Animal models of hepatic targeted infection of multilocular echinococcosis larvae, their construction methods and applications

Abstract

This invention provides an animal model of hepatic echinococcosis caused by *Echinococcus multilocularis*, its construction method, and its applications. The construction method mainly includes preserving the protoscolex, extracting the protoscolex of *Echinococcus multilocularis*, and injecting the preserved protoscolex into C57BL / 6N mice. The resulting mouse model can be used to observe the infection and development of hepatic echinococcosis, and to screen and / or evaluate hepatic echinococcosis drugs. The advantages of this invention are that it explores the growth pattern of *Echinococcus multilocularis* and its impact on the hepatic vascular system from a novel perspective. Furthermore, addressing the problems of complex and high-risk traditional modeling methods, it proposes an optimized liver-targeted inoculation pathway and establishes a standardized pathogen treatment process, ensuring infection success rate and animal survival rate while reducing experimental interference factors.

Description

Technical Field

[0001] This invention belongs to the field of animal models for drug screening, specifically the animal model of hepatic targeted infection by multilocular echinococcosis, its construction method and application. Background Technology

[0002] Alveolar echinococcosis (AE) is a common hepatic echinococcosis. Previously considered a benign, self-limiting disease, it has been misdiagnosed due to its malignant biological behavior. The lesions exhibit infiltrative growth, resembling malignant tumor metastasis and invasion of surrounding organs (such as the diaphragm and lungs). It can spread to other organs through infiltration, blood, or lymphatic circulation. Without timely treatment, the 10-year mortality rate can reach 94%, hence the name "parasitic cancer." Early stages are often asymptomatic, with patients typically presenting at an advanced stage. Radical hepatectomy is currently the preferred and only curative treatment for advanced AE. However, due to its malignant tumor-like infiltrative growth, achieving a negative margin (R0 resection, no parasite residue) is difficult, leading to residual lesions and invasion of important intrahepatic vascular structures such as the first and second hepatic hilum, inferior vena cava, and vital bile ducts. This can cause serious complications such as obstructive jaundice, biliary tract infection, portal hypertension, Budd-Chiari syndrome, cirrhosis, and liver failure. Current surgical methods for staged radical resection of hepatic echinococcosis, ex vivo liver resection plus autologous liver transplantation, allogeneic liver transplantation, and palliative treatments such as albendazole drug therapy and radiofrequency ablation have limitations, including high surgical difficulty, inability to address invasion of important vascular systems, inability to completely remove lesions, peritoneal implantation of protocercariae or germinal layer cells, and recurrence. How to further improve the quality of life and surgical methods for advanced-stage patients remains a challenge. Clinical findings suggest that the parasite and surrounding compressed vascular systems can be separated intraoperatively without damaging important vascular systems, and blood supply is restored after the compression of the vessel walls is relieved. This indicates that multilocular echinococcosis larvae may not grow invasively but rather compressively, similar to granular echinococcosis (Eg). Whether it is possible to temporarily relieve the compression and invasion of surrounding important vascular systems (Glisson system, hepatic venous system) by the lesions in advanced-stage patients, alleviate symptoms, and provide a window of opportunity for subsequent conservative treatment is a problem that needs to be solved.

[0003] To gain a deeper understanding of the biological characteristics of alveolar echinococcosis (AE), thereby improving the diagnosis and treatment of AE patients and enhancing their prognosis, we need to analyze not only clinical patient data but also, crucially, animal experiments. In AE research, establishing a suitable animal model of Em infection is paramount. Currently, researchers commonly choose rodents to establish liver Em infection models, employing various inoculation methods, including abdominal paracentesis, percutaneous liver biopsy, open liver biopsy, transmuscular liver biopsy, and portal vein injection. However, each method has its advantages and disadvantages: 1) Although paracentesis is simple to perform, has fewer postoperative infections, and a high animal survival rate, the infection rate of liver implantation is low, lacks specificity, and cannot accurately simulate the situation where Em originates in the liver.

[0004] 2) Percutaneous liver biopsy is a simple procedure with few postoperative complications and a high survival rate. However, the liver cannot be precisely located, and the puncture needle may accidentally enter the pleural cavity, causing pneumothorax or even respiratory failure, or inadvertently damage major intrahepatic blood vessels and bile ducts, leading to complications. Although imaging assistance can improve the accuracy of localization, this method still requires professional technicians to perform.

[0005] 3) Open liver biopsy can simulate the primary infection of Em in the liver, but during the injection process, the parasite may be injected into the blood vessels of the liver parenchyma, leading to serious complications such as vascular embolism and massive hemorrhage of the liver parenchyma. The animals suffer greater damage and there is a risk of postoperative infection and intestinal obstruction.

[0006] 4) The procedure of liver biopsy through the abdominal wall muscle layer after incision is relatively simple and suitable for smaller experimental animals such as mice. This is because the abdominal wall muscle layer of mice is thin, which can avoid major abdominal blood vessels, reduce the risk of accidental entry into the thoracic cavity, and result in a higher inoculation rate, less infection, and less likelihood of intestinal obstruction. However, this method is limited to small animals.

