Method for constructing transvaginal uterus orthotopic choriocarcinoma tumorigenesis animal model

Abstract

The invention provides a method for constructing a transvaginal uterus in-situ choriocarcinoma tumorigenesis animal model, and belongs to the technical field of biomedicine. The method for constructing the transvaginal uterus in-situ choriocarcinoma tumorigenesis animal model comprises the following steps: S1, preparing and marking cells; s2, nude mouse model construction operation: performing in-situ inoculation through a vaginal uterus, and feeding nude mice in an SPF-level environment; a self-made mouse vagina fixator is matched with a 100 microliter microsyringe, and 50 microliter cell suspension is injected into the vagina; and S3, model monitoring and evaluation. The model is used for accurately simulating the complete pathological process of in-situ occurrence, development, local invasion and hematogenous metastasis of human choriocarcinoma in the uterus, and a key experimental platform is provided for deeply researching the pathogenesis and metastasis rule of the choriocarcinoma and evaluating a novel treatment strategy; operation is not needed, operation is easy and convenient, animal wounds and the death rate are reduced, the experiment cost is reduced, and meanwhile the experiment efficiency and repeatability are improved.

Description

Technical Field

[0001] This application relates to the biomedical field, and more specifically, to a method for constructing a transvaginal animal model of choriocarcinoma in situ of the uterus. Background Technology

[0002] Currently, choriocarcinoma is a highly malignant trophoblastic tumor originating from trophoblastic cells of gestational chorioretinopathy, exhibiting extremely strong invasiveness and early hematogenous metastasis. Establishing animal models that accurately mimic the biological behavior of choriocarcinoma is crucial for studying its pathogenesis, metastatic patterns, and evaluating treatment strategies. Currently, the main types of choriocarcinoma tumorigenesis models used in research include the following:

[0003] 1. Subcutaneous transplantation tumor model

[0004] Construction method: JEG3 cells (5×10⁻⁶) derived from human choriocarcinoma tissue were used. 6 –1×10 7 (1 per mouse) was injected subcutaneously into immunodeficient mice (such as nude mice).

[0005] Features: Tumor formation rate is as high as 100%, with a short latency period (tumors can be palpated 7-10 days after injection). Early-stage tumors exhibit rapid exponential growth, are soft in texture, and have clear borders; peak volume is reached in approximately 3 weeks (approximately 250 mm). 3 It is characterized by significant angiogenesis; growth slows in later stages, and a necrotic area may appear in the center. At 21 days, the tumor weighs approximately 0.25g. It continuously secretes βhCG, with serum βhCG levels peaking at 14-21 days post-tumor formation and then declining. Pathologically, it retains typical choriocarcinoma features (multinucleated giant cells, atypical trophoblastic cells). Although it exhibits local invasiveness, it primarily manifests as localized growth.

[0006] Limitations: Because it is completely separated from the uterine microenvironment, the tumor grows only locally and rarely metastasizes, making it impossible to simulate the critical hematogenous metastasis process in clinical practice.

[0007] 2. Tail vein injection transfer model

[0008] Construction method: 2×10⁻⁶ mmol / L was injected into 4-week-old female athymic nude mice (BALB / c nude mice) via the tail vein. 6 One well-active (>90%) JEG3 cell line was used to simulate distant metastasis.

[0009] Features: Highly effective in establishing a metastasis model primarily involving lung metastases (metastasis rate >90%). Short latency period (micrometastases appear 67 days after injection). Lung metastases cover 40-50% of the lung surface area at day 21, and can subsequently spread to the ovaries and uterus; metastatic tumors retain typical choriocarcinoma morphology (multinucleated giant cells, atypical trophoblastic cells). Serum βhCG levels peak at 14-21 days. Mouse survival ≤50 days.

[0010] Limitations: Although it can efficiently induce lung metastasis, this method forces hematogenous dissemination, bypassing the natural metastasis cascade process of primary lesion evolution, in situ invasion, and escape into the circulatory system. Therefore, it is not suitable for studying the invasion mechanism of the primary lesion.

[0011] 3. Uterine in situ tumor model

[0012] Construction method: The uterine horn of 6-week-old female NOD-SCID mice was surgically exposed, and 2.5 × 10⁻⁶ mmol / L mice were injected. 5 A suspension of MTX-resistant JEG3R cells mixed with 1:1 Matrigel.

