动物造模,药效评价,病毒性角膜炎 z-bio 2024-08-02 369

　　The most common viral keratitis is herpes simplex virus keratitis. HSK is a blinding eye disease caused by herpes simplex virus infection, which seriously endangers patients' visual function. Its clinical characteristics are mainly characterized by a long course of disease, repeated attacks, and ultimately leading to corneal leukoplakia, corneal neovascularization, corneal ulcers, and even perforation, which seriously endanger patients' visual function. The clinical basic research on HSK is currently mainly conducted through the establishment of HSK animal models. The main modeling methods include corneal scratch inoculation and ultraviolet B-light irradiation.

　　[Modeling Method] Healthy BALB/c mice, regardless of gender, weighing 18-20g, with no eye diseases such as conjunctivitis or keratitis in both eyes, were selected. Type I herpes simplex virus (HSV-1), with a pre use titer of 2 × 1000000pfu/ml. Administer 2.5g/L chloramphenicol eye drops to both eyes of mice twice a day, and inoculate after one week. Stain the mouse cornea with 10g/L fluorescein sodium before virus inoculation, and examine the corneal condition under a slit lamp microscope to rule out the presence of corneal or conjunctival lesions. Anesthetized mice were injected intraperitoneally with chloral hydrate. Under a microscope, the tip of the back of the No.5 surgical blade was scratched in the shape of a "#" on the cornea of the right eye, preferably penetrating the corneal epithelial layer. Then, 5 μ l of DMEM containing 2 × 1000000pfu HSV-1 virus was dropped onto the surface of the cornea of the right eye using a pipette. After closing the eyelids, gently massage for 30 seconds to ensure that the virus solution fully contacts the cornea. On the day after modeling, continue to administer 2.5g/L chloramphenicol eye drops twice a day for two consecutive weeks. Starting from the next day, the cornea was stained with 10g/L fluorescein sodium and examined under a slit lamp microscope for 3 consecutive days. At the same time, tears from the corneal surface of the right eye were collected using a cotton swab for detection of human embryonic kidney epithelial cells (HEK293T) to determine the presence of virus replication.

　　For the above model animals, 1g/L acyclovir eye drops were added to the right eye on the basis of 2.5g/L chloramphenicol eye drops twice a day for two consecutive weeks to promote the healing of acute epithelial keratitis in HSK model mice and establish latent infection of the virus in the trigeminal ganglion or cornea, thus establishing a HSK latent infection model.

　　Seven weeks after virus inoculation and confirmed by PCR detection of corneal and trigeminal ganglion samples as a latent HSK infection mouse model, the right eye surface was anesthetized and exposed to ultraviolet radiation with a wavelength of 302nm for 3 minutes. The power of the ultraviolet transmitter is 1.4mW/square centimeter, and the irradiation intensity is 250MJ/square centimeter. Starting from the day after irradiation with UV-B light, the cornea was observed under a slit lamp microscope for 7 consecutive days after staining with 10g/L fluorescein sodium. Then, HEK293T cells were cultured by wiping the right cornea with a cotton swab dipped in DMEM to observe the pathological changes and determine whether a successful HSK recurrence model had been established.

【 Model Characteristics 】 After virus inoculation, the cornea shows punctate, dendritic, or map like lesions, and the corneal wiping fluid shows histopathological changes in HEK293T cells, manifested as HEK293T cells becoming larger, rounder, and grape shaped aggregation changes, and causing some cells that originally adhered to the wall to detach and float in DMEM. This is considered as primary infection of HSK. If acute epithelial keratitis disappears and the virus is detected positive by PCR in the cornea and trigeminal ganglion, it is determined to establish a latent HSK infection model. If the cornea shows punctate, dendritic, or map like corneal lesions again after exposure to ultraviolet B light, and CPE appears in corneal wiping culture, it is determined as the establishment of HSK recurrence model.
This method is simple, has a high modeling rate, and can establish HSK experimental animal models at different infection stages. Both rabbits and mice can be used to establish this model, but some studies have found that although rabbits are easy to establish latent infection models, they are prone to spontaneous recurrence. However, the HSK latent infection model established in mice is not prone to spontaneous recurrence. The 302nm ultraviolet irradiation induced recurrence of latent infection in mouse models is not only simple, effective, and easily damaged, but also closer to the environmental conditions of human HSK recurrence, which has a good enlightening effect on further in-depth research on the influence of environmental factors on human HSK recurrence and its prevention and treatment.