepatica;

[0021] In the figure: (A) Blank control group, infection intensity: 0. (B) Low-intensity infection group, infection intensity: 500 eggs / mouse. (C) Medium-intensity infection group, infection intensity: 1000 eggs / mouse. (D) High-intensity infection group, infection intensity: 1500 eggs / mouse.

[0022] Figure 4 Comparison chart of the egg rates between the mouse model infected with Capillaria hepatica and normal mice;

[0023] Figure 5 Comparison chart of the activities of serum ALT and AST between the mouse model infected with Capillaria hepatica and normal mice;

[0024] Figure 6 Comparison chart of the sperm rates between the mouse model infected with Capillaria hepatica and normal mice;

[0025] Figure 7 Comparison chart of sperm deformities between the mouse model infected with Capillaria hepatica and normal mice;

[0026] Figure 8 Actual picture of the types of sperm deformities in mice.

[0027] Figure 9 H&E staining results of the testis tissues of mice in each group and the rate of sperm-free ducts in the testes of mice;

[0028] In the figure: (A) Testis section of the blank control group; (B) Testis section of the Capillaria hepatica-infected group; (C) Testis section of the retinol-supplemented group; (D) Testis section of the Capillaria hepatica-infected plus retinol-supplemented group; (E) Rate of sperm-free ducts in the testes of mice.

[0029] Figure 10 H&E staining results (40 times) of the testes of mice in each group;

[0030] In the figure: (A) Blank control group; (B) Capillaria hepatica-infected group, black arrows indicate sperm with separated heads and tails; (C) Retinol-supplemented group; (D) Capillaria hepatica-infected plus retinol-supplemented group.

[0031] Figure 11 Test results of the secretion of hormones GnRH, FSH, LH, and testosterone in the testicular axis (HPG axis) of mice in each group;

[0032] In the figure: (A) Concentration of luteinizing hormone in mouse serum; (B) Concentration of follicle-stimulating hormone in mouse serum; (C) Concentration of testosterone in mouse serum; (D) Concentration of gonadotropin-releasing hormone in mouse serum. Detailed implementation mode

[0033] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0034] Example 1: Establishment of a Capillaria hepatica infection model

[0035] Experimental animals

[0036] Purchase healthy male white mice at 6 - 8 weeks old, with a body weight of 30 ± 5 g. Breeding conditions: single-cage breeding, temperature controlled at about 23 °C, light 12L:12D. Mice in each group drink water and eat freely.

[0037] Collect eggs by artificial digestion method:

[0038] Using aseptic dissection operation, completely remove the diseased liver of the Capillaria hepatica-positive white mice, put it into an EP tube, weigh it, and record. Finely cut the diseased liver in physiological saline at 4 °C, mix it with artificial gastric juice (containing 1% (w / v) pepsin, 0.7% (v / v) hydrochloric acid, and 0.9% (w / v) sodium chloride) at a ratio of 1 g:10 mL, and oscillate and digest in a constant temperature shaker at 42 °C at 120 rpm for 4 - 6 h. Remove larger residual liver tissues through a 100-mesh sieve, let the filtrate naturally precipitate for 30 min, centrifuge and wash, divide the remaining sediment into 1.5 mL ep tubes, centrifuge at 1500×g in gradients for 3 times, 5 min each time, discard the supernatant, and then wash repeatedly with distilled water 2 to 3 times until it is clear to obtain a clear solution containing Capillaria hepatica eggs, collect the eggs, and label. Make a smear, count under the microscope, and reserve for use.

[0039] Egg culture:

[0040] Prepare petri dishes, absorbent cotton, and filter paper. Tear the absorbent cotton and lay a single layer at the bottom of the petri dish. Cut the filter paper into a circular shape the same size as the bottom of the petri dish and place it on the absorbent cotton. Then, use a 1 mL pipette to add 1% formaldehyde to each petri dish to moisten the filter paper. After that, evenly drip the solution containing Capillaria hepatica eggs on the filter paper, cover the lid of the petri dish, mark the date, parasite species, and 1% formaldehyde, and place it in an incubator at 30°C for cultivation. Start microscopic examination two days after cultivation: Use a pipette to suck a small amount of the solution and drop it on a glass slide, cover it with a coverslip, and observe the eggs under a microscope at 100X magnification. Add 1% formaldehyde every two days and observe the development of the eggs under the microscope until they develop into infective eggs.