[0007] 5) While portal vein injection can effectively simulate the natural infection route of *E. m.*, it is more complex than open liver biopsy. This method uses *E. m.* protoscolex as the source of infection, rather than the oncocytic larvae released from eggs in natural infection. Therefore, it cannot fully simulate the interaction between the gastrointestinal tract and the parasite from the oral cavity to the portal vein. Furthermore, portal vein puncture is difficult and involves multiple steps, increasing the risk of postoperative infection and bleeding in experimental animals. Locating the portal vein often requires freeing the intestinal segment, increasing the risk of intestinal obstruction or necrosis and reducing postoperative survival. Simultaneously, the small portal vein makes it difficult for larger *E. m.* tissues to pass through, reducing the concentration of the protoscolex suspension and thus affecting the inoculation success rate. Therefore, portal vein injection requires a high level of operator skill, and the inoculation result depends to some extent on the operator's proficiency, making it relatively complex for beginners.

[0008] In summary, developing simple methods for creating animal models of Echinococcus multilocularis infection, and obtaining animal models with high infection rates while ensuring animal survival, are urgent problems to be solved in this field. This also aims to provide a powerful tool for drug screening and disease progression monitoring. Summary of the Invention

[0009] This application aims to solve the problems existing in the above background technology and provides a method for constructing a multilocular echinococcosis liver-targeted infection animal model, including the following steps: (1) Protoscolex preservation: Select SPF-grade long-clawed gerbils with a rat age of 7 weeks and a weight of 40±4g, and inject echinococcosis protoscolex into the peritoneal cavity with a 1 ml syringe for preservation. (2) Extraction of Echinococcus multilocularis prostomum: Echinococcus multilocularis vesicle tissue was extracted from the long-clawed gerbil in step (1), and the Echinococcus multilocularis prostomum was purified. 3) Establishment of C57BL / 6N mouse liver echinococcosis model: 4-6 week old female C57BL / 6N mice were randomly divided into blank control group, 30d infection group, 60d infection group, 90d infection group and 120d infection group. The prostomum of the multilocular echinococcosis obtained in step (2) was injected into the C57BL / 6N mice.

[0010] Furthermore, in step (1), after intraperitoneal injection of Echinococcus prostomyces, long-clawed gerbils are fed for more than 6 months to extract multilocular Echinococcus prostomyces.

[0011] Furthermore, the method for extracting Echinococcus multilocularis larvae in step (2) includes the following steps: (a) After the Echinococcus multilocularis larvae are completely removed, they are placed in a culture dish, and the vesicles are carefully dissected with sterile ophthalmic scissors and forceps. The remaining peritoneal tissue is removed, and the vesicles are washed 2-3 times with PBS buffer until the washing solution is translucent and bloodless. Then, the vesicles are transferred to clean PBS buffer. (b) Take the tissue cleaned in step (a) and carefully separate the vesicles in a clean bench to avoid breaking the vesicles too much; (c) Use tweezers to pick up the separated vesicles and place them into a glass grinder; (d) Grind the tissue thoroughly until it becomes a homogenate. The grinding speed should be controlled at 20-40 rpm and the ambient temperature should be controlled at 0-10℃. The specific speed should be such that the temperature of the ground tissue does not exceed 10℃. The grinding force should be such that the vesicles break and release the protostomes. Avoid excessive force that may cause the grinder to break. During the grinding process, PBS buffer can be added in small amounts multiple times, 1-2 ml each time, to control the grinding temperature. (e) After grinding the vesicle tissue from step (d) into a tissue homogenate, slowly pour it into a 100-mesh sieve and rinse the sieve with PBS buffer. (f) Place a 50mL beaker under the sieve to collect and store the washed-off protostomes; (g) Wait for the liquid in the beaker to settle and separate into layers: a white precipitate at the bottom, white flocculent matter floating in the middle, and a clear liquid at the top; (h) After the supernatant is clear, discard the supernatant and floating matter, keep the precipitate, add PBS at a volume ratio of precipitate:PBS = 1:1, and continue to wash away white flocculent matter and other impurities. Repeat steps (g)-(h) 1-2 times, collect the precipitate and transfer it to a 50mL centrifuge tube. (i) Pour the precipitate obtained in step (h) into a 200-mesh cell filter sieve, place the sieve upwards on the mouth of a 100 mL beaker, and use a pipette to gently rinse the precipitate with PBS to speed up filtration until all the precipitate has been filtered, using 1-2 mL of PBS each time. (j) The protostomes remain on the filter screen. The 200-mesh cell filter screen is inverted and placed in a 100mL clean beaker. PBS is added and filtered in reverse. The white precipitate remaining on the filter screen can be seen flowing into the beaker below. Because of the small pore size of the 200-mesh filter, protoscolex cannot pass through the mesh and remains on the filter. Invert the 200-mesh cell filter onto a clean 100mL beaker, and gently rinse the filter with 1-2mL of PBS each time until the white precipitate remaining on the filter indicates that all the protoscolex has flowed into the beaker below. (k) After the liquid in the beaker settles and separates into layers, discard the supernatant and keep the precipitate, which contains protoscolex of multilocular Echinococcus larvae.

