Application of reagent for promoting miR-21 expression in preparation of medicine for preventing and/or treating diabetic keratopathy
A technology of mir-21 and 1.mir-21, which is applied in the field of medicines and reagents to promote the expression of miR-21, can solve the problems of unclear effect and mechanism of diabetic keratopathy.
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Embodiment 1
[0054] Establishment of type I diabetes mouse model:
[0055] 1) Male C57 mice aged 6-8 weeks were used, and they were routinely fed for one week after purchase, and the body weight and blood glucose of each mouse were recorded before injection; WT and miR-21KO male mice were also fed until 6-8 weeks to induce the model.
[0056] 2) The mice to be intraperitoneally injected with STZ were fasted for 12 hours.
[0057] 3) Prepare the current STZ-citrate solution, the final concentration is 7.5mg / mL, calculate the injection volume according to the dose of 60mg / kg per mouse, and use 1 ml insulin injection for intraperitoneal injection of mice, pay attention to the injection process It should be protected from light, put in food after the injection, replace the drinking water with 5% glucose, and replace it with normal water before fasting.
[0058] 4) Control mice of the same age were only injected intraperitoneally with the same volume of citrate buffer.
[0059] 5) According t...
Embodiment 2
[0065] Establishment of mouse corneal epithelial injury repair model:
[0066] 1) Select diabetic mice successfully induced in Example 1 and normal mice of the same age, and record blood glucose and body weight; for WT and miR-21KO diabetic mice, select mice with similar blood glucose and body weight for the experiment.
[0067] 2) General anesthesia for mice: extract 0.3ml of the prepared anesthetic, and inject intraperitoneally into the left lower abdomen of the mice.
[0068] 3) The right eye is the experimental eye, and the left eye does not undergo any treatment. After the mouse is anesthetized, the beard and eyelashes are cut off with the corneosclera, and the part around the experimental eye is disinfected with 0.5% Aner's iodine skin disinfectant, which is dripped into the conjunctival sac Topical anesthesia was performed with 1 drop of Alcaine eye drops.
[0069] 4) Use a 2.5mm diameter trephine to gently press and rotate the center of the mouse cornea to leave an in...
Embodiment 3
[0075] Nerve staining of whole mouse corneal smear
[0076] 1) Take miR-21KO normal mice, WT normal mice, WT diabetic mouse model (DMWT) and miR-21KO diabetic mouse model (DM miR-21KO) as the experimental objects, and kill the mice by conventional methods, and group them according to The eyeballs were taken out and put into EP tubes filled with Zamboni's fixative solution, and fixed on ice for 1 hour.
[0077] 2) Cut the corneal tissue. Note that during the cutting process, the fixative solution should completely submerge the eyeball, with the scleral limbus 0.5mm, try to remove excess iris, and continue to fix on ice for 1 hour (24-well plate, rinse with a 1ml gun when transferring the cornea, To prevent accumulation of endothelial deposits); all the front half of the eyeball can be cut off together and put into the fixative solution, and then the iris is cleaned one by one (to avoid corneal shrinkage, and the iris is easy to clean).
[0078] 3) Wash the cornea 3 times with ...
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