Establishment method of mouse infection model of alveolar echinococcosis
Through ultrasound-assisted injection of vesicular echinococcal head joints in the mouse liver parenchyma, the problems of instability and inaccurate evaluation of existing vesicular echinococcal infection models were solved, efficient non-invasive dynamic monitoring and higher success rates were achieved, and a reliable platform for supporting drug development was achieved.
Patent Information
- Application Number
- CN202510871198.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-26
- Publication Date
- 2025-08-12
AI Technical Summary
The existing animal model establishment method for mice infected with vesiculococcusia has problems such as low infection success rate and unstable, uncontrollable cyst growth rate and site, and subjective lack of dynamic monitoring of live organs, which affects the repeatability and clinical transformation value of the experiment.
Ultrasound-assisted methods were used to accurately inject viscous echinococcal heads into the liver parenchyma of mice to avoid invasive operations, achieve non-invasive dynamic monitoring, improve the success rate and survival rate of infection, and reduce damage to the liver and other organs.
It significantly improves the success rate and survival rate of infected mouse models, shortens operating time, reduces experimental errors, realizes non-invasive dynamic monitoring of cysts and more realistic pathological simulation, and provides a reliable platform for drug development.
Smart Images
Figure CN120458766A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of animal model construction, in particular to a method for constructing a mouse model of alveolar echinococcosis infection. Background Art
[0002] Alveolar echinococcosis is a serious zoonotic parasitic disease caused by the larvae of Echinococcus cysticercus. Its transmission routes include contact with foxes or wild dogs, and ingestion of cyst eggs when skinning fox fur. The clinical treatment of alveolar echinococcosis faces three major challenges: (1) The cure rate of surgical resection is extremely low, with only 30% of patients being able to undergo surgery, and the postoperative recurrence rate exceeds 50%; (2) Drug treatment options are limited. Albendazole, the only proven effective drug, requires long-term use and has significant drug resistance and hepatotoxicity problems; (3) There is a lack of effective treatment options for patients in the late stage, metastatic lesions do not respond well to existing treatments, and there is a lack of targeted drugs. At present, the research on the immune escape mechanism of the larvae, host microenvironment interaction and drug resistance mechanism is still difficult, and interdisciplinary joint research is urgently needed to break through the bottleneck of diagnosis, treatment and prevention and control.
[0003] Currently, experimental studies of alveolar larvae mainly focus on the exploration of intermediate host infection mechanisms, that is, the development process of larvae in intermediate hosts (such as rodents and livestock), and the study of definitive host transmission dynamics, that is, the survival and transmission routes of worm eggs in the environment (such as fecal contamination of water sources or food). In these experimental studies, C57BL / 6 mice are commonly used as animal models. However, existing methods for establishing mouse animal models of alveolar larvae infection have many drawbacks, including low and unstable infection success rates (affected by worm egg activity and host immune differences), uncontrollable cyst growth rate and location (random spread to extrahepatic organs), and subjective evaluation methods (reliance on terminal dissection and lack of dynamic monitoring in vivo). These limitations affect the reproducibility of experiments and their clinical translational value. Therefore, future research should focus on optimizing modeling methods and improving the consistency and reproducibility of animal models. Summary of the Invention
[0004] The present invention aims to provide a method for constructing a mouse model of hepatic alveolar Echinococcus infection to address the aforementioned problems of the prior art. The ultrasound-assisted modeling method of the present invention avoids the mechanical damage to liver tissue and nonspecific inflammation caused by invasive procedures, enabling noninvasive dynamic monitoring, effectively improving the success rate and survival rate of modeling, shortening the operation time, and reducing damage to the liver and other organs, thereby minimizing experimental error.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The present invention provides a method for constructing a mouse infection model of alveolar echinococcosis, comprising the following steps: selecting female mice aged 6-8 weeks, anesthetizing them, removing the epidermal hair on the chest and abdomen, locating the left lobe of the mouse liver under the guidance of an ultrasound probe, and directly injecting the scolices of the alveolar echinococcosis with good vitality into the liver parenchyma under the assistance of ultrasound probe observation;
[0007] The scolex of the alveolar echinococcosis is extracted from gerbils infected with alveolar echinococcosis.