[0012] Furthermore, the final purification of the protoscolex of the multilocular Echinococcus larvae in step (2) includes: (I) transferring the precipitate from the beaker to a culture dish, adding PBS, shaking the culture dish, tilting the culture dish to one side and letting it stand, and seeing the white cysts appear on the dry side of the bottom of the dish, and picking out the impurities with tweezers; (II) When the liquid differentiation layer appears, aspirate the upper layer of clear, colorless PBS solution along the edge of the liquid, and remove the suspended vesicle skin at the same time; (III) Repeat steps (I) and (II) until the impurities are completely removed, at which point a pure proto-pro ...

[0013] Furthermore, step (2) also includes calculating the final purified protoscolex viability: (I) Under a microscope, take 5 μL of purified protoscolex for counting and add an equal amount of 0.1% eosin staining solution for staining; (II) By eosin staining, the protoscolex with good activity showed abstinence, while the dead insect body stained red. The average value of the three visual fields was selected as the overall survival rate estimate. Activity rate = (Number of rejected parasites / Total number of parasites) × 100%; (III) The purified active protostomes with an activity of not less than 95% were evenly distributed into T25 / T75 cell culture dishes and placed in a CO2 incubator for subsequent use.

[0014] Furthermore, in step (3), the blank control group was: approximately 1 ml of PBS phosphate buffer was injected subcapsulated into the liver of C57BL / 6N mice using a 1 ml disposable insulin syringe; Experimental group: Take the protoscolex of multilocular echinococcosis with an activity ≥95% obtained in step (2), and inject 1 mL of protoscolex suspension into the subcapsular membrane of the liver of C57BL / 6N mice; Both the blank control group and the experimental group were raised in an SPF-grade barrier facility under constant temperature and humidity.

[0015] Therefore, the multilocular echinococcosis liver-targeted infection animal model constructed using the above method is also within the scope of protection of this invention.

[0016] The animal model described above can be used to: (I) observe the infection and development process of hepatic echinococcosis; (II) Screening for drugs to treat hepatic echinococcosis; (III) Evaluation of drugs for hepatic echinococcosis.

[0017] Furthermore, based on the following in vitro detection indicators, we will evaluate the infection and progression of hepatic echinococcosis, screen for hepatic echinococcosis drugs, and evaluate the efficacy of hepatic echinococcosis drugs: (I) Serum ELISA was used to detect the levels of ALT and AST in the serum of C57BL / 6N model mice in each group; (II) HE pathological observation of blood vessel morphology in C57BL / 6N model mice; (III) IHC staining of morphological and pathological changes in blood vessels and bile ducts in C57BL / 6N model mice; (IV) Distribution of collagen fibers in lesions, expression of fibrosis in blood vessel walls, Masson and Sirius Red staining in C57BL / 6N model mice; (V)C57BL / 6N model mice localization of Echinococcus multilocularis larvae PAS staining.

[0018] In summary, the beneficial effects of the present invention obtained by adopting the above technical solutions are as follows: (1) The present invention explores the growth mode of hepatic echinococcosis and its influence on the hepatic vascular system from a completely new perspective, and provides an animal model construction method for studying the growth mode of hepatic echinococcosis, and performs multi-dimensional model verification, etc.

[0019] (2) In view of the problems that traditional methods for establishing liver echinococcosis models are complicated and risky, a method of subcapsular injection of 1ml disposable insulin needle is proposed to optimize the liver targeted inoculation route, reduce liver trauma, and ensure infection success rate and animal survival rate.

[0020] (3) The method of subcapsular injection of Em protoscolex suspension into the liver of C57BL / 6N model mice through open abdominal surgery has the following significant advantages: First, this method can effectively avoid direct contact between the experimenters and Em eggs, thereby ensuring the safety of the experimenters, reducing the risk in the process of establishing the primary infection model, and reducing the stringent requirements on laboratory level; Second, this model makes the Em infection occur precisely in the liver, rather than in the abdominal cavity or subcutaneous tissue, which highly simulates the real situation of Em originating in the liver; Third, the experimenters can reasonably control the dosage of infection, and further study the specific effects of dosage, time and other factors on the process of Em infection; Fourth, using Em protoscolex suspension instead of homogenate for injection can significantly reduce the occurrence of complications such as portal vein obstruction, and improve the stability and reliability of the model. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the process for constructing an animal model according to the present invention; Figure 2 Aseptic dissection of a long-clawed gerbil; Figure 3 A diagram illustrating the establishment of a C57BL / 6N mouse model of hydatid disease in the liver capsule via injection. Figure 4 A graph showing the serum ALT and AST levels in different infection groups of C57BL / 6N mice detected by ELISA; Figure 5 HE staining images of blood vessels and bile ducts in liver tissues of C57BL / 6N mice from different infection groups; Figure 6 HE staining of blood vessels and bile ducts in gerbil liver tissue; Figure 7 Image showing strong positive expression of CD34 in the veins of gerbils and various infection groups; Figure 8 Image showing strong positive expression of α-SMA in the veins of gerbils and various infection groups; Figure 9 Image showing strong positive expression of CK7 in the bile ducts of gerbils and various infection groups; Figure 10 Image showing strong positive expression of CK19 in the bile ducts of gerbils and various infection groups; Figure 11 Masson staining image of collagen fibers in liver tissue of C57BL / 6N mouse infection group; Figure 12 Sirius Red staining of collagen fibers in liver tissue of C57BL / 6N mice infected group; Figure 13 PAS staining of the cuticle of parasites in the C57BL / 6N mouse infection group. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0023] Example: All materials used in this invention are commercially available. Experimental materials involved include: (1) Long-clawed gerbils were purchased from the Center for Disease Control and Prevention. C57BL / 6N mice were purchased from Henan Skbes Biotechnology Co., Ltd.

