Hepatocapillary nematode parasite infected Bush vole model as well as construction method and application thereof

Abstract

The invention relates to the field of biomedicine, in particular to a Bush vole model infected by hepatic capillary nematode parasites and a construction method and application of the Bush vole model, and the construction method of the model comprises the following steps: separating and collecting hepatic capillary nematode eggs from naturally infected Bush vole livers; 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 parasite load can be accurately controlled through oral infection, then the Bush vole model with different parasite loads for hepatic capillary nematode parasite infection can be obtained, and the model can be used for researching the pathogenesis of hepatic capillary nematode and evaluating the curative effects of anti-parasite drugs and immunomodulators, and has important scientific research and clinical application values.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to a model of Hepatic capillaria parasite-infected Bush voles, a construction method thereof, and an application thereof. Background Art

[0002] The theory of reproductive cost mediated by parasites reveals that the resource allocation trade-off between reproduction and the immune system is the core mechanism regulating the survival and reproductive strategies of hosts. Although this phenomenon has been widely verified at the individual level, its population ecological effects and regulatory mechanisms remain unclear. Existing studies on Hepatic capillaria-infected Bush voles have significant limitations: Although the animal model constructed by natural infection with metacercaria can achieve pathogen colonization, it cannot effectively reflect the true impact of parasites on host population dynamics, and its regulatory role is often masked by environmental factor interference. Summary of the Invention

[0003] To solve the above problems, the present invention provides a model of Hepatic capillaria parasite-infected Bush voles, a construction method thereof, and an application thereof. This model can effectively decouple confounding factors, reveal the regulatory rules of parasite infection on host population fluctuations, and provide a new research platform for elucidating the ecological and evolutionary mechanisms of pathogen-host interactions.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A construction method of a model of Hepatic capillaria parasite-infected Bush voles includes the following steps:

[0006] S1. Isolate and collect Hepatic capillaria eggs from the liver of naturally infected Bush voles by artificial digestion method; specifically:

[0007] 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% (w / v) pepsin, 0.7% (v / v) hydrochloric acid, and 0.9% (w / v) sodium chloride at a ratio of 1:10 (w / v);

[0008] b. Enzymatic digestion and separation stage: Oscillate and digest in a constant temperature shaker at 42 ± 0.5°C at 120 rpm for 5 ± 0.5 hours;

[0009] c. Purification stage: Remove larger residual liver tissue 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 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 high-purity egg suspension.

[0010] S2. Place the obtained Hepatic capillaria eggs in a filter paper culture system containing 1% formaldehyde, and culture them at a constant temperature of 30°C until they become infective eggs;

[0011] S3. Infect experimental animals with infective-stage eggs via gavage, with an inoculation dose of 500 - 1500 eggs per animal.

[0012] Furthermore, the screening criteria for the experimental animals are as follows: healthy male striped field mice, 6 - 8 weeks old, weighing 30 ± 5 g, and individually caged for 1 week for adaptation.

[0013] The present invention also provides a model of hepatic capillariasis-infected striped field mice, which is constructed by the above-mentioned method for constructing a model of hepatic capillariasis-infected striped field mice and can be used to reveal the host immune-reproductive interaction mechanism under parasite infection conditions.

[0014] Specifically, the model of hepatic capillariasis-infected striped field mice described in the present invention can be used to systematically monitor humoral immunity indexes: dynamically detect the serum IgG titer and the expression profiles of Th1 / Th2 cytokines IL-6 and IL-10 by ELISA.

[0015] The model of hepatic capillariasis-infected striped field mice described in the present invention can be used to analyze the neuroendocrine regulation mechanism: detect the expression levels of key nodes GnRH, FSH, and LH of the HPG axis and the kinetics of testosterone synthesis by qPCR and radioimmunoassay.

[0016] The model of hepatic capillariasis-infected striped field mice described in the present invention can be used to evaluate nutritional intervention strategies: establish a retinol gradient supplementation model (0 - 5000 IU / kg), and evaluate its regulatory effects on the regeneration of reproductive epithelial cells and the balance of T lymphocyte subsets by histopathology and flow cytometry.

