Application of m6A-modification-related gene ALKBH5 in aspect of promoting repair of nerve axon injuries
A technology for damage repair and peripheral nerve injury, applied in the field of biomedical research, can solve problems such as loss of nerve function, incomplete elucidation, and difficult treatment of long-distance peripheral nerve injury
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Embodiment 1
[0043] Example 1. Detection of expression and localization of ALKBH5
[0044] (1), DRG sample protein extraction and Western blot
[0045] After the rat sciatic nerve crush model was prepared, the L4-L6 dorsal root ganglion (DRG) tissue on the side of the sciatic nerve crush was taken at 0h, 1d and 3d after operation, and the tissue protein sample was prepared by the tissue sample protein extraction kit: different time The spotted L4 and L5 DRG tissues were transferred from the cryovials to 1.5 ml RNase-free EP tubes with RNase-free tips. Add 150ul tissue lysate to the tube, add protease inhibitors, use a homogenizer to beat the tissue into a single-cell suspension, place it on ice for 15 minutes, and then centrifuge at 12000 rpm for 10 minutes at 4°C (involving protein sample preparation operation centrifugation temperature Both are 4). Carefully take out the centrifuge tube, draw the supernatant into a new EP tube, use the protein concentration detection kit to detect th...
Embodiment 2
[0049] Embodiment 2, the effect of interfering with the expression of ALKBH5 on the growth of neuron axons
[0050] (3) Isolation and culture of DRG neurons
[0051] Prepare the dissection solution and put it into a small dish, add double antibodies (penicillin and streptomycin) and place it on ice to pre-cool. Anesthetize 3 rats intraperitoneally, use surgical scissors to cut the skin from the tail along the spine to the head, carefully remove the entire spine, open the lamina from the neck, and use micro forceps to pull out all DRG tissues and put them into the dissection solution . After all the DRG tissues were taken out, the dissection fluid was discarded, and the tissues were rinsed twice with cell-grade PBS to remove excess tissues and blood. Discard the PBS, add 2ml of collagenase (3mg / ml), and transfer the tissue and digestion solution to a 5ml centrifuge tube. The tissue was fully shredded with micro scissors, placed in a cell culture incubator, and digested for...
Embodiment 3
[0056] Example 3, the effect of interfering with the expression of ALKBH5 on the growth of sciatic nerve axons
[0057] (6) Interfering with intrathecal injection of virus
[0058] Experimental animals SD rats are provided by the Experimental Animal Center, 15 per batch, adult male rats, about 200g. The experimental mice were randomly divided into 3 groups, 5 in each group. Before the operation, the rats were first anesthetized in the abdominal cavity, and then the rat’s back ilium was sterilized, and then the skin was exposed with surgical scissors, the muscle tissue was cut along the ilium with ophthalmic scissors, and the protruding spinous process was cut off with bone replacement scissors , and then use a small cotton ball dipped in normal saline to gently wipe off the blood and minced meat to clearly expose the intervertebral foramen of L4 and L5 DRG. The 2 / 8 type AAV virus packaged with shRNA was uniformly injected with a micro-injector. Injected into the interverte...
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