[0024] (2) Hematoxylin and eosin (HE) staining kit, modified Masson trichrome staining kit, and modified Sirius red staining kit, products of Beijing Solarbio Science & Technology Co., Ltd.

[0025] (3) Periodic acid-Schiff (PAS) staining kit, product of Shanghai Beyotime Biotechnology Co., Ltd.

[0026] (4) Mouse anti-α-SMA monoclonal antibody, rabbit anti-CD34 monoclonal antibody, rabbit anti-CK7 monoclonal antibody, rabbit anti-CK19 polyclonal antibody, products of Wuhan Boster Biological Engineering Co., Ltd.

[0027] (5) Normal sheep serum for blocking, DAB colorimetric kit, endogenous peroxidase inhibitor and enzyme-labeled goat anti-mouse / rabbit IgG polymer, products of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0028] (6) Horseradish enzyme-labeled goat anti-rabbit IgG and horseradish enzyme-labeled goat anti-mouse IgG, products of Shanghai Yuanmu Biotechnology Co., Ltd.

[0029] (7) Alanine aminotransferase (ALT / GPT) test kit and aspartate aminotransferase (AST / GOT) test kit, products of Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0030] Specific steps: as follows Figure 1 As shown: (1) Preservation of Protoscolex: Fifty SPF-grade long-clawed gerbils, half male and half female, were purchased from the Center for Disease Control and Prevention and housed at the Experimental Animal Center of Shihezi University School of Medicine. Gerbils approximately 7 weeks old and weighing between 40±4g were selected. Protoscolex larvae were drawn from a 1 ml syringe and injected intraperitoneally for preservation. A feeling of emptiness during needle insertion indicated that the needle had entered the peritoneal cavity. The syringe was then lifted upwards to inject the protoscolex into the peritoneal cavity.

[0031] (2) Extraction of the protoscolex of Echinococcus multilocularis: Dissection of long-clawed gerbils: Long-clawed gerbils infected with Echinococcus multilocularis larvae for more than 6 months were euthanized near an alcohol lamp and soaked in 75% alcohol for at least 30 minutes to kill bacteria on the surface of the gerbils. The gerbils were placed on a clean foam board. After ensuring that the alcohol on the surface of the gerbils had completely evaporated, the limbs were fixed to the sterile foam board with medical tape. After fully exposing the abdomen, the central abdominal skin was lifted with sterile surgical forceps. The abdominal cavity was opened by making an incision along the midline of the abdomen with scissors, covering the area from the thoracic cavity to the pelvic cavity. The skin and subcutaneous fascia may be dissected to a moderate degree to ensure complete exposure of the Echinococcus multilocularis larvae tissue. The Echinococcus multilocularis larvae tissue was carefully separated from the surrounding organs with forceps, ensuring the integrity of the Echinococcus multilocularis larvae tissue while minimizing damage to the surrounding liver tissue to reduce the risk of bleeding and prevent blood from contaminating the Echinococcus multilocularis larvae tissue. Grind the Echinococcus multilocularis larvae and screen the homogenate: After completely removing the Echinococcus multilocularis larvae, place them in a culture dish. Carefully dissect the vesicles using sterile ophthalmic scissors and forceps, removing any remaining peritoneal tissue. Wash 2-3 times with PBS buffer until the washing solution is translucent and bloodless, then transfer to clean PBS buffer. Take the washed tissue and carefully separate the vesicles in a laminar flow hood, avoiding excessive fragmentation. Pick up some of the dissected vesicles with forceps and place them in a glass grinder. During grinding, add small amounts of PBS buffer (1-2 ml each time) multiple times to evenly mix the vesicle tissue and avoid excessively rapid grinding that could generate heat, affecting protoscolex survival and causing the grinder to break. Previous studies have found that excessive grinding may result in a large number of tiny broken vesicles in the homogenate, increasing the impurity content of the protoscolex. Therefore, after appropriately grinding the Echinococcus multilocularis larvae into a tissue homogenate, slowly pour it into a 100-mesh sieve. Place a 50 mL beaker under the sieve to collect and store the rinsed protoscolex. Use a pipette to gently rinse the precipitate with PBS to accelerate filtration until all precipitate has been filtered out. Since the protoscolex is smaller than the cystic membrane, the protoscolex can pass through the filter mesh into the beaker, while the larger cystic membrane remains on the filter mesh. Wait for the liquid in the beaker to settle and separate into layers; you will see a lower layer of white precipitate, a middle layer of floating white flocculent matter, and an upper layer of clear liquid. Once the supernatant is clear, discard the supernatant and floating matter, keeping the precipitate. Add PBS at a volume ratio of (precipitate:PBS = 1:1) to continue washing away the white flocculent matter and other impurities. Repeat this process 1-2 times. Collect the precipitate and transfer it to a 50mL centrifuge tube. Pour the precipitate into a 200-mesh cell filter sieve, placing the sieve upwards over the mouth of a 100mL beaker. Use a pipette to gently rinse the precipitate with PBS to accelerate filtration until all precipitate has been filtered out, using 1-2mL of PBS each time.

