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CN-121987760-A - Animal model construction method for simulating various stages from HPV continuous infection to cervical cancer under natural infection state and application thereof

CN121987760ACN 121987760 ACN121987760 ACN 121987760ACN-121987760-A

Abstract

The invention belongs to the technical field of medical animal model construction, in particular to a construction method and application of an animal model for simulating various stages from HPV continuous infection to cervical cancer under a natural infection state, the method uses a drug immunosuppressant for promoting HPV infection to combine MmuPV to perform lower genital tract infection modeling of mice, the constructed animal model has stable and uniform papilloma virus infection, high modeling rate and low mortality, the method can completely reproduce the whole process from HPV continuous infection to cervical cancer change, has short modeling time, controllable process and good reproducibility, accords with clinical practice, improves the efficiency and quality of model construction, provides an ideal animal model for the subsequent research of an oncogenic mechanism of HPV infection and a virus host interaction mechanism, and can assist in developing and screening clinical treatment methods for preventing and treating cervical lesions at each stage, promote clinical transformation, research and development of new drugs and the like.

Inventors

  • XIAO JING
  • CHEN MINGLI
  • CHEN XIAOFENG
  • HU LINGJUAN
  • LI GENG
  • ZHANG JIAONA

Assignees

  • 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院）

Dates

Publication Date
20260508
Application Date
20251230

Claims (10)

  1. 1. The animal model construction method for simulating each stage from HPV continuous infection to cervical cancer under the natural infection state is characterized by comprising the following steps: Step 1, inoculating MmuPV virus particles at the tail of an immunodeficiency animal to naturally grow papilloma, collecting tumor tissues, homogenizing and centrifuging to obtain supernatant, and preparing a MmuPV virus library; step 2, carrying out gradient dilution on MmuPV plasmid corresponding to MmuPV virus, constructing a standard curve between the virus copy number and the CT value of qPCR, and calculating the virus copy number; Step 3, immunosuppressant is given to animals with normal immune functions, and continuous administration is carried out after toxin is counteracted; step 4, adopting MmuPV to infect the lower genital tract of the animal in step 3 to perform modeling; and 5, measuring the virus load of the vaginal lavage fluid every week after the second week after infection to track and record the infection change state of the animal, periodically killing the animal to perform HE staining and judging cervical vagina lesion level, and monitoring the cervical vagina lesion level of the mice by detecting the virus load in the vaginal lavage fluid every week.
  2. 2. The method of constructing an animal model for simulating the stages of HPV persistent infection to cervical cancer in a naturally infected state according to claim 1, wherein the immunodeficient animal in step 1 is a T lymphocyte-deleted and/or B lymphocyte-deleted mouse.
  3. 3. The method for constructing an animal model for simulating the stages from HPV continuous infection to cervical cancer according to claim 1, wherein the initial concentration of 100-200 ng/. Mu.L is used for carrying out gradient dilution of MmuPV < 1 > plasmids in the step 2, a standard curve between the virus copy number and the CT value of qPCR is constructed, and the corresponding CT value of the virus liquid is substituted into the formula to obtain the corresponding virus copy number, thereby realizing the quantification of the virus load.
  4. 4. The method for constructing an animal model for simulating the stages from HPV persistent infection to cervical cancer according to claim 3, wherein the virus solution is obtained by placing collected papilloma tissues in a sterile tube, adding PBS and grinding beads, breaking the tissues, centrifuging, transferring the supernatant to another sterile tube, and filtering the supernatant by using a pinhole filter before inoculating the supernatant to an animal with normal immune function.
  5. 5. The method of constructing an animal model for simulating the stages from HPV persistent infection to cervical cancer under natural infection according to any one of claims 1 to 3, wherein the immunosuppressant in step 3 is cyclosporin.
  6. 6. The method for constructing an animal model for simulating the stages from HPV persistent infection to cervical cancer according to claim 5, wherein the step 4 of constructing the model is performed by synchronizing the animal of step 3 with a progestogen to the interphase, then damaging the lower genital tract of the animal, promoting the invasion of virus into the basal layer, and then injecting MmuPV virus solution for attack of the lower genital tract.
  7. 7. The method for constructing an animal model for simulating the stages from HPV persistent infection to cervical cancer according to claim 6, wherein the step of damaging the lower genital tract of the animal comprises the step of damaging the lower genital tract with a dental floss bar and 4% nonoxynol.
  8. 8. An animal model constructed by the method for constructing an animal model simulating the stages from HPV persistent infection to cervical cancer under the natural infection state of any one of claims 1 to 7.
  9. 9. Use of the animal model of claim 8 as a basic research animal model for exploring HPV infection, transmission, oncogenic mechanisms, virus-host immunointeractions.
  10. 10. Use of the animal model of claim 8 for developing and evaluating clinical interventions for the prevention and treatment of HPV infection cervical lesions at various stages.

