Application of umbilical blood platelet mitochondria in preparation of drug for treating autoimmune diseases
A technology for immune diseases and platelets, which is applied to blood/immune system cells, drug combinations, allergic diseases, etc., can solve problems such as insufficient effect and large side effects, and achieve a complex storage method, small side effects, and obvious therapeutic effects. Effect
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Embodiment 1
[0039] Preparation and Preservation of Cord Blood Platelet Mitochondria
[0040] S1. Take 20mL of cord blood and slowly add it to the lymphocyte separation medium (cord blood volume: lymphocyte separation medium volume = 1:1), centrifuge, centrifugation conditions: 20°C, 400g~1610g, 25min, start acceleration: 0, end Hourly deceleration: 0. After the centrifugation, the cord blood is stratified, and the supernatant above the peripheral blood mononuclear cell layer (PBMC) is sucked, and the action is slow and gentle, and the PBMC should not be sucked as much as possible. The start acceleration of the above centrifugation: 0, the end deceleration: 0. Only when the acceleration and deceleration are 0, can the PBMC be separated, also known as the buffy coat. If there is acceleration or deceleration, the buffy coat cells will be centrifuged to the bottom of the test tube, resulting in separation failure, and PBMC cells cannot be obtained. In this way, PBMC cells can be obtained an...
Embodiment 2
[0054] 1. Obtaining peripheral blood mononuclear cells
[0055] 1. Collect 5ml of peripheral blood in a heparin anticoagulant tube, slowly add the blood to the lymphocyte separation medium (peripheral blood volume: lymphocyte separation medium volume = 1:1), centrifuge, centrifugation conditions: 20 ° C, 400g ~ 1610g , 25min, start acceleration: 0, end deceleration: 0. After centrifugation, the cord blood is stratified, and the layer of peripheral blood mononuclear cells (PBMC for short) is sucked with a straw, and the action is slow and gentle, and as little lymphocyte fluid as possible is sucked.
[0056] 2. Continue to centrifuge the PBMC layer, centrifugation conditions: 20°C, 580g, 10min, start acceleration: +8, end deceleration: -9. Take 5ml~10ml 10× red blood cell lysate and 45ml sterilized water to prepare 1× red blood cell lysate. After the centrifugation is completed, remove the supernatant, add 1× red cracking solution for lysis for 2 minutes to 10 minutes, add 0....
Embodiment 3
[0073] 1. Isolation of donor mouse spleen cells
[0074] 1. Euthanize the donor mouse in the carbon dioxide chamber, then dislocate the neck; spray the abdomen with 70% ethanol;
[0075] 2. Make an incision at the abdomen level, peel off the skin to expose the peritoneum, use forceps to keep the peritoneum away from internal organs, and make an incision on the left abdominal peritoneum to expose and remove the spleen;
[0076] 3. Put the spleen into the serum-free culture medium (X-VIVO 15w / o Gentor Phenol Red from Lonza Company) in a 10ml petri dish, remove and discard the excess tissue in the spleen;
[0077] 4. Crush the spleen using 2 sterile glass slides to make a single cell suspension; filter the cell suspension through a 70 μm filter into a 50 ml sterile centrifuge, and rinse the filter with 5 ml of serum-free culture medium;
[0078] 5. Centrifuge the cells, centrifugation conditions: 300g, 10min, start acceleration: +8, end deceleration: -9, discard the supernatant;...
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