动物造模,单纯疱疹病毒感染模型 z-bio 2021-12-27 447

　　Herpes simplex virus (herpes simplex virus, HSV) belongs to the herpes virus family, and its genome is linear double-stranded DNA. The population infection rate is as high as 80%. Herpes simplex virus can be divided into type 1 herpes simplex virus (HSV-1) and type 2 herpes simplex virus (HSV-2). The two are different in biology, natural history, biochemical composition and immunological characteristics. The infection is the same. HSV-1 is usually transmitted through oral secretions and often lurks in the trigeminal nerve and upper cervical ganglion of the human body. On the other hand, almost all HSV-2 is transmitted through sexual contact, usually lurking in the nerve root area of the human body. HSV can cause a variety of diseases, including herpes encephalitis, herpetic keratitis, genital herpes, neonatal infections and fetal teratogenicity. Due to the wide host range of the herpes virus, many animals, including rabbits, guinea pigs, rats and mice, can replicate the animal model of HSV-1 latent infection through herpes simplex virus infection. Currently, there are many vaccination methods for HSV latent infection models, such as intranasal vaccination, toe vaccination, corneal scraping vaccination, tail vein vaccination and tooth extraction vaccination. There are different vaccination methods for the success rate and mortality of the model. Model animals. the difference.

　　1 genital herpes model

　　(1) Copy method Wash the vulva with normal saline, wipe it several times with a dry cotton swab, then tap the vulva with a plum needle to make the vulva congestion and ooze a little water.. Clean the vulva with a cotton swab. Use a 1 ml syringe to draw 0.15 ml 10-7 times force TCID50 virus solution, insert the forced feeding needle, insert the needle into the vagina of 3-4 cm guinea pig, inject the virus into the vaginal vault, and then slowly withdraw it. Insert a gelatin sponge into the vagina to maintain the poisoned state. Drop a small amount of venom on the needle, then spread it evenly with a glass rod to allow the virus to penetrate the skin. After 3-6 days, different numbers and sizes of blisters may appear in the vagina. These blisters will fuse together and cause serious injury. The blisters are filled with slurry, the surface of the blisters is fragile, the blisters contain high-titer virus particles, and the blisters burst to form ulcers. The lymph nodes are enlarged and the virus is isolated from the vaginal swabs and tissues at the beginning of the disease.

　　(2) Model characteristics After infecting animals, HSV virus can destroy vaginal mucosa and skin, spread locally, and isolate the virus from vaginal secretions and tissues. The virus spreads from the lesion to the local lymph nodes and may spread further. You can also see that the virus has spread to the nervous system and has been latent for a long time. Latent virus is regularly activated, causing skin and mucous membranes, herpetic lesions. (3) Comparative medicine All guinea pigs after vaccination showed symptoms similar to human genital herpes, mainly manifested as redness, swelling, blisters, ulcers and abdomen of the vulva. Moreover, a potential infection can be established in the nervous system, and herpes lesions can recur spontaneously at the site of the vaccination. The foreign animal models of genital herpes currently reported in pharmacodynamic research include mice, monkeys and guinea pigs. Mice can develop HSV-2 genital infections, but they rarely show visible lesions and die easily. Vaginal HSV-2 infections in monkeys are very similar to humans, but for objective reasons, their use is limited. This infection is an ideal worm animal model because it not only shows clinical symptoms similar to human early and recurrent I, but also establishes a latent infection in the nervous system and has a lower mortality rate than mice. infection.

　　2 herpetic keratitis model

　　(1) Copy method Put the anesthetized experimental mice under a microscope, and then use a "cross"-shaped 1 ml injection needle and 5μl2 to scratch the corneal epithelium of the experimental mice. ×100000000 PFU/plus ml The virus falls on the scratched surface of the cornea. After infection, the eyes of the mice were stained with sodium fluorescein and the damage was observed under a slit lamp. 24 hours after infection, the corneal epithelium of the model mice showed varying degrees of sodium fluorescein staining, most of which showed punctate staining and damage. On the third day of infection, the area of the cornea was stained with sodium fluorescein. The number of model mice increased and the cornea became more severe. On the first day, there were less transparency and dendritic lesions, which was a typical change, indicating that the lesions were intensified; 7 days after infection, the cornea of the model mice became cloudy and lost transparency. (2) Model features Herpes simplex virus keratitis caused by herpes simplex virus is a delayed allergic disease mediated by T lymphocytes. CD4 + T lymphocytes are a mouse model that causes destruction of the interstitial layer of tissues. The main cells involved in inflammation. The virus replicates and proliferates in mouse corneal epithelial cells, prevents host cell protein biosynthesis, destroys tight junctions between cells, destroys cells and releases virus particles. The virus particles spread deeply, causing persistent infection and blindness. (3) Comparative medicine Herpes simplex virus can cause keratitis in mice, not only is it prone to latent infection, but the possibility of recurrence is also less. The mouse model of herpes simplex keratitis can be used to study the mechanism of cytokines and adhesion molecules in herpes simplex keratitis. Corresponding antibodies can also be used in therapeutic research to guide clinical treatment. The mouse model is not as ideal as the rabbit model and lacks the spontaneous release of the virus and disease recurrence, but corneal inflammation occurs shortly after the mouse is infected. This is similar to humans. Herpetic keratitis.

