动物模型,视网膜脱离 z-bo 2020-08-08 506

　　(1) Replication method ①Cultivation of rabbit skin fibroblasts: Take a New Zealand rabbit weighing about 2kg, shave one of its buttocks, and disinfect the skin with 75% ethanol; use an ophthalmic corneal trephine to make an 8mm diameter incision into the dermis. Separate and remove the button-shaped skin tissue with scissors, rinse it several times in sterile saline, and then cut it into 1mm3 pieces in a small beaker; spread it into a 6-well 35mm plastic culture plate and add appropriate amount of culture medium (RPMI1640). The culture medium, containing 15% calf serum and penicillin (100 U/ml each), was cultured at 37° C. with 5% CO2. Change the culture medium twice a week. Generally, on the 7th to 10th day, the fibroblasts are attached to the bottom of the culture plate and then subculture can be carried out. After 0.25% trypsin digestion, pipetting and dispersing with D-Hanks solution and centrifugation (1000r/min, centrifugation for 5min), the cells were divided into 200ml glass culture flasks for culture. ②Preparation of cell suspension for intravitreal injection: Digest, disperse and centrifuge the cells cultured to the wall of the bottle, and then add D-Hanks solution and pipette to make cell suspension. Take a small amount of suspension and trypan blue staining solution to mix at a ratio of about 9:1, use a hemocytometer to count live cells, and finally dilute the cell suspension to a concentration of 2.5×1,000,000 cells per 1 ml. Use a tuberculin syringe to draw 0.1ml (containing 2.5×100000 cells) for use. ③Intravitreal cell injection: Before the injection, the rabbit's eyes were dilated with 1% atropine eye drops 2 to 3 times, and ketamine (5 mg/kg body weight) was injected intramuscularly for general anesthesia. During injection, 1% methyl cellulose was dropped on the surface of the cornea and a contact lens was placed. Under the operating microscope, insert the needle 3mm behind the limbus. When the needle tip reaches the center of the vitreous body, when the needle tip reaches the center of the vitreous from the pupil area, slowly inject the cell suspension.

　　(2) Features of the model After the cells are injected into the vitreous, the ophthalmoscope is used to check immediately. It can be seen that there is dusty turbidity in the vitreous, and the injection route is the densest. On the 4th day, a small amount of cord-like or membranous proliferations formed in the vitreous. On the 7th day, the proliferation material extends to the posterior pole of the eyeball, and is connected with the optic nerve head or both sides of the medullary complex, which manifests as retinal blood vessels dilation and tortuous or wrinkles. Retinal detachment occurs around 14 days, usually as a localized detachment of the medullary heddle on both sides, and some gradually expand to complete detachment. Generally, there is no retinal hole formation.

　　(3) Comparative medicine This model method injects allogeneic skin fibroblasts into the vitreous body of rabbit eyes, and the proliferation is formed due to the continuous proliferation of the cells, and the retina is pulled to detach it. This process is very similar to the clinical features of human eye proliferative vitreoretinopathy. 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 prevention and treatment effects of drugs on proliferative vitreoretinopathy. However, the experimental conditions must be controlled when making the model. Because the rabbit eyeball is smaller than the human eyeball and the front and back diameter of the lens is larger, the vitreous cavity is relatively small; when performing vitreous injection, the needle direction must be mastered to prevent the needle from damaging the lens. Traumatic cataract affects fundus examination. After the needle tip reaches the vitreous, care should be taken not to damage the retina, so it is safer to operate the model under the operating microscope.