[0013] Characteristics: Highly mimics the in situ uterine growth and invasive characteristics of choriocarcinoma. Typical pathological features (continuous βhCG secretion). Three weeks after tumor in situ growth, uterine weight increases significantly (approximately 3-fold), and serum βhCG levels rise. Lung / ovarian metastases can also be observed.

[0014] Disadvantages: Open abdominal surgery is required for cell implantation, and the procedure is invasive, resulting in a mortality rate of approximately 28%. The non-pregnant uterus lacks the physiologically hypoxic microenvironment of the placenta, leading to relatively low transfer efficiency.

[0015] 4. In situ placental tumor formation model in pregnant mice

[0016] Construction method: In SHO-SCID immunodeficient pregnant mice (8-12 weeks of age) at 7.5 days of gestation (dpc), the second placenta (each uterine horn) was exposed via laparotomy, and 2×10⁻⁶ oz. of SHO-SCID immunodeficient mice was injected into the placental decidual layer. 6 A suspension of luciferase-labeled JEG3 cells mixed with a 1:1 Matrigel.

[0017] Features: This is the first established in situ model of choriocarcinoma during pregnancy, which effectively reproduces clinicopathological features. Rapid hematogenous metastasis: Spontaneous metastasis to the liver, lungs, spleen, and peritoneum can occur within 12 days after injection (19.5 days of gestation). Microenvironment-dependent: The high vascular density of the placenta and the physiological hypoxic environment drive tumor dissemination. Advantages of dynamic assessment: In vivo bioluminescence imaging (IVIS) can dynamically and sensitively assess the progression of primary and metastatic lesions by quantifying the photon signal of the region of interest (ROI) (sensitivity superior to serum βhCG detection); histological examination reveals infiltration of atypical trophoblastic cells within the placenta and high Ki67 expression (>80%).

[0018] Disadvantages: The procedure requires laparotomy in pregnant mice to expose the uterine horn for placental injection, resulting in a surgical miscarriage risk >30%, a long treatment period (approximately 19.5 days to the observation endpoint), and high costs. Furthermore, although choriocarcinoma primarily originates from gestational trophoblastic cells, clinically, patients rarely experience concurrent pregnancy, making this model different from actual clinical practice.

[0019] The existing technical solutions mentioned above have the following drawbacks: existing models can characterize the in situ growth and metastasis characteristics of choriocarcinoma to varying degrees, but they all have inherent limitations and cannot more accurately simulate the biological behavior of choriocarcinoma in the uterus, nor can they be used to construct animal models that are easy to operate, have a high tumor formation rate, and obvious metastatic characteristics. Summary of the Invention

[0020] To overcome the above deficiencies, this application provides a method for constructing a transvaginal uterine in situ choriocarcinoma tumor animal model, aiming to improve the simulation of the biological behavior characteristics of choriocarcinoma in the uterus.

[0021] This application provides a method for constructing a transvaginal uterine orthotopic choriocarcinoma tumorigenesis animal model, including...

[0022] S1. Cell Preparation and Labeling: JEG-3 human choriocarcinoma cells were resuscitated and passaged under standard conditions; JEG-3 cells were transfected with a transfection aid, and 72 hours after transfection, cells were screened using a medium containing 2.0 μg / mL puromycin to kill untransfected cells; the puromycin-containing medium was changed periodically until a stable JEG-3 cell line expressing Luciferase was obtained, i.e., a stable transfected cell line; the stable transfected cell line was expanded and cryopreserved for later use; for in situ seeding.

[0023] S2. Nude mouse model construction: Nude mice were in situ inoculated via vagina and uterus and housed in an SPF-grade environment; a self-made mouse vaginal fixator was used with a 100μL microsyringe to inject 50μL of cell suspension into the vagina;

[0024] S3. Model Monitoring and Evaluation:

[0025] (1) General observation: The day after inoculation, observe the nude mice’s activity, mental state, breathing, and skin color, and accurately record the weight changes.

[0026] (2) Animal in vivo imaging;

[0027] (3) Anatomy and gross observation;

[0028] (4) Tissue processing and pathological examination.

[0029] In a preferred embodiment of the present invention, the standard conditions include RPMI 1640 medium containing 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator; to achieve in vivo imaging monitoring, JEG-3 cells are transfected with lentiviral particles carrying the Luciferase gene (MOI=10), and a transfection aid is added.

[0030] In a preferred embodiment of the invention, the transfection aid includes Lipofectamine. TM3000; During the transfection process, replace the complete culture medium 6-8 hours after transfection and continue culturing.

