Cervical continuous lesion animal model and construction method thereof

Abstract

The invention provides an animal model of cervical continuous lesion and a construction method thereof, and relates to the technical field of medical animal models. The construction method of the cervix continuous lesion animal model comprises the following steps: cell perfusion: taking human cervix cell epithelial immortalized cells to prepare a cell suspension, and perfusing the cell suspension into the cervix surface and the cervix canal of a tested animal; the exogenous estrogen is continuously administrated for several days to promote the cervical lesion; constructing an animal model: collecting the cervical tissue of an animal, and making a sample slice; the animal model is constructed through intravaginal human cervical cell epithelial immortalized cell virus infection, the animal cervix shows diffuse continuous lesions, the modeling mode is innovative and successful, the blank of construction of the cervical cancer precancerous lesion animal model in China at present is made up, the model construction mode is consistent with the human body disease mode, and the model construction method has a wide application prospect. The accuracy of model construction can be better reflected, and an optimal animal model selection is provided for disease diagnosis and drug research and development in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of medical animal models, and in particular to an animal model of continuous cervical lesions and a construction method thereof. Background Art

[0002] The research progress of animal models of precancerous lesions is of great significance for understanding the pathogenesis, prevention and treatment of cancer. In recent years, with the advancement of scientific research technology, the research of animal models of precancerous lesions has made significant progress. Researchers have adopted a variety of methods to establish animal models of precancerous lesions, including chemical induction, transplanted tumor animal models, and genetically modified animal models. There is now a clear model for cervical cancer lesions, but there are few studies on the construction of animal models of cervical precancerous lesions, and there is currently no clear and successful modeling method.

[0003] Nowadays, there are several ways to construct mouse models of cervical precancerous lesions: such as transgenic mouse models, xenograft models, and skin transplant models. Among them, the lesion site of the transgenic mouse model of cervical cancer is in the cervix, and it manifests as spontaneous or induced lesions rather than exogenous or transplanted lesions. Although this model is more accurate for a variety of studies including drug development and has consistent gene expression, it is expensive. In addition, transgenic models also have limitations. Compared with humans, the mouse model induced by genes lacks the diversity of genetic mutations. Due to the different sites of infection of human papillomavirus, multiple epithelial mucosa are susceptible, so this model not only shows cervical lesions, but also has multiple lesions in the skin, penis, and head and neck, which seriously affects the survival rate of model mice. It is different from the construction of simple cervical precancerous lesions mouse model. Xenograft models require sufficient tolerance of mouse immunity, so immune dysfunction mice are usually selected; xenograft models cannot be used as the preferred model for research on cervical cancer and immunity and local microenvironment. In the construction of the skin transplant model, the lesion site is easily affected by other factors and does not show an obvious continuous pathological process. In addition, mice have a low tolerance to surgical trauma, and differential gene skin transplants often heal poorly in model mice. This operation has high technical requirements for the operator and requires the support of equipment and technology such as super microsurgery. Summary of the invention

[0004] The problem solved by the present invention is how to construct an animal model that is consistent with the process of cervical lesions in the human body. The modeling method is innovative and successful, filling the gap in the current domestic construction of animal models for cervical precancerous lesions. The model construction method is consistent with the way humans get sick, and can better reflect the accuracy of model construction, providing the best animal model selection for future disease diagnosis and drug development.

[0005] In order to solve the above problems, the present invention provides an animal model of cervical continuous lesions and a construction method thereof.

[0006] In a first aspect, the present invention provides a method for constructing an animal model of cervical continuous lesions, comprising the following steps: S1. Perfusion cells: immortalized human cervical epithelial cells are prepared into a cell suspension, and the cell suspension is perfused into the cervical surface and endocervical canal of the test animal; S2. Promoting lesions: Administering exogenous estrogen for several consecutive days to promote the occurrence of cervical lesions; S3. Construct an animal model: collect cervical tissue from animals and make sample slices.

[0007] Optionally, before step S1, the step S0. Preparation before modeling: mice or rats were selected as test animals and raised.

[0008] Optionally, step S0 includes: administering a progestogen drug to the test animal.

[0009] Optionally, step S0 includes: administering immunosuppressive drugs to the test animal.

[0010] Optionally, step S0 includes: injecting 5% nonoxynol-9 (N-9) into the vagina of the test animal to destroy the cervical epithelial layer in the vagina.

[0011] Optionally, step S0 includes: before the test animal is perfused with the cell suspension, flushing the vagina of the test animal with PBS buffer.