Description

Animal model construction method for simulating various stages from HPV continuous infection to cervical cancer under natural infection state and application thereof Technical Field The invention belongs to the technical field of medical animal model construction, and particularly relates to an animal model construction method for simulating various stages from HPV continuous infection to cervical cancer change under a natural infection state and application thereof. Background The fourth place of global female malignant tumor with High incidence of cervical cancer, persistent infection of High-risk papillomavirus (High-risk Human Papillomaviruse, HR-HPV) is the main cause of cervical cancer, and a specific and effective treatment means for HPV infection is lacking at present. Since cervical lesions develop slowly for up to ten years, and clinically early stage HPV infection and no obvious symptoms are seen in patients at the precancerous lesion stage, it is extremely difficult to clinically realize continuous samples of tracking patients from normal cervical to HPV persistent infection to precancerous lesions and cervical cancer, which limits systematic research on cervical cancer pathogenesis. Therefore, the development of an animal model which successfully simulates the whole process from HPV infection to cervical cancer is important for further exploring the pathogenesis of the disease and evaluating the clinical control method of each stage of disease progression. Papillomaviruses isolated from large mammals in the past have certain limitations in the specific experimental operation process, and the past laboratory mouse animal models also cannot simulate all lesion stages of HPV infection carcinogenesis in human natural infection states. Specifically, due to species-specific tropism of papillomaviruses, viruses isolated from animals such as dogs, rabbits and cattle can study mechanisms such as virus replication, but further application is limited due to large individual differences of animals, limited available reagents, excessive experimental cost and the like, and the traditional HPV transgenic mice can be used for researching the key effects of oncoproteins such as E6, E7 and the like, however, the modeling method of the HPV transgenic mice has low clinical similarity, cannot simulate a series of oncogenic processes after natural infection of human beings with viruses, is expensive, and needs to find a better alternative model. Murine papillomavirus (MmuPV 1) was isolated in 2011, and subsequent attempts by researchers in the oral cavity, skin, anus and lower genital tract opened a new way for studying the mechanisms of natural infection and carcinogenesis of PV in laboratory mice, however, past MmuPV1 infected lower genital tract animal models have limitations such as excessively long construction time and clinical practical inconsistencies. Spurgeon et al (A Novel In Vivo Infection Model To Study Papillomavirus-Mediated Disease of the Female Reproductive Tract. mBio. 2019 Mar 5;10(2):e00180-19.) successfully constructed pathological stage models such as cervical intraepithelial neoplasia level 1 (CERVICAL INTRAEPITHELIAL neoplasia, CIN 1), CIN2, CIN3, cervical squamous cell carcinoma (cervical squamous cell carcinoma, CSCC) by adopting methods such as MmuPV1, combined estrogens (Estrogen, E2), ultraviolet irradiation (Ultraviolet radiation B, UVB) and the like, but the construction of the inter-group models is nonuniform, the construction time is overlong and the like. In addition, the long-term use of exogenous estrogen for modeling cervical tumor is also controversial, estrogen acts on a plurality of target organs such as uterus, breast, ovary, pituitary gland and the like mainly through nuclear receptor estrogen receptor alpha and estrogen receptor beta, continuous exposure of exogenous estrogen at high level can obviously increase the risk of onset of breast cancer, endometrial cancer and ovarian cancer, cervical cancer is clinically used as a non-hormone-dependent tumor, the onset process is not close to the increase of estrogen level, and non-specific side reaction caused by long-term application of estrogen to animal models cannot be ignored. Atkins et al (Monitoring mouse papillomavirus-associated cancer development using longitudinal Pap smear screening. mBio. 2024 Aug 14;15(8):e0142024.) used MmuPV to infect the lower genital tract in NU/J mice carrying NU mutation in one Foxn1 allele, developed CIN3 to SCC 5.5 months after infection, and appeared SCC in all mice at 8 months of infection, however, the further application was limited by the excessively long modeling time and the expensive cost and difficult availability of mice. Therefore, in view of the limitations of the existing HPV infection laboratory animal models, it is necessary to establish an animal model which has short modeling time, less operation times, stable HPV infection, good reproducibility, low mortality, and is clini