A kind of adsc exosome and its preparation method and application
A technology of exosomes and drugs, applied in the field of human exosomes preparation, to achieve the effect of promoting the generation of hair follicles, easy to accept, and mild treatment methods
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Embodiment 1
[0037] Example 1 Adipose-derived mesenchymal stem cell exosomes promote wound healing rate
[0038] 1. Experimental grouping
[0039] Control group: normal saline; positive group: wound medicine (Kangfuxin solution); treatment group: human adipose-derived mesenchymal stem cell exosomes.
[0040] group control group positive group treatment group Quantity (only) 30 30 30
[0041] 2. Establishment method of skin full-thickness skin injury model
[0042] (1) weighing, intraperitoneal injection of chloral hydrate 350mg / kg, mice are anesthetized;
[0043] (2) The hair on the back of the mouse was removed with shears to expose the bare skin;
[0044] (3) Cut a circular incision with a diameter of 1 cm on the back to damage the dermis of the skin;
[0045] (4) Subcutaneously inject 0.5ml sugar saline into mice after operation to replenish energy;
[0046] (5) The postoperative mice were kept warm under a heating lamp.
[0047] 3. Measurement of mouse...
Embodiment 2
[0052] Example 2 Expression of PCNA in the skin of mouse injury site
[0053] 1. Extraction of total protein from the damaged skin of mice
[0054]The mice were sacrificed by neck breaking at 3, 7, 14, and 21 days respectively, and routine aseptic dissection was carried out to separate the skin and extract the total protein of the skin. The specific method is as follows:
[0055] (1) Preparation: Make ice (turn on the ON button of the ice maker); clean the mortar and pestle and dry them in an oven; prepare 1.5mL EP tubes; take out RIPA, BSA5mg / mL and 4×loading from -20°C buffer, and thawed at 4°C;
[0056] (2) Pre-cool the mortar and pestle in an ice box, weigh the skin of the mouse, put it into the pre-cooled mortar, cut it into pieces, add 1 mL of RIPA to grind it, collect the tissue fluid into an EP tube, and put it in Pre-cool in an ice box to prevent protein degradation at high temperature;
[0057] (3) Vortex once every 20 minutes for a total of 2 hours. During this ...
Embodiment 3
[0107] Expression of Caveolin-1 in the mouse skin tissue of embodiment 3
[0108] 1. Western blotting method as in Example 2
[0109]2. Immunohistochemistry
[0110] ① Dewaxing and rehydration, the process is as follows: xylene Ⅰ 10min; xylene Ⅱ 10min; xylene + ethanol 10min; absolute ethanol 5min; absolute ethanol 5min; 95% ethanol 5min; Ethanol 5min; PBS wash 3 times, 5min each time;
[0111] ② Add 3% H dropwise 2 o 2 Prepare it now, incubate in a water bath at 37°C for 30 minutes to remove endogenous peroxidase, otherwise it will cause background staining other than the antigen; wash 3 times with PBS, 5 minutes each time;
[0112] ③Heat the citric acid buffer solution in microwave to boiling water, then repair the antigen in microwave for 10 minutes on low heat, let cool to room temperature; wash with PBS 3 times, 5 minutes each time;
[0113] ④ Add 0.1% TritonX-100 dropwise, incubate in 37°C water bath for 10 minutes; wash with PBS 3 times, 3 minutes each time;
[01...
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