[0031] In a preferred embodiment of the present invention, the nude mice obtained are 4-week-old female BALB / c (nu / nu); before inoculation, JEG-3 cells are digested, centrifuged, and resuspended in sterile PBS or culture medium, and the cell concentration is precisely adjusted to 4 × 10⁻⁶. 7 Cells / mL; 50 μL of this suspension was used for inoculation of each nude mouse.

[0032] In a preferred embodiment of the present invention, a self-made mouse vaginal fixator is used to expose and fix the vaginal opening; a 100μL microsyringe is fitted with a 30G fine needle. This completely eliminates the need for open abdominal surgery required for traditional construction of uterine in situ tumor models, avoiding problems such as large surgical trauma, slow recovery, and susceptibility to infection. The use of the self-made mouse vaginal fixator in conjunction with the microsyringe allows for precise operation, with the "breakthrough sensation" at the cervix serving as a key positioning marker to ensure accurate injection site placement, achieving a high success rate of non-invasive in situ inoculation.

[0033] During the general observation period, hCG colloidal gold rapid diagnostic test strips can be used to test the urine of nude mice to help determine the tumor formation status. Among the disease progression indicators, weight loss, emaciation, poor activity, deep and rapid breathing, pale skin, and cachexia are important.

[0034] In a preferred embodiment of the present invention, the procedure for transvaginal in situ uterine inoculation includes:

[0035] a. Place the nude mouse in the tail vein injection stand, with its prone position and tail facing the operator;

[0036] b. Gently insert the homemade vaginal fixator into the vagina of the nude mouse, and open the vaginal opening appropriately to fully expose the vaginal entrance;

[0037] c. Slowly and gently insert the needle of a microsyringe containing 50 μL of cell suspension into the vagina through a vaginal fixator; the 50 μL of cell suspension is equivalent to 2 × 10⁻⁶ cells / mL. 6 One cell / animal;

[0038] d. Stop advancing the needle once it has passed through the cervix and entered the uterine cavity;

[0039] e. Slowly inject the entire 50 μL cell suspension over a period of at least 30 seconds;

[0040] f. After the injection is complete, keep the syringe in place for about 30 seconds, then slowly withdraw the syringe and retainer;

[0041] g. If no cell suspension flows out of the vaginal opening, the injection is considered successful;

[0042] h. Return the nude mice to their cages and observe them as usual.

[0043] In a preferred embodiment of the present invention, during the process of inserting the microsyringe needle into the vagina through the vaginal fixator, the operator carefully senses the changes in resistance as the needle advances. When a clear "breakthrough" is felt, it usually indicates that the needle has passed through the cervix and entered the uterine cavity. Based on the breakthrough point, the syringe is slowly advanced to a depth of about 1 cm to ensure that the injection site is in the uterine cavity or near the cervical-uterine body junction.

[0044] In a preferred embodiment of the present invention, the in vivo imaging, targeting the Luciferase marker group, employs the following steps:

[0045] a. Perform in vivo imaging at predetermined observation time points or before and after drug intervention;

[0046] b. Anesthesia: Intraperitoneal injection of anesthetic agent, dose is 0.2mL / 10g body weight;

[0047] c. Substrate injection: After the nude mice are fully anesthetized, inject 15 mg / mL of D-fluorescein sodium working solution into the peritoneum at a dose of 10 μL / g body weight.

[0048] d. Imaging: 10 minutes after the substrate is injected, when the fluorescence signal reaches its strongest stable plateau, the nude mouse is placed in a small animal in vivo imaging system for optical imaging.

[0049] e. Analysis: Using complementary software, such as CarestreamMI, image acquisition, quantitative analysis, and image merging were performed to assess the light signal intensity of uterine lesions in situ, reflecting the tumor burden, and whether there were metastatic signals throughout the body.

[0050] In a preferred embodiment of the present invention, the dissection and gross observation specifically include the following steps:

[0051] a. Sacrifice the nude rat at the predetermined destination or when the animal is near death;

[0052] b. The abdominal and thoracic cavities are opened along the midline of the abdomen for a complete systemic dissection;

[0053] c. Visually observe and record:

[0054] Observe whether there is fluid or blood accumulation in the abdominal cavity and pleural cavity and its characteristics.