[0017] The present invention can precisely control the parasite load through oral infection, and thus a model of hepatic capillariasis-infected striped field mice with different parasite loads can be obtained. This model can be used to study the pathogenic mechanism of hepatic capillariasis, evaluate the efficacy of anti-parasitic 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. Moreover, this model has the advantages of simple operation, good repeatability, and reliable experimental results, providing an important experimental tool for the study of the pathological mechanism of hepatic capillariasis infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To capture the location and living environment of striped field mice;

[0019] Figure 2 Flow chart for establishing a model of hepatic capillariasis-infected striped field mice;

[0020] Figure 3Magnified views of various parts of the adult Capillaria hepatica;

[0021] Figure 4 Incubation of Capillaria hepatica eggs;

[0022] Figure 5 Conditions of the liver of Microtus brandti before and after infection;

[0023] Figure 6 ELISA results of the serum of Microtus brandti;

[0024] Figure 7 Results of the egg burden test of the liver;

[0025] Figure 8 Results of the quantitative RT-PCR analysis of the expression of genes related to spermatogenesis in the testis; Specific implementation manners

[0026] 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.

[0027] Example 1: Establishment of a Capillaria hepatica infection model

[0028] Experimental animals

[0029] Capture healthy male Microtus brandti 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. Each group of mice drinks water and eats freely.

[0030] Collect eggs by the artificial digestion method:

[0031] Using aseptic dissection operation, completely remove the diseased liver of the Capillaria hepatica-positive Microtus brandti, 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 ± 0.5°C at 120 rpm for 5 ± 0.5 h. Remove larger residual liver tissues through a 100-mesh sieve, let the filtrate naturally precipitate for 30 min, centrifuge and wash, and divide the remaining sediment into 1.5-mL ep tubes, centrifuge at 1500×g 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.

[0032] Egg culture:

[0033] Prepare culture dishes, absorbent cotton, and filter paper. Tear the absorbent cotton and lay a single layer at the bottom of the culture dish. Cut the filter paper into a circular shape the same size as the bottom of the culture dish and place it on the absorbent cotton. Then, use a 1 mL pipette to add 1% formaldehyde to each culture dish to wet the filter paper. Subsequently, evenly drip the clarified solution containing Capillaria hepatica eggs on the filter paper, cover the lid of the culture dish, mark the date, parasite species, and 1% formaldehyde, and place it in an incubator at 30 °C for culture. Start microscopic examination two days after culturing: Use a pipette to aspirate a small amount of the solution and drop it on a glass slide, cover it with a coverslip, and observe the eggs under a 100X field of view of the microscope. Add 1% formaldehyde every two days thereafter and observe the development of the eggs under the microscope until they develop into infective eggs.

[0034] Infection experiment:

[0035] After one week of single-cage adaptive feeding of healthy male Mastomys natalensis, they were randomly divided into four groups, with 4 in each group: blank control group (non-infected group), Ch1 (low-intensity infection group), Ch2 (medium-intensity infection group), and Ch3 (high-intensity infection group). Infect the Mastomys natalensis with eggs cultured to contain infective larvae through the gavage route, with infection doses of 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. A suitable breeding environment can reduce the impact of environmental factors on experimental animals, and daily observation helps to promptly detect physiological changes after infection. The results showed that there were 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) decreased, indicating that the infected mice reduced their carcass weights, decreased the energy required for themselves, and invested more energy in immunity.

[0036] Experimental example 2:

[0037] On the basis of Example 1, detect immune and inflammation-related proteins in the serum of Mastomys natalensis by ELISA:

[0038] Six weeks after the start of the experiment, take 1 mL of fresh heart blood from each Mastomys natalensis and place it in a centrifuge tube for centrifugation at 3000 rpm for 10 min to prepare serum for detecting the concentrations of immunoglobulin G (IgG), interleukin 6 (IL-6), and interleukin 10 (IL-10) in the serum of Mastomys natalensis by ELISA.

[0039] The ELISA results of the serum of Mastomys natalensis are as Figure 6As shown: The serum immune albumin (IgG) of the Bushtail voles in the group infected with Capillaria hepatica was significantly increased compared with that of the normal Bushtail voles, indicating that parasite infection promotes the body to produce an immune response. In addition, interleukin 6 and interleukin 10 were also significantly increased in the serum of the infected Bushtail voles, suggesting that infection promotes the inflammatory response.

