Preparation and application of adipose-derived stem cell secretion multiple-factor carrier exosome for promoting skin wound healing
A technology of adipose stem cells and human adipose stem cells, which is applied in artificial cell constructs, skin diseases, animal cells, etc., can solve the problems of long-term safe storage and transportation, difficult clinical application, etc., and achieve good purity, best clinical application value, and yield high effect
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[0039] The first object of the present invention is to provide a method for preparing exosome secreted by adipose stem cells to promote skin wound healing, the method comprising:
[0040] Step 1, get the human adipose stem cells of the third generation to the sixth generation in the logarithmic growth phase, after the cells are fused to 80%, discard the original culture solution (the volume of each component in the original culture solution is: 88% human fat Special basal medium for stem cells, 10% fetal bovine serum, 1% penicillin / streptomycin, 1% glutamine), phosphate buffer (the pH of phosphate buffer is 7.2-7.4, each The content of the ingredients is NaCl137mmol / L, KCl2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L) after washing 2-3 times, change the serum-free culture medium (the volume of each component in the serum-free culture medium is: 98% special basal medium for human adipose stem cells, 1% penicillin / streptomycin, 1% glutamine Amide) culture, collect cultur...
Embodiment 1
[0054] The exosome secreted by adipose stem cells can promote the migration and proliferation of normal skin fibroblasts and affect collagen synthesis:
[0055] (1), CCK-8 proliferation experiment: Human fibroblasts were mixed with 2×10 3 Inoculate in 96-well plate, after 3 hours, after the cells are completely adhered to the wall, change the serum-free medium, and use different concentrations of exosome (0ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) and 50% cultured fat The conditioned medium CM of stem cells was used to treat the cells, and each group had five duplicate wells, which were continuously detected for five days. Take out the 96-well plate on day 1, 2, 3, 4, and 5 respectively, add 10ul of CCK-8 solution to each well, incubate at 37°C for 2 hours, detect the absorbance value at 450nm wavelength under a microplate reader, observe and compare the proliferation of cells in each well Condition. It was found that exosome can promote its proliferation with the extension of time...
Embodiment 2
[0059] The exosome secreted by adipose stem cells can be homed to the skin around the wound to participate in wound repair:
[0060] BALB / c male mice aged 6-8 weeks were selected to construct a skin wound model. After the mice were anesthetized, the back of the mice near the tail (avoiding the heart) was disinfected. After the hair was shaved and the skin was prepared, a full-thickness skin wound with a size of 2cm×1.5cm was made. The wounded mice were randomly divided into 3 groups: exosome experimental group, dye negative control group, and PBS negative control group. In the experiment, take 1ml of exosome suspension with a final concentration of 1ug / ul (exosome is dissolved in PBS), add 1ul fluorescent dye DIR to the suspension, incubate and wash the exosome according to the instructions of the manufacturer, and use it as the exosome experimental group. Take 1ul fluorescent dye and dissolve it in 1ml of PBS was used as the dye solution negative control group, and 1ml of PB...
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