Preparation of fibroblast extracellular vesicles and application of fibroblast extracellular vesicles in beauty and medicines
A technology of vesicles and monoclonal antibodies, applied in animal cells, cosmetic preparations, dressing preparations, etc., can solve the problems of low activity of exosomes, easy aging of fibroblasts, low yield, etc., and achieve anti-aging activity The effect of good and good market application value
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Embodiment 1
[0034] Example 1 Preparation of P53 monoclonal antibody
[0035] Recombinant human p53 protein (abcam, product number ab84768, sequence shown in SEQ ID NO: 1) was used as the immunogen. The recombinant protein was thoroughly mixed with an equal volume of Freund's complete adjuvant, and BALB / c mice were subcutaneously injected with 50 μg / mice of the recombinant protein. Four weeks after the initial immunization, multiple subcutaneous injections on the back were given to boost the immunization, and 4 weeks later, the immunization was boosted again, with 50 μg of the immunogen per animal each time. Seven to ten days after the third immunization, blood was collected from the tail vein to measure the serum antibody titer by ELISA. Four days before fusion, the mouse with the highest antibody titer was selected and boosted by intraperitoneal injection of 150 μg of unadjuvanted antigen.
[0036] Aseptically take spleen cells of booster immunized BALB / c mice, use 50% polyethylene gly...
Embodiment 23
[0038] Example 2 3E4 monoclonal antibody affinity, subtype identification, sequence analysis and specificity identification
[0039] Using the AMC sensor, the purified 3E4 antibody was diluted to 10ug / ml with PBST, and the recombinant protein was serially diluted with PBST: 444.4nmol / ml, 222.2nmol / ml, 111.1nmol / ml, 55.6nmol / ml, 27.8nmol / ml, 0nmol / ml; operation process: equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM The pH 1.69 GLY solution and buffer 3 were used to regenerate the sensor and output data. The results are shown in Table 1. (KD stands for equilibrium dissociation constant or affinity)
[0040] Table 1 Affinity results of 3E4 monoclonal antibody
[0041] Antibody name KD (M) 3E4 monoclonal antibody 3.42E-09
[0042] It can be seen from the results in Table 1 that the 3E4 monoclonal a...
Embodiment 3
[0051] Example 3 Cultivation of fibroblasts
[0052] Fibroblast HSF was cultured in DMEM (containing 10% fetal bovine serum, 1% double antibody mixture) medium at 37°C, 5% CO 2 Incubator culture, the cells grow to more than 80%, rinse with PBS for 2~3 times, add 1 mL of 0.25% trypsin to digest at room temperature for 30~60s, tap the bottle wall to make the cells fall off, add 2 times the volume of fetal bovine serum for culture Digestion was terminated with a gun tube, pipetted evenly, centrifuged at 1000 r / min for 8 min, removed the supernatant, precipitated and added an appropriate amount of complete medium to make a single-cell suspension, adjusted the concentration, and used the obtained HSF in logarithmic growth phase in good condition for experiment. Take the HSF of the logarithmic growth phase as 1 × 10 5 Cells / mL were inoculated into a 96-well culture plate, with 90 μL per well, and placed in a cell incubator at 37°C, 5% CO. 2 After 24 hours of culture, the experime...
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