[0040] Experimental Example 3:

[0041] Six weeks after the start of the experiment, the blank control group and the infected group of Bushtail voles were dissected, and the pathological characteristics of the Bushtail voles were observed:

[0042] After dissecting the blank control group and the infected group of Bushtail voles, the pathological characteristics of the Bushtail voles were observed:

[0043] The autopsy diagram of the Bushtail vole liver is as Figure 5 shown: The surface of the liver in the blank control group was smooth, with a normal dark red color; after infection with Capillaria hepatica, the liver was enlarged, and a large number of lesion areas could be seen on the surface with the naked eye, showing punctate pearl-like white granules or nodules. When the liver was cut 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.

[0044] Under the light microscope, it can be clearly seen that the hepatic lobule structure of the Bushtail voles in the blank control group is complete, the hepatocytes are arranged neatly in cords, radiating from the central vein to the surroundings, the cell nuclei are large and round, without degeneration and necrosis, and there is no infiltration of inflammatory cells. In the liver of the infected group of Bushtail voles, Capillaria hepatica worms and eggs can be seen. A large number of eggs aggregate in a mass, and a large number of eosinophilic granulocytes and lymphocytes infiltrate in the mass. Fibroblasts proliferate around the mass to form a cyst, and the hepatocytes show focal necrosis, and some cells also show vacuolar degeneration. Capillaria hepatica parasitizes and lays eggs in the hepatic sinusoids of the host, causing varying degrees of dilation.

[0045] Experimental Example 4:

[0046] Six weeks after the start of the experiment, blood was drawn from the Bushtail vole model in the infected group for testing, 1 mL was taken from each Bushtail vole, and the effects of Capillaria hepatica on the HPG axis and sex hormone secretion were observed.

[0047] It was observed that the serum GnRH level of the mice in the Ch group was significantly increased, while the contents of FSH and LH in the serum were both significantly decreased, and finally the level of the male hormone testosterone (T) in the mice was significantly decreased. These results indicate that worm infection can affect the level of male sex hormones, may lead to abnormal spermatogenesis in the testis, and ultimately affect the reproductive ability of the mice.

[0048] Experimental Example 5:

[0049] Six weeks after the start of the experiment, the Brandt's voles in each infected group were dissected to observe the effects of different infection intensities of Brandt's voles on the liver.

[0050] 5.1 Degree of liver lesions

[0051] In the Brandt's voles in each infected group, lesions could be observed in different parts of the liver lobe after infection with Capillaria hepatica. 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 damage was significantly aggravated.

[0052] 5.2 Egg load in the liver

[0053] Parasite load is a measure of the number and virulence of parasites carried by the host organism. The number of Capillaria hepatica eggs in the livers of Brandt's voles in each infected group was tested, and the results are shown in Figure 7 as follows: In different infected groups, with the increase in infection intensity, the egg count of Capillaria hepatica in each gram of mouse liver also gradually increased. 5.3 Changes in liver function indexes in the sera of infected mice

[0054] ALT and AST are commonly used indicators reflecting liver parenchymal damage. The 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. After detecting the concentrations of AST and ALT in the sera of Brandt's voles in each infected group, it was found that the levels of serum ALT and AST in the infected group of Brandt's voles were significantly higher than those in the blank control group.

[0055] Experimental Example 6:

[0056] 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, and was divided into a Capillaria hepatica non-infected group and an infected group: the non-infected group was Con (blank control group), RE group (retinol administration group); the infected group was Ch (Capillaria hepatica infected group), 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.

[0057] 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 and feeding processes among the groups.

[0058] From the 1st day to the 3rd day of the experiment, the infected group of mice was challenged 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 RE group (retinol administration group) and the Con (blank control group) were gavaged with 100 μl of PBS. After culturing the mice for four weeks, the RE group (retinol administration group) and the RE+Ch group (Capillaria hepatica infection plus retinol supplementation group) began to be supplemented with retinol. Each mouse in the administration group was gavaged with 100 μl of soybean oil containing 50 mg / kg of retinol, while the other two groups were gavaged with 100 μl of soybean oil in the same manner, once every two days. After continuous gavage for two weeks, samples were collected.

[0059] 1) Using the tissue pathological section technique, observe the sperm indexes of Microtus brandti.