[0055] Observe the uterus, ovaries, and fallopian tubes: uterine shape, size, position, color, whether the uterine horns are bulging, and whether the blood vessels in the uterine wall are congested; whether there are nodules on the surface of the ovaries;

[0056] Observe the lungs: size, color, presence or absence of nodules;

[0057] Observe the liver: its shape, color, and presence of nodules;

[0058] Observe the brain: whether the surface blood vessels are congested and whether there are nodules;

[0059] Observe other organs for any abnormalities;

[0060] d. Focus on separating and collecting target organs such as the uterus and its adnexa, lungs, liver, and brain.

[0061] In a preferred embodiment of the present invention, the tissue processing and pathological examination steps are as follows:

[0062] a. Immediately fix the target organ in a 4% paraformaldehyde solution for 24 hours;

[0063] b. After fixation, the tissue is dehydrated, cleared, impregnated with paraffin, and embedded in paraffin.

[0064] c. Paraffin blocks are continuously sliced ​​to a thickness of 4-5 μm;

[0065] d. The sections are stained and analyzed as follows: routinely observe tissue morphological changes, tumor structure, necrosis, inflammation, depth of invasion, and metastatic lesions; or use immunohistochemical staining: use specific antibodies to stain choriocarcinoma cells to specifically label them and observe the invasive behavior and metastasis of tumor cells.

[0066] Beneficial effects:

[0067] 1. This model successfully simulates the complete pathological process of choriocarcinoma from in situ growth and local invasion to hematogenous metastasis, providing a crucial experimental platform for in-depth research into the pathogenesis and metastasis patterns of choriocarcinoma, as well as for evaluating novel treatment strategies. In vivo imaging technology allows for dynamic monitoring of tumor progression, and combined with pathological analysis, it enables in-depth exploration of the biological behavior of choriocarcinoma, possessing significant scientific research and clinical application value.

[0068] 2. This model is used to accurately simulate the complete pathological process of human choriocarcinoma in situ occurrence, development, local invasion and hematogenous metastasis in the uterus, providing a key experimental platform for in-depth research on the pathogenesis and metastasis patterns of choriocarcinoma and evaluation of new treatment strategies. Compared with the traditional open surgery model, this method does not require surgery, is simple to operate, reduces animal trauma and mortality, lowers experimental costs, and improves experimental efficiency and reproducibility. Attached Figure Description

[0069] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0070] Figure 1 This is a schematic diagram illustrating the construction of a transvaginal choriocarcinoma in situ uterine tumor animal model provided by the embodiments of this application;

[0071] Figure 2 Anatomical images of the uterus at different times after JEG3 cell inoculation, provided for embodiments of this application;

[0072] Figure 3 Gross anatomical diagrams of various isolated organs from 28-day-old nude mice provided for embodiments of this application;

[0073] Figure 4 HE-stained pathological image of a uterine section 28 days after in situ vaginal inoculation of JEG3 choriocarcinoma cells, provided as an embodiment of this application.

[0074] Figure 5 This is a small animal imaging image taken 28 days after nude mice were in situ injected with Luciferase-JEG3 cells via the vagina and uterus, as provided in the embodiments of this application. Detailed Implementation

[0075] The technical solutions of the embodiments of this application will now be described with reference to the accompanying drawings. Please refer to... Figure 1-Figure 5 This invention provides a method for constructing a transvaginal uterine choriocarcinoma tumorigenesis animal model, including...

[0076] S1. Cell Preparation and Labeling: JEG-3 human choriocarcinoma cells were resuscitated and passaged under standard conditions; JEG-3 cells were transfected with a transfection aid, and 72 hours after transfection, cells were screened using a medium containing 2.0 μg / mL puromycin to kill untransfected cells; the puromycin-containing medium was changed periodically until a stable JEG-3 cell line expressing Luciferase was obtained, i.e., a stable transfected cell line; the stable transfected cell line was expanded and cryopreserved for later use; for in situ seeding.

[0077] S2. Nude mouse model construction: Nude mice were in situ inoculated via vagina and uterus and housed in an SPF-grade environment; a self-made mouse vaginal fixator was used with a 100μL microsyringe to inject 50μL of cell suspension into the vagina;

[0078] S3. Model Monitoring and Evaluation:

[0079] (1) General observation: The day after inoculation, observe the nude mice’s activity, mental state, breathing, and skin color, and accurately record the weight changes.

[0080] (2) Animal in vivo imaging;

[0081] (3) Anatomy and gross observation;

[0082] (4) Tissue processing and pathological examination.