[0008] Furthermore, the scolices of the alveolar Echinococcus are diluted with PBS to a concentration of 30,000 / mL.
[0009] Furthermore, the injection dose of the alveolar Echinococcus scolex is 0.1 mL.
[0010] Furthermore, the gerbils were infected with alveolar Echinococcus for 90 days.
[0011] Furthermore, the method for extracting the scolex of the alveolar Echinococcus is as follows:
[0012] Under sterile conditions, alveolar echinococcosis tissue in the abdominal cavity of the gerbil is obtained, and the protoscolex suspension is obtained after being chopped, ground and sieve-filtered in a PBS solution containing penicillin and streptomycin. The protoscolex suspension is washed, precipitated and purified to obtain the scolex of the alveolar echinococcosis.
[0013] Furthermore, the volume ratio of penicillin and streptomycin to PBS is 1:100.
[0014] Furthermore, the sieve is an 80-mesh sieve.
[0015] Furthermore, after the injection was completed, the mice continued to be fed for 60-90 days.
[0016] The present invention discloses the following technical effects:
[0017] Compared with traditional surgical modeling, the ultrasound-assisted modeling method of the present invention avoids mechanical damage to liver tissue and the generation of nonspecific inflammation caused by invasive operations, realizes non-invasive dynamic monitoring, effectively improves the success rate and survival rate of modeling, shortens the operation time, and at the same time, reduces damage to the liver and other organs, and reduces experimental errors.
[0018] By precisely locating the cyst inoculation site, the present invention can not only study local microenvironmental characteristics (such as angiogenesis and immune infiltration), but also achieve longitudinal evaluation of the same animal (including cyst volume and degree of fibrosis) through dynamic ultrasound monitoring, more realistically simulating the pathological process of clinical alveolar echinococcosis, and providing a reliable platform for drug development. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0020] Figure 1 This is the alveolar Echinococcus scolex observed under a 40× microscope after eosin staining;
[0021] Figure 2 Figure 1 shows mouse livers at different times of infection with alveolar Echinococcus cysts;
[0022] Figure 3 Ultrasound images of mouse liver at different times of infection with alveolar Echinococcus. DETAILED DESCRIPTION
[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.
[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0028] Example
[0029] 1. Experimental Methods
[0030] First, two experimental groups were established, with 15 female C57BL6 mice (6-8 weeks old, weighing 18-22g) in each group. Group A was surgically injected with scolex into the liver capsule to create the model, and group B was infected by injecting the protoscolex of Echinococcus into the liver capsule of C57BL / 6 mice under ultrasound guidance.
[0031] 1. Specific steps for constructing the mouse model in Group A
[0032] (1) Extraction of alveolar Echinococcus scolex
[0033] A gerbil artificially infected with alveolar Echinococcus for 90 days was anesthetized and then killed by cervical dislocation after the anesthesia took effect. To ensure the sterility of the extracted tissue, the killed gerbil was immersed in a beaker of 75% alcohol for 10 minutes.
[0034] The mouse limbs were fixed, and the alveolar echinococcosis tissue was extracted from the gerbil's abdominal cavity under sterile conditions. First, the gerbil's abdominal skin was cut open, and the peritoneum was exposed by blunt separation. The peritoneum was cut open along the midline of the abdomen and the lesion tissue was extracted.
[0035] Extract the alveolar echinococcosis tissue and dispense it into a sterile 10 cm cell culture dish containing a double-antibody (penicillin + streptomycin) PBS solution (PBS: double-antibody solution volume ratio is 100:1). Grind the diseased tissue on a clean bench. First, place the diseased tissue in a tissue grinder, cut it into pieces with tissue scissors, and grind it into a white translucent homogenate. Filter it with an 80-mesh sieve to remove excess tissue sediment.
[0036] Take the filtered protoscoleces and place them in a culture dish. Wash and precipitate the obtained alveolar echinococcosis several times to improve the purity. Remove the supernatant and the protoscoleces with poor activity. Take 10 μL of the suspension from the high-purity alveolar echinococcosis and drop it on a glass slide. Stain the alveolar echinococcosis with 0.1% eosin solution, count and determine the activity. Select the protoscoleces of the highly active alveolar echinococcosis for subsequent experiments ( Figure 1 ).