[0060] Through sperm counting, it was found that the testicular index of the infected group decreased slightly compared with the blank control group (non-infected group). Obvious abnormal sperm were observed in the Ch group. The sperm were smeared and stained, and the sperm morphology was analyzed. It was found that there were a large number of abnormally shaped sperm in both groups. Statistical analysis showed that head abnormalities accounted for the main abnormal type, and the tail morphology was basically normal. Most of the head abnormalities were sperm with separated heads and tails.

[0061] 2) RT-PCR detection of related gene expression

[0062] 2.1 Tissue RNA extraction

[0063] (1) Pretreatment: Add 2 - 3 small steel beads and 1 ml of Trizol to each tube of tissue, and break it with a tissue grinder. (Generally, 40 mg of tissue. If there is too much tissue, there will be interference from genomic DNA.) The processed homogenate is stored at -80 °C, and RNA is extracted as soon as possible.

[0064] (2) Vortex the ground tissue homogenate, balance the samples, and transfer 500 - 1000 μl of the supernatant to a new 1.5 mL RNase-free EP tube after centrifuging at 12000 rpm for 10 min.

[0065] (3) Add 200 μl of chloroform, gently invert up and down 6 times to mix evenly, and let it stand at room temperature for 15 minutes to separate layers.

[0066] (5) Pre-cool the centrifuge in advance at 4 °C, centrifuge at 12000 rpm for 15 minutes, and transfer 500 μl of the upper aqueous phase to a new RNase-free 1.5 mL EP tube.

[0067] (6) Add 500 μl of isopropanol (pre-cooled), gently invert up and down 5 times, and let it stand for 5 minutes. Then centrifuge at 12000 rmp at 4 °C for 10 minutes, and pour off the supernatant.

[0068] (7) Add 1 mL of DEPC water to prepare 75% ethanol (pre-cooled), gently pipette up and down to mix well, centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0069] (8) Discard the supernatant, slowly aspirate the supernatant around the pellet with a pipette until it is completely clean, and dry at room temperature for 15 minutes.

[0070] (9) Add 30 μL of DEPC water, dissolve the RNA at room temperature for 10 minutes, centrifuge clockwise, detect the RNA concentration and purity and record, and store the sample at -80°C.

[0071] 2.2 Reverse transcription of cDNA

[0072] Operate according to the instructions in the Promega reverse transcription kit, and use the extracted tissue RNA as a template for reverse transcription.

[0073] The first component system is 5 μL, including 3 μL of template RNA, 1 μL of Random Primers, and 1 μL of Oligo(dT). After mixing evenly, place it in a metal bath at 70°C for heat shock for 5 minutes, then immediately transfer it to ice and incubate on ice for 5 minutes.

[0074] Add 6 μL of Nuclease-free Water, 2.5 μL of MgCl2, 4 μL of Reverse Transcription 5×Buffer, 1 μL of dNTP Mixture, 0.5 μL of Ribonuclease Inhibitor, and 1 μL of AMV Reverse Transcriptase to the first component, for a total reaction system of 20 μL.

[0075] After mixing well, place it in a PCR instrument for reverse transcription. The reverse transcription product obtained after the reaction can be stored in a -20°C refrigerator or used immediately.

[0076] Reaction conditions: 25°C for 5 minutes; 42°C for 60 minutes; 70°C for 15 minutes

[0077] 2.3 Primer synthesis

[0078] Design primers with the molecules related to retinol metabolism and spermatogenesis in mouse testis as the target genes. The primers are all from the published literature in Pubmed, and the fragment size is 20 - 23 bp. Verify the feasibility of the primers: Download the genomic sequences of the corresponding genes in mouse (Mus musculus) from the NCBI database respectively, use the Snap gene software to align the sequences with the primers, select mouse GAPDH as the internal reference gene, normalize the samples, and set the annealing temperature to 60°C.

[0079] Using the cDNA obtained by reverse transcription as a template, RT-PCR amplification was performed. When detecting the target gene for each sample, an internal reference gene needs to be set as a control at the same time. The prepared reaction solution (containing 2X Taq Pro Universal SYBR qPCR Master Mix, DEPC water, upstream and downstream primers) was mixed evenly, and after instantaneous centrifugation and mixing, it was aliquoted into an eight-strip row, 19 μL per well. Then, 1 μL of the template cDNA to be detected was added to the corresponding wells respectively. Finally, after mixing evenly, it was placed in a fluorescence quantitative PCR instrument.