[0083] In a specific embodiment of the present invention, the standard conditions include RPMI1640 medium containing 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator; to achieve in vivo imaging monitoring, JEG-3 cells are transfected with lentiviral particles carrying the Luciferase gene (MOI=10), and transfection aids are added.

[0084] In a specific embodiment of the present invention, the transfection aid includes Lipofectamine. TM 3000; During the transfection process, replace the complete culture medium 6-8 hours after transfection and continue culturing.

[0085] In a specific embodiment of the present invention, the nude mice obtained were 4-week-old female BALB / c (nu / nu); before inoculation, JEG-3 cells were digested, centrifuged, and resuspended in sterile PBS or culture medium, and the cell concentration was precisely adjusted to 4 × 10⁻⁶. 7 Cells / mL; 50 μL of this suspension was used for inoculation of each nude mouse.

[0086] In a specific embodiment of the present invention, a self-made mouse vaginal fixator is used to expose and fix the vaginal opening; a 100μL microsyringe is equipped with a 30G fine needle. Figure 1 The diagram illustrates the process of transvaginal in situ inoculation of JEG3 cells into the uterus, including key steps such as nude mouse fixation, use of a vaginal fixator, and microinjector operation. This method completely eliminates the need for open surgery required in traditional methods of constructing uterine in situ tumor models, avoiding problems such as significant surgical trauma, slow recovery, and susceptibility to infection. A self-made mouse vaginal fixator, combined with a microinjector, is used for precise manipulation. The "breakthrough sensation" at the cervix is ​​used as a key positioning marker to ensure accurate injection site placement, achieving a high success rate of non-invasive in situ inoculation.

[0087] During the general observation period, hCG colloidal gold rapid diagnostic test strips can be used to test the urine of nude mice to help determine the tumor formation status. Among the disease progression indicators, weight loss, emaciation, poor activity, deep and rapid breathing, pale skin, and cachexia are important.

[0088] In a specific embodiment of the present invention, the procedure for transvaginal in situ uterine inoculation includes:

[0089] a. Place the nude mouse in the tail vein injection stand, with its prone position and tail facing the operator;

[0090] b. Gently insert the homemade vaginal fixator into the vagina of the nude mouse, and open the vaginal opening appropriately to fully expose the vaginal entrance;

[0091] c. Slowly and gently insert the needle of a microsyringe containing 50 μL of cell suspension into the vagina through a vaginal fixator; the 50 μL of cell suspension is equivalent to 2 × 10⁻⁶ cells / mL. 6 One cell / animal;

[0092] Cell suspension preparation: LucJEG3 cells in logarithmic growth phase were digested with 0.25% trypsin, and digestion was terminated with 10% fetal bovine serum culture medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in sterile PBS. The cells were counted and the cell concentration was adjusted to 4 × 10⁶ cells / mL. 7 cells / mL.

[0093] d. Stop advancing the needle once it has passed through the cervix and entered the uterine cavity;

[0094] e. Slowly inject the entire 50 μL cell suspension over a period of at least 30 seconds;

[0095] f. After the injection is complete, keep the syringe in place for about 30 seconds, then slowly withdraw the syringe and retainer;

[0096] g. If no cell suspension flows out of the vaginal opening, the injection is considered successful;

[0097] h. Return the nude mice to their cages and observe them as usual.

[0098] In a specific embodiment of the present invention, during the process of inserting the micro-syringe needle into the vagina through the vaginal fixator, the operator carefully senses the change in resistance as the needle advances. When a clear "breakthrough" is felt, it usually indicates that the needle has passed through the cervix and entered the uterine cavity. Based on the breakthrough point, the syringe is slowly advanced to a depth of about 1 cm to ensure that the injection site is in the uterine cavity or near the cervical-uterine body junction.

[0099] In a specific embodiment of the present invention, the in vivo imaging, targeting the Luciferase marker group, employs the following steps:

[0100] a. Perform in vivo imaging at predetermined observation time points or before and after drug intervention;

[0101] b. Anesthesia: Intraperitoneal injection of anesthetic agent, dose is 0.2mL / 10g body weight;

[0102] c. Substrate injection: After the nude mice were fully anesthetized, they were intraperitoneally injected with 15 mg / mL of D-luciferase sodium working solution at a dose of 10 μL / g body weight; luciferase labeling: JEG3 cells were transfected with lentivirus carrying the Luciferase gene (purchased from Vector Laboratories). At transfection, cells were seeded in 6-well plates, and when the cell confluence reached 50%, lentivirus particles (MOI = 10) and 8 μg / mL Lipofectamine were added. TM 3000, mix gently. After 6 hours, replace with fresh medium and continue culturing. After 72 hours, add medium containing 2.0 μg / mL puromycin for selection. Change the medium every 3 days until a stable JEG3 cell line expressing Luciferase (LucJEG3) is obtained.