[0012] Optionally, the exogenous estrogen in step S2 is administered for more than 3 months.

[0013] Optionally, in step S3, the cervical tissue is fixed in 4% paraformaldehyde, embedded in paraffin and sliced ​​continuously throughout the cervical tissue to obtain sample slices.

[0014] Optionally, the sample sections are stained with hematoxylin-eosin and subjected to histopathological analysis.

[0015] On the other hand, the present invention provides an animal model of continuous cervical lesions established by the method for constructing an animal model of continuous cervical lesions as described above.

[0016] The beneficial effects of the animal model of continuous cervical lesions and the construction method thereof of the present invention are: The animal model was constructed by intravaginal human cervical epithelial immortalized cell virus infection, and the local cervical lesions of animals saved a lot of manpower and material resources and time, improved the stability of lesion location and lesion nature, and enhanced the convenience of detection; Use progestin drugs such as medroxyprogesterone acetate to promote the shedding of cervical epithelial cells, destroy the animal cervical epithelium, expose the cervical epithelial basement membrane, and make epithelial immortalized cells act directly on the already destroyed cervix as much as possible; The use of immunosuppressive drugs such as cyclophosphamide can reduce the immune function of animals and increase the success rate of virus implantation; Administering exogenous estrogen for several consecutive days can promote the occurrence of cervical lesions and improve the success rate of model construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A flowchart of the method for constructing an animal model of continuous cervical lesions of the present invention; Figure 2 These are magnified images under a microscope of cervical lesions of the model animal in Example 1 at different time points. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be interpreted as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not intended to limit the scope of protection of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by those skilled in the art to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific implementation methods and are not intended to limit this application; The term "including" and its variations used in this article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, the meaning of "multiple" is two or more.

[0020] In view of the problems existing in the above-mentioned related technologies, this embodiment provides an animal model of continuous cervical lesions and a construction method thereof, which relates to the field of medical detection technology.

[0021] like Figure 1 As shown, the present invention provides a method for constructing an animal model of continuous cervical lesions, comprising the following steps: S1. Perfusion cells: immortalized human cervical epithelial cells are prepared into a cell suspension, and the cell suspension is perfused into the cervical surface and endocervical canal of the test animal; S2. Promoting lesions: Administering exogenous estrogen for several consecutive days to promote the occurrence of cervical lesions; S3. Construct an animal model: collect cervical tissue from animals and make sample slices.

[0022] In this way, an animal model is constructed by intravaginal viral infection of immortalized human cervical epithelial cells, and local cervical lesions in animals are detected, which saves a lot of manpower and material resources and time, improves the stability of the lesion location and nature, and enhances the convenience of detection.

[0023] Specifically, the concentration of the cell suspension prepared from immortalized human cervical epithelial cells was set at 2 x 10 6 After the animal is anesthetized, the vagina is dilated to fully expose the cervix, and the cell suspension is infused on the surface of the cervix and the endocervical canal. To prevent cell flow and enhance cell adhesion, the animal is placed in a tilted position with the head low and the tail high for 5 minutes after the operation to promote absorption.

[0024] Optionally, before step S1, the step S0. Preparation before modeling: mice or rats were selected as test animals and raised.

[0025] C57BL / 6 mice (8-10 weeks old, black) were selected for mice, and SD rats (white) were selected for rats. The breeding conditions were as follows: relative humidity in the laboratory was 50-70%, the room temperature of the animals was 22-26°C, lighting was continuous for 12 hours day and night, and water was freely available. This animal experiment was approved by the Animal Ethics Committee of Heilongjiang University of Chinese Medicine, with the approval number: 2022101102.

[0026] Optionally, step S0 includes: administering a progestogen drug to the test animal.

[0027] In this way, progestogen drugs are used to promote the shedding of cervical epithelial cells, destroy the animal cervical epithelium, expose the cervical epithelial basement membrane, and make epithelial immortalized cells act directly on the already destroyed cervix as much as possible. Progestogen drugs can be selected from medroxyprogesterone acetate and the like.

[0028] Specifically, 5 days before the perfusion cell experiment, mice were administered 3 mg of medroxyprogesterone acetate per day, and rats were administered 6 mg of medroxyprogesterone acetate per day.

[0029] Optionally, step S0 includes: administering immunosuppressive drugs to the test animal.