[0041] Infection experiment:

[0042] After one week of single-cage adaptive feeding of healthy male mice, they were randomly divided into four groups: con (blank control group), Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group), with 4 mice in each group. Infect the mice by gavage with the eggs cultured to contain infective larvae, and the infection doses are 500 per mouse in the Ch1 group, 1000 per mouse in the Ch2 group, and 1500 per mouse in the Ch3 group. Observe and record the appearance, behavior, diet, and drinking water of the mice daily, and regularly measure their body weights. The results show that there are no significant differences in body weight and food intake among the groups, while the carcass rates of Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group) decrease, indicating that the infected mice reduce their carcass weights, reduce the energy required for themselves, and invest more energy in immunity.

[0043] Example 2:

[0044] Based on Example 1, detect the immune and inflammation-related proteins in the serum of mice by ELISA:

[0045] Six weeks after the start of the experiment, take 1 mL of fresh heart blood from each mouse and place it in a centrifuge tube for centrifugation at 3000 rpm for 10 min to prepare serum. Detect the concentrations of immunoglobulin G (IgG), interleukin 6 (IL-6), and interleukin 10 (IL-10) in the serum of mice by ELISA.

[0046] The mouse serum ELISA is as Figure 2 shown: The serum immune albumin (IgG) of the mice infected with Capillaria hepatica is significantly increased compared with that of normal mice, indicating that parasite infection promotes the body to produce an immune response. In addition, interleukin 6 and interleukin 10 are also significantly increased in the serum of the infected mice, suggesting that infection promotes the inflammatory response.

[0047] Example 3:

[0048] Six weeks after the start of the experiment, the blank control group and the infected group of mice were dissected, and the pathological characteristics of the mice were observed:

[0049] The pathological sections of the livers of mice infected with Capillaria hepatica are as Figure 3 shown: The surface of the liver in the blank control group (con) was smooth, with a normal dark red color; the livers of Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group) were enlarged, and a large number of pathological areas could be seen on the surface with the naked eye, presenting punctate pearl-like white granules or nodules. When the liver was cut open and divided with scissors, it was found that the liver tissue of the infected group was severely fibrotic and became brittle compared with the normal liver of the control group.

[0050] Under the light microscope, it could be clearly seen that the hepatic lobule structure of the mice in the blank control group (con) was intact, the hepatocytes were arranged neatly in cords, radiating from the central vein to the surrounding in a radial pattern, the cell nuclei were large and round, without degeneration or necrosis, and there was no infiltration of inflammatory cells. However, the bodies and eggs of Capillaria hepatica could be seen in the livers of the mice in Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group). A large number of eggs aggregated in masses, and a large number of eosinophilic granulocytes and lymphocytes infiltrated in the masses. Fibroblasts proliferated around the masses to form cysts, and there were focal necrosis of hepatocytes, and some cells also showed vacuolar degeneration. Capillaria hepatica parasitized and laid eggs in the hepatic sinusoids of the host, causing different degrees of dilation.

[0051] Example 4:

[0052] Six weeks after the start of the experiment, the mice in each infected group were dissected separately, and the effects of different infection intensities of mice on the liver were observed.

[0053] 4.1 Degree of liver lesions

[0054] After the mice in each infected group were infected with Capillaria hepatica, lesions could be observed in different parts of the liver lobes. As the infection intensity increased, the area of liver lesions increased with the increase in the infection intensity of Capillaria hepatica, and the degree of liver injury was significantly aggravated.

[0055] 4.2 Egg burden in the liver

[0056] Parasite burden is a measure of the number and virulence of parasites carried by the host organism. The test results of the number of Capillaria hepatica eggs in the livers of the mice in each infected group are as Figure 4 shown: Among different infected groups, the egg count of Capillaria hepatica per gram of mouse liver in Ch1 (low-intensity infection group) was significantly higher than that in Ch2 (medium-intensity infection group), and the egg count of Capillaria hepatica per gram of mouse liver in Ch2 (medium-intensity infection group) was significantly higher than that in Ch3 (high-intensity infection group). It can be seen that as the infection intensity increases, the egg count of Capillaria hepatica per gram of mouse liver also gradually increases.

[0057] 4.3 Changes in Liver Function Indexes in the Serum of Infected Mice

[0058] ALT and AST are common indicators reflecting liver parenchymal damage. An increase in the activities of serum AST and ALT indicates that inflammation causes an increase in the permeability of the liver cell membrane, and AST and ALT enter the plasma. The concentrations of AST and ALT in the serum of the mice in each infected group were measured, and the results are as Figure 5 shown: The levels of serum ALT and AST in the mice of Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group) were significantly higher than those in the blank control group (con).

