动物造模,视网膜脱离动物模型 z-bio 2022-01-05 531

　　(1) Replication method (1) Rabbit skin fibroblast culture: Take a New Zealand rabbit weighing about 2 kg, shave off one of the buttocks, and disinfect the skin with 75% ethanol. Use the eye's corneal cyclome to make an 8 mm diameter incision. Separate and remove the button-shaped skin tissue with scissors, rinse it with sterile saline several times, cut into 1 mm 3 in a small beaker, and spread it on a 6-well 35 mm plastic culture plate. Add an appropriate amount of medium (RPMI1640). Cultures containing 15% calf serum and penicillin (100 U/ml each) were cultured at 37°C and 5% CO2. Change the media twice a week. Generally, from day 7 to day 10, fibroblasts can be attached to the bottom of the culture plate and subcultured. After 0.25% trypsinization, pipetting, dispersion with D-Hanks solution and centrifugation (1000/min, centrifugation for 5 minutes), the cells were cultured in 200 ml glass culture flasks.

　　(2) Preparation of cell suspension for intravitreal injection: After digesting, dispersing and centrifuging the cultured cells to the bottle wall, add D-Hanks solution and pipette to homogenize the cell suspension. Mix a small amount of the trypan blue stained suspension at a ratio of about 9:1, count the live cells using a hemocytometer, and finally add the cell suspension to a concentration of 2.5 x 1,000,000 cells per milliliter. Use a tuberculin syringe to aspirate 0.1 ml (containing 2.5 x 100,000 cells) for later use.

　　(3) Intravitreal injection: Before injection, dilate rabbit eyes with 1% atropine eye drops 2-3 times, and intramuscularly inject ketamine (5mg/kg body weight) for general anesthesia. During the injection, 1% methyl cellulose was dropped onto the surface of the cornea, and contact lenses were placed. Under a surgical microscope, insert the needle 3 mm behind the corneal valve annulus. When it reaches the center of the vitreous from the pupil area, cut the needle tip upward and slowly inject the cell suspension.

　　(4) Model features After the cells are injected into the vitreous body, immediately check with ophthalmoscope. You will see that the densest vitreous in the injection path has a lot of dust and turbidity. On the 4th day, a small amount of string-like or film-like growth formed on the vitreous. On day 7, the proliferation extends to the posterior pole of the eye and connects to both sides of the optic disc or myeloid complex. It manifests as dilation and entanglement or wrinkles of retinal blood vessels. Retinal detachment usually occurs about 14 days after the partial detachment of the bilateral medullary heddle, and some gradually expand to complete the detachment. Normally, no retinal holes are formed.

　　(5) Comparative medicine This model method injects allogeneic skin fibroblasts into the vitreous of rabbit eyes. As the cells continue to proliferate, they proliferate and pull and detach the retina. This process is very similar to the clinical features of proliferative vitreoretinopathy in the human eye. The incidence of traction retinal detachment directly reflects the severity of proliferative vitreoretinopathy, and can also be used as an indirect quantitative indicator to measure the efficacy of drugs for the disease. Therefore, this model method provides an ideal animal model for studying the preventive and therapeutic effects of drugs on proliferative vitreoretinopathy. However, the experimental conditions need to be controlled when creating the model. The rabbit eyeball is smaller than the human eyeball, and the front and back diameter of the lens is larger, so the vitreous cavity is relatively small. When performing vitreous injection, the needle direction must be grasped to prevent damage to the lens. Traumatic cataract will affect the fundus examination. After the needle tip reaches the vitreous, care must be taken not to damage the retina. This makes it safer to manipulate the model using the surgical microscope.