[0032] Because of the small pore size of the 200-mesh sieve, protoscolex cannot pass through the mesh and remains on the filter. Invert the 200-mesh cell filter onto a clean 100mL beaker. Gently rinse the sieve with 1-2mL of PBS using a pipette until only white precipitate remains on the filter; then let all the precipitate flow into the beaker below. After the liquid in the beaker has separated into layers, discard the supernatant and retain the precipitate, keeping a portion of the upper layer. Screening and purification of *Echinococcus multilocularis* protoscolex: Transfer the precipitate from the beaker to a petri dish, add PBS, shake the dish, and tilt it to one side to allow it to stand. White cysts will appear on the dry area at the bottom of the dish; remove the impurities with tweezers. When the liquid differentiation layer appears, aspirate the upper layer of clear, colorless PBS solution along the edge of the liquid, simultaneously removing the suspended cysts. Add PBS to the petri dish and continue the above operation until the impurities are almost completely eliminated. At this point, a pure protoscolex precipitate can be obtained. If some floating matter remains, the surface precipitate can be discarded because this part contains few protoscolex but abundant calcareous bodies (calcareous bodies are small, white, sand-like particles, slightly smaller than the protoscolex, and relatively easy to identify). Proscolex viability was calculated: Under a microscope, 5 μL of purified proscolex was counted and stained with an equal volume of 0.1% eosin. Eosin staining revealed that viable proscolex exhibited abstention, while dead proscolex stained red. The average of three fields of view was used to estimate the overall survival rate. Survival rate = (number of abstention proscolex / total number of proscolex) × 100%. Purified viable proscolex with at least 95% viability were evenly distributed into T25 / T75 cell culture dishes and placed in a CO2 incubator for later use. Discarded gerbils were cleaned, autoclaved, and disinfected by soaking in 20% benzalkonium chloride solution, properly wrapped, and then autoclaved to prevent residual proscolex from contaminating the surrounding environment.

[0033] (3) Establishment of the C57BL / 6N mouse model of echinococcosis in the liver: 4-6 week old female C57BL / 6N mice were randomly divided into a blank control group (n = 8), a 30-day infection group (n = 8), a 60-day infection group (n = 8), a 90-day infection group (n = 8), and a 120-day infection group (n = 8). In the blank control group, mice were anesthetized (intraperitoneal injection of 5% chloral hydrate), and the liver was exposed after laparotomy. Approximately 1 ml of PBS phosphate buffer was injected under the liver capsule using a 1 ml disposable insulin needle. White cysts were observed on the surface of the liver. After hemostasis with gelatin sponge, the abdomen was sutured and the mice were revived. Protoscolex of Echinococcus multilocularis with an activity ≥95% were obtained from the above-mentioned peritoneally preserved gerbils. Each infection group was injected with 1 ml of protoscolex suspension (approximately 600 protoscolex) using the same method. After surgery, mice in each group were kept warm and revived in SPF-grade barrier facilities with constant temperature and humidity.

[0034] (4) Mice in each infection group were euthanized and weighed after blood was collected from their eyeballs at the corresponding infection time points. One mouse in the blank control group was euthanized at each time point. The livers were removed and the growth of the livers and Em in each group was observed. The livers were fixed in paraformaldehyde.