　　3 herpes facial nerve palsy model

　　(1) Copy method Collect 4 weeks old BAlb/cAJcl mice weighing 18-18 g, and anesthetize by intraperitoneal injection of 50 mg/kg body weight of sodium pentobarbital. The auricle and the front 2/3 of the right tongue were respectively inoculated with 6.7-10000000 PFU/ml of 25μl HSV, and the left bone PBS was used as a control. Within 6-9 days after vaccination, the right facial nerve was paralyzed, and the symptoms continued to recover spontaneously for 3-7 days. Confirmation of HE staining: facial nerve edema and inflammatory changes. The facial nerve of the temporal bone is obviously edema, and there is no gap between the facial nerve canals. (2) Model features HSV-1 can induce Bell's acute transient facial nerve palsy in model mice. HSV antigen has been detected in the facial nerve, reproductive ganglion and facial nerve nucleus of mice. The facial nerve can cause facial nerve palsy. Staining with FITC-labeled anti-gC monoclonal antibody can cause the geniculate ganglion, root decline and the appearance of immuno-positive cells in the facial nucleus, which leads to retrograde transport of the virus through axons, that is, the response shown by humoral and cellular immunity. .. (3) The histopathological changes of the facial nerve of comparative medical model animals are similar to that of human bell palsy, that is, severe nerve swelling, vacuolar degeneration, and infiltration of inflammatory cells in the facial nerve and facial nerve nucleus. The paralyzed side is the proximal reproductive ganglia, which is relatively serious, especially in the migration of nerve roots in the proximal brain tissue. This model lays the foundation for animal testing for more detailed research on the etiology and clinical treatment of Bell's palsy.

　　4 herpes encephalitis model

　　(1) Copy method KM mice weighing 15-18 g. First, the herpes simplex virus (HSV-1) is placed in HeLa cells and cultured for 48 hours. If 50% of HeLa cells show enlarged, round or confluent lesions, collect virus titer and determine TCID50=10. -5th power; virus culture and intracranial inoculation of mice. The inoculated virus concentration is TCID50 = 10-3, the inoculation volume is 0.03 ml/mouse, the injection site is the line between the upper right corner of the eye and the root of the ear, the needle entrance is 2-3 mm, and there is a sense of error. At the predetermined time for model creation and observation, the mouse brain tissue was processed for intracranial HSV-1 DNA determination, brain tissue was collected for routine observation, and normal tissue sectioning was performed, fixed with 10% formaldehyde solution for HE staining . Observe under an optical microscope. After fixing a part of the brain tissue with 2% glutaraldehyde, ultrathin sections were prepared with an electron microscope and observed with a transmission electron microscope. (2) Model characteristics After HSV-1 inoculation, model animals showed normal disease characteristics. Three to five days after vaccination, symptoms of various severity begin to appear, including weight loss, shoulder shrugs, curly hair, and quadriplegia. And the reaction is slower, some of them suffer from drowsiness and hemiplegia, discomfort and death. The HSV-infected mice began to show symptoms on the 4th day, and 70% of the animals became sick on the 5th day, usually survived within 7 days, and died within 2 days after the onset. When using PCR to detect HSV-1 DNA in mouse brain, the positive rate reached 100%. After inoculation with the virus, the brain tissue of the model mice can be seen with the naked eye, with obvious congestion and edema, and ventricular hemorrhage. When observed under a microscope, the mouse cerebral cortex cells are arranged disorderly. Form and vacuole. Observing the ultra-fine structure through electron microscopy, it was found that there were mitochondrial degeneration and necrosis in the brain tissue of model mice, enlarged vesicles, vacuole formation, and mature and immature virus particles. The modeling is simple, the disease characteristics are clear, the mouse mortality rate after infection is high, and the survival period is short.

　　(3) Comparative medicine. Humans are the natural host of herpes simplex virus. The infection rate of HSE in the population can be as high as 80%. The main clinical cause of herpes simplex viral encephalitis (HSE) in children is herpes. Herpes simplex virus type 1. Due to the severe clinical manifestations of HSE in children, it is usually possible to endanger the lives of children, and even the survivors have serious sequelae. At present, the incidence of HSE in children is not yet fully understood, and it is very important to establish an ideal HSE animal model for research. After inoculating and infecting mice with HSV-1 virus, the virus spreads to the human brain through the olfactory nerve without viremia. The clinical symptoms of model animals are similar to human herpes encephalitis. The mouse HSE model established in this way is suitable for research on the etiology, prevention and treatment of HSE.