[0103] d. Imaging: 10 minutes after the substrate is injected, when the fluorescence signal reaches its strongest stable plateau, the nude mouse is placed in a small animal in vivo imaging system for optical imaging.

[0104] e. Analysis: Using complementary software, such as CarestreamMI, image acquisition, quantitative analysis, and image merging were performed to assess the light signal intensity of uterine lesions in situ, reflecting the tumor burden, and whether there were metastatic signals throughout the body.

[0105] By combining luciferase gene labeling technology and a small animal in vivo imaging system, the growth of uterine in situ tumors and potential systemic metastasis can be monitored non-invasively, in real time, and dynamically at different time points after model construction, providing a dynamic assessment method for studying the development and metastasis of choriocarcinoma.

[0106] Figure 2 As shown, the uterine anatomy at different time points after JEG3 cell inoculation is illustrated. In A, 7 days after inoculation, a unilateral uterine horn cyst-like protrusion is shown; in B, 14 days after inoculation, uterine congestion and obvious blood vessels are shown; and in C, 28 days after inoculation, a purplish-blue swollen mass of the uterus is shown.

[0107] In a specific embodiment of the present invention, the dissection and gross observation specifically include the following steps:

[0108] a. Sacrifice the nude rat at the predetermined destination or when the animal is near death;

[0109] b. The abdominal and thoracic cavities are opened along the midline of the abdomen for a complete systemic dissection.

[0110] c. Visually observe and record:

[0111] Observe whether there is fluid or blood accumulation in the abdominal cavity and pleural cavity and its characteristics.

[0112] Observe the uterus, ovaries, and fallopian tubes: uterine shape, size, position, color, whether the uterine horns are bulging, and whether the blood vessels in the uterine wall are congested; whether there are nodules on the surface of the ovaries;

[0113] Observe the lungs: size, color, presence or absence of nodules;

[0114] Observe the liver: its shape, color, and presence of nodules;

[0115] Observe the brain: whether the surface blood vessels are congested and whether there are nodules;

[0116] Observe other organs for any abnormalities;

[0117] d. Focus on separating and collecting target organs such as the uterus and its adnexa, lungs, liver, and brain.

[0118] Figure 3 The image shows gross anatomical diagrams of various organs extracted from nude mice at 28 days of age. A represents bloody ascites; B represents purplish-blue metastatic nodules on the surface of the ovary; C represents the liver; D represents the lungs (with visible metastatic nodules); E represents the brain; and F represents a uterine tumor in situ.

[0119] In a specific embodiment of the present invention, the tissue processing and pathological examination steps are as follows:

[0120] a. Immediately fix the target organ in a 4% paraformaldehyde solution for 24 hours;

[0121] b. After fixation, the tissue is dehydrated, cleared, impregnated with paraffin, and embedded in paraffin.

[0122] c. Paraffin blocks are continuously sliced ​​to a thickness of 4-5 μm;

[0123] d. The sections are stained and analyzed as follows: routinely observe tissue morphological changes, tumor structure, necrosis, inflammation, depth of invasion, and metastatic lesions; or use immunohistochemical staining: use specific antibodies to stain choriocarcinoma cells to specifically label them and observe the invasive behavior and metastasis of tumor cells.

[0124] like Figure 4 As shown, HE staining pathological images of uterine sections 28 days after JEG3 choriocarcinoma cells were orthotopically inoculated into the vagina show the typical "biphasic growth" pattern, "blood lake" structure, and tumor cell infiltration into the myometrium of the uterus.

[0125] Figure 5 Images were captured using a small animal imaging system 28 days after nude mice were inoculated into the uterus via the vagina. The images showed the fluorescence signal distribution of the uterine tumor and metastatic lesions throughout the body, and the tumor burden and metastasis were quantitatively analyzed.

[0126] The working principle of this method for constructing a transvaginal in situ choriocarcinoma tumor animal model is as follows: Through detailed temporal pathological analysis, it was clearly revealed and confirmed for the first time in an animal model that choriocarcinoma cells inoculated vaginally into the uterine cavity do not directly form expansive lesions within the uterine cavity. Instead, they first infiltrate and break through the endometrial layer, entering the subendometrial stroma and even the myometrium, before proliferating in situ to form choriocarcinoma lesions with a typical "blood lake" structure. This discovery is of great significance for understanding the early invasive behavior of choriocarcinoma.