[0035] (5) Multi-dimensional model validation of data indicators: The levels of ALT and AST in the serum of C57BL / 6N model mice were detected by ELISA in each group: After anesthetizing the C57 mice, the mice were fixed with the left hand, and the eyeballs were compressed to make them protrude as much as possible. The whiskers and hair around the eyeballs were trimmed with the right hand to prevent hemolysis. The eyeballs were removed with curved forceps, and blood was immediately collected into a 1.5mL EP tube. The abdomen and heart of the mouse could be pressed to accelerate blood flow and pump it out. After the blood had drained, the mouse was euthanized by dislocation. If there was insufficient blood, both eyeballs could be removed. After standing at room temperature for 2 hours to allow the blood to coagulate and separate into layers, the tubes were centrifuged (without shaking) at 3000rpm / min, 4℃, for 10-15 minutes. The supernatant was collected as serum. The serum was transferred to centrifuge tubes and stored at -80℃. ALT / AST matrix solution preheated to 37℃; 2,4-dinitrophenylhydrazine solution; 0.4 mol / L sodium hydroxide solution (diluted to 4 mol / L sodium hydroxide solution: double-distilled water = 1:9 before use, and prepared as needed); take a 96-well plate, set up a measurement well and a control well for each sample, and add 20 μL of matrix solution to each well. Add 5 μL of the sample to be tested to the measurement well. Gently shake the plate to mix, and incubate at 37℃ for 30 min. Add 20 μL of 2,4-dinitrophenylhydrazine solution to each measurement well and control well, and add 5 μL of the sample to each control well. Gently shake the plate to mix, and incubate at 37℃ for 20 min. Add 200 μL of 0.4 mol / L sodium hydroxide solution to each measurement well and control well. Gently shake the 96-well plate to mix, and incubate at room temperature for 15 min. Measure the OD value of each well using a microplate reader at 505 nm (ALT) / 510 nm (AST). Refer to the standard curve using the absolute OD value (OD value of measurement well minus OD value of control well) to obtain the corresponding ALT / AST activity value. ALT / AST activity (U / L) = ALT / AST activity (Kammen units) obtained by substituting into the standard curve × 0.482 × N. (0.482: conversion from Kamen units to U / L; N: dilution factor of the sample before testing).

[0036] HE pathological observation of blood vessel morphology in C57 mice and gerbils: After fixation, liver tissue was dehydrated with a gradient of alcohols, cleared with xylene, embedded in paraffin, and sectioned. White slides were baked at 60℃, dewaxed and dehydrated in xylene and a gradient of ethanols, rinsed with running water, stained with hematoxylin for 3-5 min, and rinsed with running water until the water flow was clear. The slides were then immersed in 1% hydrochloric acid-ethanol solution for 2-3 s, soaked in water for 10 min to regain blue color, stained with 0.5% eosin for 5 min, and rinsed with running water until the water flow was clear. The sections were placed in a gradient of ethanol for dehydration and cleared with xylene. The slides were mounted with neutral resin solution and observed and photographed under a microscope.

[0037] IHC staining for morphological and pathological changes of blood vessels and bile ducts in C57 mice and gerbils: Tissue sections were dewaxed and hydrated, then rinsed with running water. Sections were placed in preheated sodium citrate buffer and heated to boiling, then cooled to room temperature in a fume hood and washed with PBS. Endogenous peroxidase was added and incubated in the dark. 1% nonspecific antigen serum blocking agent was added and incubated in the dark. Primary antibodies (α-SMA, CD34, CK7, and CK19 antibodies, all at a working concentration of 1:400) were added and incubated at 4°C for 12–16 h. Goat anti-rabbit / mouse secondary antibody was added and incubated at 37°C for 30 min. Staining was terminated after DAB development. Hematoxylin was stained onto the nuclei for 3 min, followed by rinsing with running water. Differentiation was performed with 1% hydrochloric acid ethanol for 2–3 s, followed by rinsing with running water. The sections were dehydrated with graded alcohols, cleared with xylene, and mounted.

[0038] Masson staining: C57 mice and gerbils showed collagen fiber distribution and vascular wall fibrosis expression in lesions. Tissue sections were dewaxed and hydrated, then rinsed with running water. Mordant was applied for 1 hour, followed by rinsing with running water. Azurite blue was added for 2–3 minutes, followed by rinsing with running water. Mayer's hematoxylin staining solution was applied for 2–3 minutes, followed by rinsing with distilled water. Differentiation was performed with acidic ethanol for a few seconds, followed by rinsing with distilled water. Ponceau S and fuchsin staining solutions were applied for 10 minutes, followed by rinsing with distilled water. Phosphomolybdic acid was added for 10 minutes, then discarded, followed by aniline blue staining for 5 minutes. The aniline blue was rinsed with a weak acid solution, followed by 2 minutes of weak acid treatment. The sections were then dehydrated, cleared, and mounted as before.

[0039] The distribution of collagen fibers and expression of vascular wall fibrosis in C57 mice and gerbils lesions were observed. Sirius Red staining: Tissue sections were routinely dewaxed and hydrated, then rinsed with running water. Iron hematoxylin staining solution was prepared. Iron hematoxylin staining solution was applied for 5-10 min, followed by rinsing with double-distilled water for 10-20 s. The sections were then rinsed with tap water for 5-10 min. Sirius Red staining solution was applied for 15-30 min, followed by rinsing with running water. The sections were then dehydrated, cleared, and mounted as described above.

[0040] PAS staining for localization of Echinococcus multilocularis larvae in C57 mice and gerbils: Tissue sections were dewaxed, hydrated, and washed as described above. Treatment with periodic acid solution for 10 min, followed by washing with distilled water. 100 μL of Schiff reagent was added, and the sections were incubated at 37°C in the dark for 30-60 min, then washed with distilled water. Hematoxylin staining was performed, followed by differentiation with distilled water and hydrochloric acid-ethanol solution to achieve blue reversion. The sections were then dehydrated with a gradient of alcohols, cleared with xylene, and mounted.