[0080] Reaction conditions:

[0081]

[0082] After the reaction, the 2-△△Ct method was used for calculation to perform relative quantitative analysis on the mRNA expression level of the detected gene.

[0083] The results of quantitative RT-PCR analysis of the expression of genes related to spermatogenesis in the testis are as Figure 8 shown. In the Capillaria hepatica infection group, the expression level of the testicular Stra8 gene decreased significantly, while it recovered somewhat in the infection plus retinol supplementation group. Compared with the retinol supplementation group, the expression level of the 17α-hydroxylase (Cyp17A1) gene decreased significantly in the Capillaria hepatica infection group, with statistical significance, and retinol supplementation after Capillaria hepatica infection could significantly increase the expression of this gene. In addition, the results showed that there was no significant difference in the expression of the spermatogenesis-related 6 (SPATA6) gene among the groups, but compared with the blank control (con) group and the retinol supplementation (re) group, the expression level in the infection (ch) group showed a lower level. The above results were consistent with the results of mouse sperm count and sperm morphology observation, that is, compared with the retinol supplementation group, Capillaria hepatica infection could significantly down-regulate the expression of the Stra8 gene and Cyp17A1 gene related to spermatogenesis, and to a certain extent reduce the expression of the Spata6 gene, while the gene expression increased after retinol supplementation.

[0084] 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 Bush vole model infected with Capillaria hepatica parasites, characterized in that: The following steps are involved: S1. Isolate and collect Capillaria hepatica eggs from the liver of naturally infected Bush voles; S2, placing the obtained Capillaria hepatica eggs in a filter paper culture system containing 1% formaldehyde, and culturing them at a constant temperature of 30° C. until the eggs reach the infection stage; S3. Infectious worm eggs are inoculated into the experimental animals by oral gavage, with the inoculation dose being 500 to 1500 worm eggs per animal.

2. The method for constructing a Bush Vole model infected with Capillaria hepatica parasites according to claim 1, characterized in that: The step S1 is to separate and collect Capillaria hepatica eggs by artificial digestion, and comprises the following steps: a. Tissue pretreatment stage: The infected liver tissue was finely minced in 4°C physiological saline and mixed 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); b. Enzymatic separation stage: Digestion was carried out at 42±0.5℃ in a constant temperature shaker at 120 rpm for 5±0.5 hours; c. Purification stage: Remove larger residual liver tissue through a 100-mesh sieve, allow the filtrate to settle naturally for 30 minutes, and wash by centrifugation. The remaining sediment is divided into 1.5 mL eppendorf tubes and centrifuged at 1500×g for 3 times, each time for 5 minutes. Discard the supernatant and wash repeatedly with distilled water for 2 to 3 times until clarified to obtain a high-purity egg suspension.

3. The method for constructing a Bush Vole model infected with Capillaria hepatica parasites according to claim 1, characterized in that: The screening criteria for the experimental animals are: healthy male Bush voles, 6 to 8 weeks old, weighing 30±5g, and adaptively raised in a single cage for 1 week.

4. A Bush vole model infected with Capillaria hepatica parasites, characterized in that: The method for constructing a Bush vole model infected with Capillaria hepatica parasites as described in any one of claims 1 to 3 is used to construct the model.

5. The use of a Bush Vole model infected with Capillaria hepatica parasites as claimed in claim 4, characterized in that: Used to reveal the host immune-reproductive interaction mechanism under parasitic infection conditions.

6. The use of a Bush Vole model infected with Capillaria hepatica parasites as claimed in claim 5, characterized in that: Used for systematic monitoring of humoral immune indicators: ELISA method is used to dynamically detect serum IgG titer and Th1 / Th2 cytokine IL-6 and IL-10 expression profiles.

7. The use of a Bush Vole model infected with Capillaria hepatica parasites as claimed in claim 5, characterized in that: Used for analysis of neuroendocrine regulatory mechanisms: qPCR and radioimmunoassay were used to detect the expression levels of GnRH, FSH, LH, key nodes of the HPG axis, and the dynamics of testosterone synthesis.

8. The use of a Bush Vole model infected with Capillaria hepatica parasites as claimed in claim 5, characterized in that: For the evaluation of nutritional intervention strategies: a retinol gradient supplementation model (0-5000 IU / kg) was established, and its regulatory effects on germinal epithelial cell regeneration and T lymphocyte subset balance were evaluated by histopathology and flow cytometry.

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