[0030] In this way, the use of immunosuppressive drugs can reduce the immune function of animals and increase the success rate of virus implantation. Immunosuppressive drugs can be selected from monoclonal anti-CD3 antibodies or cyclophosphamide.

[0031] Specifically, 4 days before the perfusion cell experiment, mice were orally administered 0.1 mg / mouse and rats were orally administered 0.2 mg / mouse daily.

[0032] Optionally, step S0 includes: injecting 5% nonoxynol-9 (N-9) into the vagina of the test animal to destroy the cervical epithelial layer in the vagina.

[0033] In this way, nonoxynol-9 (N-9) again destroyed the cervical epithelial layer and promoted the fusion of implanted cells and cervical basement membrane cells.

[0034] Specifically, 6 hours before the cell perfusion experiment, 5% nonoxynol-9 (N-9) was perfused into the vagina of the test animals. Afterwards, a disposable rubber plug was used to block the vaginal opening to prevent the drug solution from leaking out, and the animals were placed in a tilted position with the head low and the tail high for 5 minutes.

[0035] Optionally, step S0 includes: before the test animal is perfused with the cell suspension, flushing the vagina of the test animal with PBS buffer.

[0036] Optionally, the exogenous estrogen in step S2 is administered for more than 3 months.

[0037] In this way, excessive intake of exogenous estrogen can promote cervical lesions and increase the risk of cervical cancer. Studies have shown that estrogen is an important auxiliary factor in the progression of cervical precancer and carcinoma in mice. Therefore, after virus implantation, mice were given 0.05 mg of estradiol daily and rats were given 0.1 mg of estradiol daily for more than 3 months. Long-term administration of exogenous estrogen can enable animals to reach an ideal disease state.

[0038] Optionally, combined with the disease lesion status and related research, human papillomavirus (HPV) virus infection under the action of exogenous estrogen generally causes cervical squamous intraepithelial lesions in 3-4 months and cervical cancer in about 5-7 months. Therefore, the observation time of this experiment is planned to be: observe once every 14 days in the first month, and observe once every 7 days from the second month until the lesions are clear. At the same time, the cervix of the model mouse that only completed vaginal washing on the first day without subsequent epithelial destruction and cell perfusion was selected as a control, and compared with the cervix of the model mouse 126 days after modeling to observe the scope and degree of the lesions. Reasonable time planning can detect lesions in a more timely manner.

[0039] Optionally, in step S3, the cervical tissue is fixed in 4% paraformaldehyde, embedded in paraffin and sliced ​​continuously throughout the cervical tissue to obtain sample slices.

[0040] Specifically, the mice are killed by cervical dislocation under anesthesia, and cervical tissues are collected. The thickness of the serial sections can be controlled at 5 μm.

[0041] Optionally, the sample sections are stained with hematoxylin-eosin and subjected to histopathological analysis.

[0042] Specifically, the criteria for histopathological grading are based on the thickness of the basal layer, the frequency of nuclear atypia and nuclear enlargement, and the extent of interstitial infiltration.

[0043] On the other hand, the present invention provides an animal model of continuous cervical lesions established by the method for constructing an animal model of continuous cervical lesions as described above.

[0044] The method for constructing an animal model of continuous cervical lesions provided by the present invention utilizes progestogen drugs to promote the shedding of cervical epithelial cells, destroy the cervical epithelium, and expose the cervical epithelial basement membrane to prepare for later experiments. At the same time, immunosuppressive drugs are used to reduce the immune function of mice and improve the success rate of virus implantation. Then, combined with H8 cell line infection and implantation, exogenous estrogen is used to induce cervical lesions in mice. A CIN model that is most similar to the clinical lesion characteristics of patients and only manifests as local cervical lesions can be quickly constructed. The disease pathological process conforms to the clinical progression from a precancerous state to an invasive cancer and a metastatic state.

[0045] Compared with the transgenic mouse CIN model, the method for constructing an animal model of continuous cervical lesions provided by the present invention is as follows: the gene-induced model not only shows cervical lesions, but also skin and head and neck lesions, which is different from the simple study of CIN. At the same time, systemic disease seriously affects the survival rate of model mice, causing a certain degree of interference in the evaluation of drug safety and effectiveness, and it is expensive, with a high loss rate, and is often manifested as multi-point disease. The price of the present invention is relatively controllable; it is most similar to the clinical CIN lesion characteristics, and only manifests as local cervical lesions; the survival rate of mice is high.