[0127] The experiment determined and limited the JEG3 cell suspension used for in situ inoculation, with an inoculation volume of 50 μL per nude mouse. This combination of parameters ensured a high tumorigenesis rate, with uterine lesions observed as early as 7 days post-inoculation, and drove tumor progression to the typical cachexia endpoint within approximately 28 days, providing a clear observation window for efficacy evaluation.

[0128] The primary uterine lesion exhibited typical histological features of choriocarcinoma, including biphasic growth of trophoblastic cells, central ischemic necrosis, "blood lake" structure, and vascular remodeling and vascular mimicry. Pathology confirmed that the tumor destroyed the entire endometrial layer and infiltrated the deep myometrium to the serosa.

[0129] Local invasion and metastasis: Ovarian metastasis is visible as purplish-blue nodules on the surface of the ovary, with an incidence of 100% (at 28 days); metastasis to organs such as the lungs and liver can also be observed, and the metastatic characteristics are highly similar to those of clinical choriocarcinoma.

[0130] Seven days after inoculation: In all dissected nude mice (3 / 3), cystic outward protrusions could be observed in the unilateral uterine horn, and tumor formation could be identified with the naked eye, with a tumor formation rate of 100%. The nude mice were generally in good condition, and their weight did not change significantly.

[0131] Fourteen days post-inoculation: The survival rate of nude mice was 100%, and their activity level was good. Gross anatomical examination revealed bilateral uterine congestion and red coloration, with significant congestion of blood vessels in the lateral uterine walls. Some nude mice still showed bulging of the uterine horns. This stage suggests that the tumor is in an active growth phase and a stage of vascular invasion.

[0132] 28 days post-inoculation: Nude mice entered a state of cachexia (extreme emaciation, poor activity, rapid and deep breathing, and pale skin). Mortality increased significantly. Dissection of surviving nude mice revealed a unilateral, purplish-blue, swollen mass protruding into the abdominal cavity at the uterine horn, with 100% accompanied by purplish-blue metastatic nodules on the surface of both ovaries. Furthermore, 75% developed massive hemorrhagic pleural effusion, and 100% developed hemorrhagic ascites. This stage marks the late stage of the disease, accompanied by widespread metastasis and systemic failure.

[0133] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

Claims

1. A method for constructing a transvaginal animal model of choriocarcinoma in situ in the uterus, characterized in that, include S1. Cell Preparation and Labeling: JEG-3 human choriocarcinoma cells were resuscitated and passaged under standard conditions; JEG-3 cells were transfected with a transfection aid, and 72 hours after transfection, cells were screened using a medium containing 2.0 μg / mL puromycin to kill untransfected cells; the puromycin-containing medium was changed periodically until a stable JEG-3 cell line expressing Luciferase was obtained, i.e., a stable transfected cell line; the stable transfected cell line was expanded and cryopreserved for later use; for in situ seeding. S2. Nude mouse model construction: Nude mice were in situ inoculated via vagina and uterus and housed in an SPF-grade environment; a self-made mouse vaginal fixator was used with a 100μL microsyringe to inject 50μL of cell suspension into the vagina; S3. Model Monitoring and Evaluation: (1) General observation: The day after inoculation, observe the nude mice’s activity, mental state, breathing, and skin color, and accurately record the weight changes. (2) Animal in vivo imaging; (3) Anatomy and gross observation; (4) Tissue processing and pathological examination.

2. The method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 1, characterized in that, The standard conditions include culture medium containing 10% fetal bovine serum, incubated at 37°C in a 5% CO2 incubator; to achieve in vivo imaging monitoring, JEG-3 cells were transfected with lentiviral particles carrying the Luciferase gene, and a transfection aid was added.

3. The method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 1, characterized in that, The transfection aid includes Lipofectamine. TM 3000; During the transfection process, replace the complete culture medium 6-8 hours after transfection and continue culturing.

4. The method for constructing a transvaginal uterine choriocarcinoma tumor animal model according to claim 1, characterized in that, The nude mice used were 4-week-old female BALB / c mice; before inoculation, JEG-3 cells were digested, centrifuged, and resuspended in sterile PBS or culture medium, and the cell concentration was precisely adjusted to 4 × 10⁻⁶. 7 Cells / mL; 50 μL of this suspension was used for inoculation of each nude mouse.