[0059] Example 5:

[0060] After the experimental mice were adaptively fed in the animal house for one week, the formal experiment began. This experiment was divided into 4 groups, with 6 mice in each group, divided into the non-infected group and the infected group of Capillaria hepatica: the non-infected groups were Con (blank control group) and RE group (retinol administration group); the infected groups were Ch (Capillaria hepatica infection group) and RE+Ch group (Capillaria hepatica infection plus retinol supplementation group). Since retinol is extremely insoluble in water, soybean oil was selected as the solvent to dissolve retinol.

[0061] During the experiment, the status of the mice was observed every day to see if it was normal. All the mice were fed under the same environmental conditions, and there was no mutual contact during the drinking water and feeding processes among the groups.

[0062] From the 1st day to the 3rd day of the experiment, the infected mice were fed with the infective eggs of Capillaria hepatica by gavage at 4 pm every day. The infection intensity was 1500 eggs, and each mouse was gavaged with 100 μl in total. At the same time, the non-infected group was gavaged with 100 μl of PBS. After the mice were cultured for four weeks, retinol was supplemented to the RE group and the RE+Ch group. Each mouse in the administration group was gavaged with 100 μl of soybean oil containing 50 mg / kg of retinol. At the same time, the other two groups were gavaged with 100 μl of soybean oil in the same way, once every two days. After continuous gavage for two weeks, samples were collected.

[0063] (1) Sperm Count

[0064] 1. Take the unilateral intact epididymis of the mouse, place it in 200 μl of PBS, cut it into pieces until there are no obvious tissue masses, and add 800 μl of PBS and blow and suck to mix well;

[0065] 2. Place it in an oven at 37 °C for 30 minutes to fully release the sperm. Place it horizontally when placing to prevent the sperm from sinking to the bottom. During this period, it needs to be mixed 2-3 times;

[0066] 3. Place it on a hot stage at 65 °C for 10 minutes to make the sperm lose their vitality. Prepare a hemocytometer, scissors, and a vortex oscillator;

[0067] 4. After vortex mixing, perform dilution in a certain ratio and adjust the dilution ratio as appropriate.

[0068] 5. Take 10 μl of the liquid and add it to a hemocytometer for counting. After counting 4 - 6 times for each mouse, take the average value.

[0069] (2) Sperm malformation rate

[0070] Use the eosin - aniline black method to stain the inactivated sperm, and statistically observe the malformed sperm under a microscope. A total of 200 sperm are counted on each slide. Record the number of sperm with head malformations (such as amorphous head, no head, multiple heads, small head, and round head, etc.), tail malformations (such as short tail, no tail, coiled tail, bent tail, and irregular tail diameter, etc.), and head - tail malformations respectively. Calculate the proportion they account for, which is the sperm malformation rate. Sperm malformation rate = (number of malformed sperm / 200) × 100%.

[0071] The operation steps are as follows:

[0072] 1. Gently shake and mix the liquid containing sperm and perform gradient dilution.

[0073] 2. Take a clean small test tube, add 10 μL of fresh semen and an equal amount of eosin staining solution, mix well and let it stand for 15 s.

[0074] 3. Add 10 μL of aniline black staining solution, mix well, and it becomes semen - eosin - aniline black staining solution. Let it stand for 15 - 30 s.

[0075] 4. Add 5 μL of the above semen - eosin - aniline black staining solution onto a glass slide to make a smear.

[0076] 5. Air - dry, examine under a microscope, and count. Observe under an oil immersion objective. The sperm are red and the background is purplish - red. Use the tissue pathological section technology to observe the sperm indexes of the mice.

[0077] The results are as Figure 6 shown. Through sperm counting, it is found that the number of sperm in the epididymis of the infection group Ch (hepatic capillariasis infection group) is slightly reduced compared with the blank control group (con). Then, smear and stain the sperm to analyze the sperm morphology, as Figure 8 shown: There are a large number of sperm with abnormal morphology in the infection group (ch). Through statistical analysis, head abnormalities account for the main abnormal type, and the tail morphology is basically normal. Among the head abnormalities, most are sperm with separated head and tail. Calculate the sperm malformation rate of each group. The results are as Figure 7 shown: Lack of retinol and hepatic capillariasis infection can cause abnormal sperm deformation in mice, especially the weakening of the head - tail connection, resulting in the separation of the head and tail of a large number of sperm in these mice.