[0046] Compared with the xenogeneic implantation mouse CIN model, the method for constructing an animal model of cervical continuous lesions provided by the present invention is different from that of the xenogeneic implantation mouse CIN model: the xenogeneic implantation model requires sufficient tolerance of mouse immunity, the mouse strain used is usually unable to evaluate the dynamic changes of the immune system, and the growth site is irrelevant to the anatomical site of CIN, and the clinical progression of precancerous lesions cannot be fully simulated. The model mice of the present invention can respond appropriately to immunotherapy and produce anti-tumor immunity; the directional expression of HPV oncogenes E6 and E7 in mouse female reproductive tract tumors is most similar to the clinical CIN lesion characteristics, and only manifests as local cervical lesions.

[0047] Compared with the skin transplantation model, the method for constructing the animal model of cervical continuous lesions provided by the present invention is as follows: the lesion site of the skin transplantation model mouse is easily affected by other factors to affect the transplantation success rate, and no obvious continuous lesion process is shown. It should also be considered that the mouse has low tolerance to surgical trauma and the differential gene skin transplantation has poor healing in the model mouse. The model of the present invention is most similar to the clinical CIN lesion characteristics. The directional expression of HPV oncogenes E6 and E7 in mouse female reproductive tract tumors only manifests as local cervical lesions; the disease pathological process is consistent with the clinical progression from precancerous state to invasive cancer and metastatic state; and surgical trauma, poor healing factors and the like are avoided.

[0048] The present invention is further described below in conjunction with specific embodiments.

[0049] Example 1, a method for constructing an animal model of continuous cervical lesions, specifically comprising the following steps: S0-1. Model mouse strain selection: black C57BL / 6 mice (8-10 weeks old) and white SD rats. C57BL / 6 mice are inbred strains, widely used in transgenic mice in genetic experiments to simulate human gene defect diseases. They are commonly used in oncology, physiology, immunology, and genetics research, and are also the most successful strains for constructing this disease. SD rats have strong resistance to diseases, but are highly sensitive to sex hormones, so they are prone to cervical lesions under hormone induction.

[0050] S0-2. Medroxyprogesterone acetate promotes the shedding of cervical epithelial cells. Starting 5 days before the start of the experiment, mice were given 3 mg of medroxyprogesterone acetate per day, and rats were given 6 mg of medroxyprogesterone acetate per day to promote the shedding of cervical epithelial cells in synchronization with the estrus period. This operation can destroy the integrity of the cervical epithelium and fully expose the cervical epithelial basement membrane, which is conducive to the binding of viral particles to cells.

[0051] S0-3. Oral administration of cyclophosphamide reduces immunity. Starting 4 days before the start of the operation, mice were given 0.1 mg of cyclophosphamide orally every day, and rats were given 0.2 mg of cyclophosphamide orally every day to reduce the immune function of the model mice and improve the success rate of virus implantation.

[0052] S0-4 and nonoxynol-9 (N-9) destroyed the cervical epithelium again. Six hours before the modeling operation, 2 ml of 5% nonoxynol-9 (N-9) was injected into the vagina to destroy the cervical epithelium and promote the fusion of implanted cells and cervical basement membrane cells.

[0053] S0-5. Clean the vagina and use PBS buffer to rinse the vagina of the test animal.

[0054] S1. Perfusion cells: immortalized human cervical epithelial cells are prepared into a cell suspension, which is then perfused into the cervical surface and endocervical canal of the test animal.

[0055] Human cervical epithelial immortalized cells (H8 cells) were used for vaginal implantation. The H8 cell line is a cell line with immortalization function established by Chinese medical researchers. It is an HPV16-positive immortalized cervical squamous epithelial cell, similar to the clinical CINII-III grade cervical intraepithelial neoplasia. Although it has the characteristics of immortalization, it will not form malignant tumors in vitro, so it was selected for this experiment.

[0056] S2. Promote lesions: Excessive intake of exogenous estrogen can promote the occurrence of cervical lesions and increase the risk of cervical cancer. Studies have shown that estrogen is an important auxiliary factor in the progression of cervical precancer and carcinoma in mice. Therefore, after virus implantation, mice are given 0.05 mg of exogenous estrogen estradiol daily and rats are given 0.1 mg of exogenous estrogen estradiol daily for more than 3 months to promote the occurrence of cervical lesions and achieve the ideal disease state.

