Application of adipose-derived mesenchymal stem cell exosome in preparation of medicines for treating adipose tissue inflammations
A fat-derived, stem cell technology, applied in the field of biomedicine, to achieve the effects of inhibiting production and release, good inhibiting effect, and simple and feasible operation
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Embodiment 1
[0045] Embodiment 1: the extraction and separation of ADSC
[0046] (1) Isolation of adipose tissue: select 10-12 weeks old C57BL / 6 male mice, anesthetize and kill the mice, open the abdominal cavity, peel off the fat in the epididymis, remove the blood vessels as much as possible, weigh the weight of the fat, and immerse it in PBS on ice placed on.
[0047] (2) Preparation of digestive juice: According to the weight of fat, 3ml of KRB buffer solution and 6mg of type Ⅰ collagenase are required for each gram of fat.
[0048] (3) Digestion of adipose tissue: Cut the stripped adipose tissue mechanically, add it to the prepared digestive solution, mix well, place it in a shaker at 37°C for 45 minutes, centrifuge at 2000rpm for 5 minutes, carefully remove the cells at the bottom and place Place on ice in KRB without enzyme.
[0049] (4) Filtration: filter the cell suspension with a 100 μm pore size copper mesh, centrifuge the filtered suspension at 2000 rpm for 5 minutes, and dis...
Embodiment 2
[0053] Example 2: Extraction and identification of ADSC-derived exosomes
[0054] ADSCs were cultured with exosome-free 10% fetal bovine serum DMEM medium (containing bFGF). Take the fourth-generation ADSC cell culture supernatant, filter it with a 0.22μm sterile filter, centrifuge at 5000g, 100K concentrated centrifuge tube at 4°C for 30 minutes, take the concentrated solution from the upper chamber into a new sterile 15ml centrifuge tube, add Exoquick-TC TM For the exosome extraction reagent, place at 4°C overnight, centrifuge at 5000g, 4°C for 30 minutes, discard the supernatant, resuspend the pellet with sterile PBS and aliquot, freeze at -80°C for storage.
[0055] The size and shape of the extracted exosomes were detected by transmission electron microscopy, and the results were as follows: figure 1 Shown; Exosome markers TSG101, CD9, CD63, HSP90 were detected by western-blot method, and further identified, the results are as follows figure 2 shown.
Embodiment 3
[0056] Example 3: In vivo detection of ADSC-derived exosomes on macrophages in adipose tissue and adipose tissue inflammation
[0057] (1) Establishment of high-fat diet-induced adipose tissue inflammation model and exosome intervention
[0058] Seven-week-old C57BL / 6 male mice were used to establish adipose tissue inflammation model. After a week of pre-high-fat diet adaptation, they were fed a high-fat diet (HFD) (containing 60% cal), and fed a normal diet (NCD) as a control group. After 12-20 weeks of high fat, intraperitoneal injection of exosomes (30μg / mouse, once every 3 days, continuous injection for 6-8 weeks), is the HFD-exosome group; intraperitoneal injection of normal saline group is the control group, namely HFD -NS. NCD-NS was observed in mice fed normal diet and injected with normal saline intraperitoneally.
[0059] ⑵ Detection of F4 / 80 in the fat tissue of the epididymis + Macrophage ratio
[0060] After the exosome intervention, adipose tissue from the e...
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