[0078] The testis sizes of the mice in the observation infection group, namely the Ch (Capillaria hepatica infection group) mice and the RE+Ch group (Capillaria hepatica infection plus retinol supplementation group), were observed and found to show no obvious abnormalities compared with those of the control mice. Through H&E staining of the testis tissues, it was observed that the testis tissues of the mice in the RE group (retinol administration group) and the RE+Ch group (Capillaria hepatica infection plus retinol supplementation group) were intact, with distinct structures, normal interstitial cells, and germ cells at various developmental stages existing in the seminiferous tubules. Spermatogonia, Sertoli cells, spermatocytes, round spermatozoa, and elongated spermatozoa were arranged in sequence from the tube wall to the lumen (C and D). However, in the seminiferous tubules of the mice lacking retinol and those infected with worms, a certain proportion of the tubules only contained spermatocytes and no late spermatids. Figure 9 (A and B). Through statistical analysis, it was found that 5.5% of the tubules in the testes of the mice lacking retinol had no late spermatids, and 11.4% of the tubules in the testes of the mice infected with worms had no late spermatids. Figure 9 (Figure E). These results indicate that both retinol deficiency and worm infection can cause meiotic arrest of spermatocytes in some seminiferous tubules of mice. Through further observation, in the seminiferous tubules of the mice supplemented with retinol and those supplemented with retinol after worm infection, the spermatozoa with complete flagella after completing metamorphosis were arranged in the center of the lumen. However, some of the spermatozoa arranged in the center of the lumen in the seminiferous tubules of the mice infected with worms lacked flagellar structures and showed abnormal morphologies. These results indicate that both retinol deficiency and worm infection can, to a certain extent, cause sperm production disorders in mice.

[0079] Example 6:

[0080] Six weeks after the start of the experiment, the mouse models in the infection group were bled for examination to observe the effects of Capillaria hepatica on the secretion of hormones GnRH, FSH, LH, and testosterone in the hypothalamic-pituitary-testicular axis (HPG axis). The results are as follows: Figure 11 As shown: Compared with the blank control group (con), the testis index of the infection group decreased slightly.

[0081] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a mouse hepatic capillaria hepatica infection model, characterized in that, It includes the following steps: S1. Isolate and collect Capillaria hepatica eggs from the liver of naturally infected mice; S2. Place the obtained Capillaria hepatica eggs in a filter paper culture system containing 1% formaldehyde and culture them to infective-stage eggs under the condition of constant temperature at 30°C; S3. Inoculate the infective-stage eggs into the experimental animals through the gavage route, and the inoculation dose is 500 - 1500 eggs per animal.

2. The construction method of a mouse liver capillaria hepatica infection model according to claim 1, characterized in that, In step S1, the Capillaria hepatica eggs are isolated and collected by the artificial digestion method, which includes the following steps: a. Tissue pretreatment stage: Finely cut the infected liver tissue in physiological saline at 4°C, and mix it with artificial gastric juice containing 1% pepsin, 0.7% hydrochloric acid and 0.9% sodium chloride at a ratio of 1 g:10 mL; b. Enzymatic digestion and separation stage: Oscillate and digest in a constant temperature shaker at 42°C at 120 rpm for 4 - 6 hours; c. Purification stage: Filter through a 100-mesh sieve and then centrifuge and wash until clear to obtain a high-purity egg suspension.

3. The construction method of a mouse hepatic capillaria hepatica infection model according to claim 1, characterized in that, The screening criteria for the experimental animals are: healthy male mice, 6 - 8 weeks old, weighing 30 ± 5 g, and adaptively raised for 1 week.

4. The construction method of a murine Capillaria hepatica infection model according to claim 1, characterized in that In step S3, gradient infection is adopted: 500 eggs per mouse in the low-infection group, 1000 eggs per mouse in the medium-infection group, and 1500 eggs per mouse in the high-infection group.

5. A mouse hepatic capillaria infection model, characterized in that, It is constructed by using the method for constructing a Capillaria hepatica parasite-infected mouse model according to any one of claims 1 - 4.

6. The application of a mouse Capillaria hepatica infection model according to claim 5, characterized in that: It is used to study the mechanism of the impact of parasite infection on the immune and reproductive trade-off of the host.

7. The application of a murine Capillaria hepatica infection model as claimed in claim 6, wherein: It is used to detect the changes in the levels of host immunoglobulin IgG and inflammatory factors IL-6 and IL-10 after infection.

8. The application of a mouse hepatic capillaria hepatica infection model according to claim 6, characterized in that: It is used to analyze the mechanism of secretion disorder of hormones GnRH, FSH, LH, and testosterone in the hypothalamus-pituitary-testicular axis.

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