[0041] (6) Data Processing: SPSS 26.0 software was used for statistical analysis in this study. Quantitative data followed a normal distribution and were expressed as mean ± standard deviation ( ). The expression indicates that if the data conforms to normality and homogeneity of variance, an independent samples t-test is used to compare two groups, and a one-way ANOVA is used to compare multiple groups. LSD and SNK tests are then performed for comparisons between two groups. If the data does not conform to normality, the Wilcoxon rank-sum test is used for comparisons between two groups, and the Kruskal-Wallis H test is used for comparisons between multiple groups. A p-value < 0.05 indicates a statistically significant difference.

[0042] Experimental results (1) such as Figure 2 As shown, aseptic dissection of a long-clawed gerbil revealed numerous multilocular echinococcosis larvae within its abdominal cavity, growing in clusters, milky white, and filling the entire cavity. Microscopic observation showed that the protoscolex was plump, with smooth edges and clearly discernible internal structures, indicating significant biological activity. To assess the activity of the protoscolex, a staining test was performed using 0.1% eosin. The results showed that active protoscolex successfully resisted dye absorption, while those with poor activity and those that had died were stained red by eosin.

[0043] (2) such as Figure 3 As shown, a C57BL / 6N mouse model of echinococcosis was constructed by injecting the liver capsule, and small white cysts appeared on the surface of the liver.

[0044] (3) such as Figure 4 As shown, the serum ALT and AST levels in each group were detected by ELISA. Compared with the normal group (without any intervention), the ALT and AST levels in the four node model groups were significantly increased (P<0.05), and the differences were statistically significant.

[0045] (4) such as Figure 5 , 6 As shown, HE staining revealed that the blood vessels and bile ducts of the infected gerbils and C57BL / 6N mice were narrowed and the walls were tortuous, while the walls were relatively intact.

[0046] (5) such as Figure 7 , 8 As shown in Figures 9 and 10, gerbils and each infected group showed strong positive expression of CD34, α-SMA in the veins and CK7 and CK19 in the bile ducts. The venous and bile duct walls showed intact and uninterrupted coloration, indicating an intact wall structure, regular bile duct walls, full lumens, and deformed lumens.

[0047] (6) For example Figure 11 As shown, Masson staining in C57BL / 6N mice from different infection groups revealed that the vein walls were predominantly composed of blue-stained collagen fibers, with sparse distribution of red-stained smooth muscle. The boundary between the lumen and the vein wall and the blue-stained loose connective tissue of the infiltrative zone was indistinct. Figure 12The Sirius Red staining results show that the vein wall is mainly composed of red-stained collagen fibers, with no yellow-stained smooth muscle observed. Yellow-stained red blood cell accumulation is visible in the lumen, and the boundary between the vein wall and the surrounding red-stained loose connective tissue is unclear.

[0048] (7) For example Figure 13 As shown, glycogen staining reveals that the cuticle of the insect body is stained dark purple. Some insect bodies are plump, the cuticle is regularly shaped, and the insect wall has not collapsed.

[0049] Therefore, the animal model of this invention can be used to observe the infection and development process of hepatic echinococcosis; screen drugs for hepatic echinococcosis; and evaluate drugs for hepatic echinococcosis.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing an animal model of liver-targeted infection by multilocular echinococcosis, characterized in that, The following steps are included: (1) Preservation of protoscolex: Select SPF-grade long-clawed gerbils with an age of 7 weeks and a weight of 40±4g, and inject the protoscolex of echinococcosis into the peritoneal cavity with a 1 ml syringe for preservation. (2) Extraction of Echinococcus multilocularis prostomum: Echinococcus multilocularis vesicle tissue was extracted from the long-clawed gerbil in step (1), and the Echinococcus multilocularis prostomum was purified. (3) Establishment of C57BL / 6N mouse liver echinococcosis model: 4-6 week old female C57BL / 6N mice were randomly divided into blank control group, 30d infection group, 60d infection group, 90d infection group and 120d infection group. The prostomum of the multilocular echinococcosis obtained in step (2) was injected into the C57BL / 6N mice.

2. The method according to claim 1, characterized in that, In step (1), after intraperitoneal injection of Echinococcus prostomyces, long-clawed gerbils were raised for more than 6 months to extract multilocular Echinococcus prostomyces.

