动物模型,病毒性角膜炎 z-bo 2020-08-03 592

　　[Modeling mechanism] Viral keratitis (viral keratitis) is the most common herpes simplex keratitis (HSK). HSK is a blinding eye disease caused by herpes simplex virus infection, which seriously affects the visual function of patients. Its clinical characteristics are mainly long-term repeated attacks, which eventually lead to corneal leukoplakia, corneal neovascularization, corneal ulcers and even perforation. It severely impairs the patient's visual function. At present, the basic clinical research of HSK is mainly carried out by establishing HSK animal models. Modeling methods mainly include corneal scratch inoculation and UV B exposure.

　　[Modeling method] Choose healthy male or female BALB/c mice, weighing 18-20 g, and both eyes are free of conjunctivitis, keratitis and other eye diseases. Herpes simplex virus type I (HSV-1), the titer before use is 2 x 1000000 pfu/ml. Two times a day and one week later, 2.5 g/L chloramphenicol eye drops were injected into the eyes of the mice. Before virus inoculation, mouse corneas were stained with 10 g/L sodium fluorescein and examined with slit lamp microscopy to exclude mice with corneal or conjunctival lesions. The mice were anesthetized by intraperitoneal injection of chloral hydrate. Under the microscope, mark a "#" mark on the cornea of the right eye with the blade of a #5 scalpel. It is recommended to penetrate the corneal epithelial layer. Next, use a pipette to add 5μl of virus containing 2×1000000pfu HSV-1.DMEM, drop it on the corneal surface of the right eye, and after closing the eyelid, gently massage for 30 seconds to completely remove the virus solution. Touch the cornea. On the second day after modeling, 2.5 g/L chloramphenicol eye drops were given twice a day for two consecutive weeks. Then, the cornea was stained with 10 g/L sodium fluorescein for 3 consecutive days from the next day, and the cornea was confirmed with a slit lamp microscope. )Detected. Check for virus replication. For the above model animals, on the basis of 2.5 g/L chloramphenicol eye drops, add 1 g/L acyclovir eye drops to the right eye to reduce the HSK model twice a day for 2 weeks. It promoted the cure of acute epithelial keratitis in mice. The virus established a latent infection in the trigeminal ganglia or cornea, and established a HSK latent infection model. Seven weeks after virus inoculation, PCR was performed on cornea and trigeminal ganglion samples to confirm that this is a mouse model of latent HSK infection. After the surface of the right eye was anesthetized, it was exposed to UV light with a wavelength of 302 m for 3 minutes. The output of the ultraviolet transmission lamp is 1.4mW/cm², and the irradiation intensity is 250MJ/cm². On the second day after ultraviolet B irradiation, 10 g/L sodium fluorescein was stained continuously for 7 days, the cornea was observed with a slit lamp microscope, the right eye cornea was wiped with a cotton swab soaked in DMEM, and HEK293T cells were cultured and observed. Cytopathological changes to determine whether the HSK recurrence model has been successfully established. [Model Features] After virus inoculation, the cornea showed punctate, dendritic or map-like lesions, and HEK293T cells showed cytopathological changes in corneal swabbing. In other words, HEK293T cells grow in the shape of grapes, round and aggregate. The original adherent growth of the cell part is separated and suspended in DMEM, which is considered to be a primary HSK infection. If the acute epithelial keratitis disappears and the PCR test virus of the cornea and trigeminal ganglion is positive, a HSK latent infection model is established. If corneal spots, dendrites or geographic keratopathy occur after irradiation with UV-B rays and the corneal wipe medium has CPE, it is considered that an HSK recurrence model has been established.

　　[Model Evaluation and Application] This method is simple and has a high modeling rate, and it can establish HSK experimental animal models at different infection periods. Although rabbits and mice can be used to establish this model, studies have shown that rabbits are easy to establish potential infection models, but tend to relapse spontaneously. In the HSK latent infection model established in mice, spontaneous recurrence will not occur. In a mouse model of latent infection, repeated irradiation of 302m ultraviolet rays is not only simple, effective and easily damaged, but also close to the environmental conditions for human HSK recurrence. This is a good stimulus to further study the impact of environmental factors on human HSK recurrence and its prevention and treatment.