5. The method for constructing a transvaginal uterine choriocarcinoma tumor animal model according to claim 4, characterized in that, A homemade mouse vaginal fixator was used to expose and fix the vaginal opening; a 100μL microsyringe was fitted with a 30G fine needle.

6. The method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 5, characterized in that, The procedure for transvaginal in situ uterine inoculation includes: a. Place the nude mouse in the tail vein injection stand, with its prone position and tail facing the operator; b. Gently insert the homemade vaginal fixator into the vagina of the nude mouse, and open the vaginal opening appropriately to fully expose the vaginal entrance; c. Slowly and gently insert the needle of a microsyringe containing 50 μL of cell suspension into the vagina through a vaginal fixator; the 50 μL of cell suspension is equivalent to 2 × 10⁻⁶ cells / mL. 6 One cell / animal; d. Stop advancing the needle once it has passed through the cervix and entered the uterine cavity; e. Slowly inject the entire 50 μL cell suspension over a period of at least 30 seconds; f. After the injection is complete, keep the syringe in place for about 30 seconds, then slowly withdraw the syringe and retainer; g. If no cell suspension flows out of the vaginal opening, the injection is considered successful; h. Return the nude mice to their cages and observe them as usual.

7. The method for constructing a transvaginal uterine choriocarcinoma tumor animal model according to claim 6, characterized in that, During the process of inserting the microsyringe needle into the vagina through the vaginal fixation device, the operator carefully senses the changes in resistance as the needle advances. When a clear "breakthrough" is felt, it usually indicates that the needle has passed through the cervix and entered the uterine cavity. Based on the breakthrough point, the syringe is slowly advanced to a depth of about 1 cm to ensure that the injection site is inside the uterine cavity or near the cervical-uterine body junction.

8. The method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 1, characterized in that, The in vivo imaging, targeting the Luciferase-labeled group, employs the following steps: a. Perform in vivo imaging at predetermined observation time points or before and after drug intervention; b. Anesthesia: Intraperitoneal injection of anesthetic agent, dose is 0.2mL / 10g body weight; c. Substrate injection: After the nude mice are fully anesthetized, inject 15 mg / mL of D-fluorescein sodium working solution into the peritoneum at a dose of 10 μL / g body weight. d. Imaging: 10 minutes after the substrate is injected, when the fluorescence signal reaches its strongest stable plateau, the nude mouse is placed in a small animal in vivo imaging system for optical imaging. e. Analysis: Using complementary software, such as CarestreamMI, image acquisition, quantitative analysis, and image merging were performed to assess the light signal intensity of uterine lesions in situ, reflecting the tumor burden, and whether there were metastatic signals throughout the body.

9. The method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 1, characterized in that, The dissection and gross observation specifically include the following steps: a. Sacrifice the nude rat at the predetermined destination or when the animal is near death; b. The abdominal and thoracic cavities are opened along the midline of the abdomen for a complete systemic dissection; c. Visually observe and record: Observe whether there is fluid or blood accumulation in the abdominal cavity and pleural cavity and its characteristics. Observe the uterus, ovaries, and fallopian tubes: uterine shape, size, position, color, whether the uterine horns are bulging, and whether the blood vessels in the uterine wall are congested; whether there are nodules on the surface of the ovaries; Observe the lungs: size, color, presence or absence of nodules; Observe the liver: its shape, color, and presence of nodules; Observe the brain: whether the surface blood vessels are congested and whether there are nodules; Observe other organs for any abnormalities; d. Focus on separating and collecting target organs such as the uterus and its adnexa, lungs, liver, and brain.

10. A method for constructing a transvaginal uterine choriocarcinoma tumor-forming animal model according to claim 1, characterized in that, The tissue processing and pathological examination steps are as follows: a. Immediately fix the target organ in a 4% paraformaldehyde solution for 24 hours; b. After fixation, the tissue is dehydrated, cleared, impregnated with paraffin, and embedded in paraffin. c. Paraffin blocks are continuously sliced ​​to a thickness of 4-5 μm; d. The sections are stained and analyzed as follows: routinely observe tissue morphological changes, tumor structure, necrosis, inflammation, depth of invasion, and metastatic lesions; or use immunohistochemical staining: use specific antibodies to stain choriocarcinoma cells to specifically label them and observe the invasive behavior and metastasis of tumor cells.

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