[0037] (2) Open the liver and inject the head segment under the liver capsule
[0038] First, viable alveolar Echinococcus protoscoleces were diluted in PBS to a concentration of 30,000 / mL. Mice from Group A were selected for modeling and anesthetized with a combination anesthetic (ketamine, diazepam, and atropine) via intraperitoneal injection. After anesthesia, the hair in the subxiphoid region was shaved and disinfected with iodine. The superficial skin was grasped with forceps, a small incision was made, and the superficial and inner layers of the skin were bluntly separated. The superficial and inner layers of the skin were then incised sequentially along the ventral midline to expose the abdominal cavity. The left lobe of the liver was completely exposed by squeezing above the xiphoid process. Using a 1mL syringe, the needle was slightly angled toward the liver capsule, puncturing the liver capsule approximately 0.5 cm deep. 0.1mL of diluted protoscoleces (approximately 3,000 protoscoleces) was injected, and a cotton swab dipped in normal saline was applied for 10 seconds. Finally, the inner and outer layers of the skin were sutured continuously using a needle holder. The skin was disinfected with iodine and kept warm on a heating pad until the mice regained consciousness.
[0039] 2. Specific steps for constructing the mouse model in Group B
[0040] (1) Extraction of alveolar Echinococcus scolex
[0041] Same as step (1) in the mouse model construction method of group A.
[0042] (2) Open the liver and inject the head segment under the liver capsule
[0043] First, viable alveolar Echinococcus protoscolices were diluted with PBS to a concentration of 30,000 / mL. Mice from Group B, used for modeling, were anesthetized with isoflurane gas. Anesthesia was completed when the corneal reflex was blunted and muscle tone decreased. After the mice were anesthetized, a 5×5 cm area of hair centered below the xiphoid process was shaved with a dermatome. The mice were placed in the lithotomy position and coupling gel was evenly applied. An ultrasound probe was placed over the liver and moved steadily to locate the left lobe of the liver.
[0044] Use a 1mL syringe to advance the needle obliquely 0.5cm. Under ultrasound, the needle tip can be observed entering the liver parenchyma (avoiding blood vessels). Inject 0.1mL of protoscolecus suspension (containing approximately 3000 protoscolecus) and a high-density shadow can be seen in the liver under B-ultrasound. Press for 10s to stop bleeding, keep warm on a heating pad, and wait for the patient to wake up.
[0045] 2. Results Analysis
[0046] 1. Surgical trauma and animal survival rate
[0047] After the mouse model was established in Group A, the overall mortality rate was 6.7% (1 / 15). One death occurred 12 hours after surgery due to sepsis caused by wound infection (blood culture detected E. coli). Autopsies of the deceased mice revealed significant peritoneal inflammation and adhesions.
[0048] After the mouse model in group B was established, there was no death case (0 / 15) and the survival rate was 100%.
[0049] Subsequent monitoring found that most mice in Group B resumed independent activities one hour after surgery, and their food intake returned to normal six hours later; their weight recovery rate was faster than that of Group A.
[0050] These results indicate that the laparotomy in Group A was more invasive, potentially leading to postoperative abdominal adhesions or infection, which resulted in the death of some mice during the observation period. In Group B, ultrasound-guided minimally invasive surgery reduced tissue damage, eliminated surgical incisions, completely avoided abdominal adhesions and wound infection, and resulted in a faster recovery.
[0051] 2. Comparison of infection success rates
[0052] The livers of mice in group A and group B were dissected at different times after infection with alveolar Echinococcus. Figure 2 As shown, liver ultrasound images are Figure 3 shown.
[0053] Ultrasound dynamic observation of the infection status of mice in each group revealed that in group A, no obvious lesions were observed on day 30 of modeling. On day 45, one mouse (6.7%) developed a tiny lesion approximately 1.1 mm in diameter. On day 60, seven mice (46.7%) developed typical cystic lesions 2-3 mm in diameter. By day 75, the modeling success rate reached 12 mice (80%), with cyst sizes varying greatly (3-24 mm). At day 90 of modeling, one mouse still had no obvious lesion, and 13 mice (86.7%) were successfully modeled. Therefore, the modeling success rates at day 45, day 60, day 75, and day 90 were 6.7%, 46.7%, 80%, and 86.7%, respectively.