3. The method according to claim 1, characterized in that, The method for extracting Echinococcus multilocularis larvae in step (2) includes the following steps: (a) After the Echinococcus multilocularis larvae are completely removed, they are placed in a culture dish, and the vesicles are carefully dissected with sterile ophthalmic scissors and forceps. The remaining peritoneal tissue is removed, and the vesicles are washed with PBS buffer 2-3 times until the washing solution is translucent and bloodless. Then the vesicles are transferred to clean PBS buffer. (b) Take the tissue cleaned in step (a) and carefully separate the vesicles in a clean bench to avoid breaking the vesicles too much; (c) Use tweezers to pick up the separated vesicles and place them into a glass grinder; (d) Grind the tissue thoroughly until it becomes a homogenate. The grinding speed should be controlled at 20-40 rpm and the ambient temperature should be controlled at 0-10℃. The specific speed should be such that the temperature of the tissue being ground does not exceed 10℃. The grinding force should be such that the vesicles break and release the protostomes. Avoid excessive force that could cause the grinder to break. During the grinding process, PBS buffer can be added in small amounts multiple times, 1-2 ml each time, to control the grinding temperature. (e) After grinding the vesicle tissue from step (d) into a tissue homogenate, slowly pour it into a 100-mesh sieve and rinse the sieve with PBS buffer. (f) Place a 50mL beaker under the sieve to collect and store the washed-off protostomes; (g) Wait for the liquid in the beaker to settle and separate into layers: a white precipitate at the bottom, white flocculent matter floating in the middle, and a clear liquid at the top; (h) After the supernatant is clear, discard the supernatant and floating matter, keep the precipitate, add PBS at a volume ratio of precipitate:PBS = 1:1, and continue to wash away white flocculent matter and other impurities. Repeat steps (g)-(h) 1-2 times, collect the precipitate and transfer it to a 50mL centrifuge tube; (i) Pour the precipitate obtained in step (h) into a 200-mesh cell filter sieve, place the sieve upwards on the mouth of a 100 mL beaker, and use a pipette to gently rinse the precipitate with PBS to speed up filtration until all the precipitate has been filtered, using 1-2 mL of PBS each time; (j) Because the 200 mesh has a small pore size, the protoscolex cannot pass through the mesh and remains on the filter. Turn the 200 mesh cell filter upside down and place it in a 100mL clean beaker. Use a pipette to draw PBS and gently rinse the filter with 1-2mL of PBS each time until the white precipitate remaining on the filter indicates that all the protoscolex has flowed into the beaker below. (k) After the liquid in the beaker settles and separates into layers, discard the supernatant and keep the precipitate, which contains protoscolex of multilocular Echinococcus larvae.

4. The method according to claim 1, characterized in that, The final purification steps of Echinococcus multilocularis protoscolex in step (2) include: (I) transferring the precipitate from the beaker to a culture dish, adding PBS, shaking the culture dish, tilting the culture dish to one side and letting it stand, and seeing white cysts appear on the dry side of the bottom of the dish, and picking out the impurities with tweezers; (II) When the liquid differentiation layer appears, aspirate the upper layer of clear, colorless PBS solution along the edge of the liquid, and remove the suspended vesicle skin at the same time; (III) Repeat steps (I) and (II) until the impurities are completely removed, at which point a pure proto-pro ...

5. The method according to claim 1, characterized in that, The step (2) also includes calculating the final purified protoscolex viability: (I) Under a microscope, take 5 μL of purified protoscolex for counting and add an equal amount of 0.1% eosin staining solution for staining; (II) By eosin staining, the protoscolex with good activity showed abstinence, while the dead insect body stained red. The average value of three visual fields was selected as the overall survival rate estimate. Activity rate = (Number of repelled parasites / Total number of parasites) × 100%; (III) The purified active protostomes with an activity of not less than 95% were evenly distributed into T25 / T75 cell culture dishes and placed in a CO2 incubator for subsequent use.

6. The method according to claim 1, characterized in that, In step (3), the blank control group was as follows: approximately 1 ml of PBS phosphate buffer was injected subcapsulated into the liver of C57BL / 6N mice using a 1 ml disposable insulin syringe; Experimental group: Take the protoscolex of multilocular echinococcosis with an activity ≥95% obtained in step (2), and inject 1 mL of protoscolex suspension into the subcapsular region of the liver of C57BL / 6N mice; Both the blank control group and the experimental group were raised in an SPF-grade barrier facility under constant temperature and humidity.

7. An animal model of liver-targeted infection by multilocular echinococcosis larvae, characterized in that, It is constructed by any of the methods described in claims 1-6.

8. Application of an animal model of hepatic targeted infection by multilocular echinococcosis larvae, characterized in that, The model will be used to: (I) observe the infection and development process of hepatic echinococcosis; (II) Screening for drugs to treat hepatic echinococcosis; (III) Evaluation of drugs for hepatic echinococcosis.

9. The application according to claim 8, characterized in that, The following in vitro detection indicators were used to evaluate the infection and progression of hepatic echinococcosis, screen for hepatic echinococcosis drugs, and evaluate these drugs: (I) Serum ELISA was used to detect the levels of ALT and AST in the serum of C57BL / 6N model mice in each group; (II) HE pathological observation of blood vessel morphology in C57BL / 6N model mice; (III) IHC staining of morphological and pathological changes in blood vessels and bile ducts in C57BL / 6N model mice; (IV) Distribution of collagen fibers in lesions and expression of vascular wall fibrosis in C57BL / 6N model mice, Masson and Sirius Red staining; (V)C57BL / 6N model mice localization of Echinococcus multilocularis larvae PAS staining.

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