[0057] Combined with the disease pathological status and related research, HPV virus infection under the action of exogenous estrogen generally causes cervical squamous intraepithelial lesions in 3-4 months and cervical cancer in about 5-7 months. Therefore, the observation time of this experiment is planned to be: every 14 days in the first month, and every 7 days starting from the second month until the lesions are confirmed (estimated to be 28 times in total).

[0058] S3. Construction of animal model: Under anesthesia, mice were killed by cervical dislocation, and cervical tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned throughout the cervix with a thickness of 5 μm. Each slide was stained with hematoxylin-eosin staining (HE staining) and histopathological analysis was performed. The criteria for histopathological grading were based on the thickness of the basal layer, the frequency of nuclear atypia and nuclear enlargement, and the presence of interstitial infiltration.

[0059] The animal model was constructed by the above method, and samples were collected at the specified time. The HE staining pathological results of the samples collected on the first day of modeling were compared with those collected on the 126th day of modeling. Figure 2 As shown in the figure, cervical lesions were observed under microscopes of different magnifications (i.e., 4X, 10X, and 40X). Among them, XD01 is the cervical tissue of mice on the first day of modeling, and it can be seen that the cervical cells are clearly stratified, the cells are arranged neatly, the nuclear-cytoplasmic ratio is normal, and the cell polarity is normal; XD126 is the cervical tissue of mice on the 126th day after modeling, Figure 2 It can be seen that the nuclei of the cells in the 1 / 3 to 2 / 3 layers below the epithelium are significantly enlarged, the nuclear-cytoplasmic ratio is increased, the nuclei are deeply stained, there are many mitotic figures, and the cell polarity is still there, which is consistent with moderate atypical hyperplasia, that is, CIN grade 2. DD01 is the cervical tissue of rats on the first day of modeling, and it can be seen that the cells are arranged neatly, the morphology is regular, and the nuclear-cytoplasmic ratio is normal; DD126 is the cervical tissue of rats on the 126th day of modeling, and it can be seen that the squamous epithelial layer is thinned, the diseased cells almost occupy the entire epithelial layer, the nuclei are abnormally enlarged, the nuclear-cytoplasmic ratio is significantly increased, the staining is darker, the nuclear mitotic figures are many, the cells show typical vacuolation, the arrangement is disordered, and there is no polarity, which is consistent with moderate to severe atypical hyperplasia, that is, CIN 2-3. It can be seen that the success and reliability of the construction method of the animal model of continuous cervical lesions of the present application.

[0060] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of continuous cervical lesions, characterized in that: The following steps are involved: S1. Perfusion of cells: immortalized human cervical epithelial cells are prepared into a cell suspension, and the cell suspension is perfused into the cervical surface and endocervical canal of the test animal; S2. Promoting lesions: Administering exogenous estrogen for several consecutive days to promote the occurrence of cervical lesions; S3. Construct an animal model: collect cervical tissue from animals and make sample slices.

2. The method for constructing an animal model of continuous cervical lesions according to claim 1, characterized in that: Before step S1, the step S0. Preparation before modeling: The test animals are selected from mice or rats and raised.

3. The method for constructing an animal model of continuous cervical lesions according to claim 2, characterized in that: The step S0 comprises: administering a progestogen drug to the test animal.

4. The method for constructing an animal model of continuous cervical lesions according to claim 2, characterized in that: The step S0 comprises: administering immunosuppressive drugs to the test animal.

5. The method for constructing an animal model of continuous cervical lesions according to claim 2, characterized in that: The step S0 comprises: injecting 5% nonoxynol-9 (N-9) into the vagina of the test animal to destroy the cervical epithelial layer.

6. The method for constructing an animal model of continuous cervical lesions according to claim 2, characterized in that: The step S0 comprises: before the test animal is perfused with the cell suspension, PBS buffer is used to flush the vagina of the test animal.

7. The method for constructing an animal model of continuous cervical lesions according to claim 1, characterized in that: The exogenous estrogen in step S2 is administered for more than 3 months.

8. The method for constructing an animal model of continuous cervical lesions according to claim 1, characterized in that: In step S3, the cervical tissue is fixed in 4% paraformaldehyde, embedded in paraffin and sliced ​​continuously throughout the cervical tissue to obtain the sample slices.

9. The method for constructing an animal model of continuous cervical lesions according to claim 8, characterized in that: The sample sections were stained with hematoxylin-eosin staining and subjected to histopathological analysis.

10. An animal model of continuous cervical lesions established by the method for constructing an animal model of continuous cervical lesions as described in any one of claims 1 to 9.

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