[0054] In contrast, in group B, one (6.7%) suspicious lesion with a diameter of 0.9 mm was observed at 30 days (later confirmed to be an alveolar echinococcosis lesion upon laparotomy). Three (20%) lesions with diameters of 1-3.5 mm developed at 45 days. Nine (60%) lesions had formed uniform cysts measuring 3-7 mm at 60 days. All 15 (100%) lesions had formed uniform cysts measuring 3-7 mm at 75 days. At 90 days, the 15 (100%) lesions in which the model was successfully established showed heterogeneity in cyst size, ranging from 3 mm to 27 mm. The success rates of model establishment were 6.7%, 20%, 60%, 100%, and 100% at 30, 45, 60, 75, and 90 days, respectively.
[0055] This indicates that Group A (traditional open abdominal injection) relied on visual positioning, which disrupted the peritoneal membrane structure, resulting in some protoscoleci not being successfully implanted into the liver parenchyma. This led to potentially large fluctuations in the infection success rate and slow lesion growth. Some mice experienced ectopic cyst growth (such as intraperitoneal spread), affecting experimental consistency. Group B (ultrasound-guided injection), assisted by real-time ultrasound imaging, ensured precise injection of the protoscoleci into the liver parenchyma, significantly improving the infection success rate (reaching 80% by 60 days), and the cysts were essentially confined to the target liver area, reducing ectopic growth.
[0056] In summary, the modeling method adopted by Group B in the embodiment of the present invention effectively improved the success rate and survival rate of modeling, shortened the operation time, and at the same time, reduced damage to the liver and other organs, narrowed the experimental error, and effectively solved the existing modeling problems of mice infected with alveolar echinococcosis.
[0057] In contrast, the traditional surgical modeling in Group A has significant limitations: its invasive procedure can easily cause mechanical damage to liver tissue and trigger nonspecific inflammation, interfering with observation of the natural course of the disease; blind puncture, which relies on the surgeon's experience, often leads to positioning errors, resulting in the formation of ectopic cysts; repeated surgeries or terminal anatomical assessments are required, making noninvasive dynamic monitoring impossible; and the operation is poorly standardized and has poor reproducibility. These differences fully demonstrate the outstanding advantages of the modeling method in Group B of the present invention in terms of minimally invasiveness, precision, and translational research value.
[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for constructing a mouse model of alveolar Echinococcus infection, characterized in that: The following steps are involved: Female mice aged 6-8 weeks were selected. After anesthesia, the epidermal hair on the chest and abdomen was removed. Under the guidance of an ultrasound probe, the left lobe of the mouse liver was located. Under the assistance of ultrasound probe observation, the scolex of the alveolar Echinococcus with good vitality was directly injected into the liver parenchyma. The scolex of the alveolar echinococcosis is extracted from gerbils infected with alveolar echinococcosis.
2. The construction method according to claim 1, characterized in that The scolices of the alveolar Echinococcus were diluted with PBS to a concentration of 30,000 / mL.
3. The construction method according to claim 2, characterized in that The injection dose of the alveolar Echinococcus scolex is 0.1 mL.
4. The construction method according to claim 1, characterized in that The gerbils were infected with alveolar Echinococcus for 90 days.
5. The construction method according to claim 1, characterized in that The method for extracting the scolex of the alveolar echinococcosis is as follows: Under sterile conditions, alveolar echinococcosis tissue in the abdominal cavity of the gerbil is obtained, and the protoscolex suspension is obtained after being chopped, ground and sieve-filtered in a PBS solution containing penicillin and streptomycin. The protoscolex suspension is washed, precipitated and purified to obtain the scolex of the alveolar echinococcosis.
6. The construction method according to claim 5, characterized in that: The volume ratio of penicillin and streptomycin to PBS is 1:
100.
7. The construction method according to claim 5, characterized in that: The sieve is an 80-mesh sieve.
8. The construction method according to claim 1, wherein: After the injections were completed, the mice continued to be fed for 60–90 days.