Retinal degeneration model and drug screening method thereof
A retinal degeneration and retinal technology, used in compound screening/testing, pharmaceutical formulations, organic active ingredients, etc., can solve the problems of destroying photoreceptor cells, difficult to reproduce, and poor repeatability.
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Embodiment 1
[0025] A lentivirus-mediated antisense mir-24 virus was injected into the subretinal space of model animals to obtain a retinal degeneration model.
[0026] The antisense sequence of antisense mir-24 is shown in SEQ ID NO.1, which is CTGTTCCTGCTGAACTGAGCCA. Cloning the antisense miR-24 sequence into a lentiviral vector to obtain a lentivirus-mediated antisense mir-24 virus is a conventional technical means in the art, and can also be purchased, that is, the antisense mir-24 virus is also commercially available. After obtaining the virus, aliquots were frozen at -80%. Take a normal adult SD rat, weighing about 200g, place it under a stereo microscope, and place a concave lens on the eyeball of the rat; use a 30gauge needle to puncture a pinhole 2mm behind the corneoscleral limbus, and then use a microsyringe equipped with a 33gauge needle Insert the needle from the pinhole to the subretinal space, and inject 3 μl (containing 5-10*10^7tu / ml) of virus. The same volume of contro...
Embodiment 2
[0051] Example 2 Immunofluorescence and nuclear layer thickness analysis were performed after subretinal injection.
[0052] After subretinal injection, nuclear layer thickness analysis and Muller cell gliosis marker detection were performed by performing immunofluorescent staining.
[0053] Immunofluorescence:
[0054] Preparation of tissue slices: Eyeballs of DM rats were carefully removed (the optic nerve existed as much as possible), and fixed in 4% paraformaldehyde for three hours. Under a dissecting microscope, cut the cornea along the 2 mm above the corneoscleral limbus, and carefully remove the lens and iris. The remaining eyeballs were dehydrated in 30% sucrose for 3 hours. Put the eyeball into the tissue embedding agent to embed, put the optic nerve on one side, be careful not to have air bubbles, and equilibrate overnight in a 4°C refrigerator. The next day, move it to a -80°C refrigerator for later use, and make a mark on the optic nerve head. After the embedde...
Embodiment 3
[0059] Subretinal injection of Anti-mir-24 caused the changes of RPE marker protein and the target protein of mir-24. Seven weeks after subretinal injection of anti-mir-24, the RPE-bruch membrane-choroid complex was isolated under a stereoscopic dissecting microscope, and wetsern blot was performed to detect the expression of RPE65 and Chil3 at the protein level
[0060] The Western Blot method is as follows
[0061] 1. To prepare the gel used for SDS-PAGE, firstly prepare the lower layer separating gel. After the preparation, immediately and slowly add it to the prepared two-layer glass plate, and add an appropriate amount of double distilled water to seal.
[0062] 2. After the lower separating gel is solidified, start to prepare the upper separating gel. Pour off the upper layer of water in step 1, add the concentrated gel, slowly insert the comb, and let it stand for solidification.
[0063] 3. After the extracted protein is boiled and denatured, add the same amount of s...
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