Collagen secretion promotion ultrashort peptide and application thereof
An ultra-short peptide and gel-promoting technology, applied in the field of biomedicine, can solve the problems of high molecular weight of collagen and limited effect of supplementing collagen, and achieve the effects of low cytotoxicity, no hemolytic activity, and obvious clearance rate.
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Embodiment 1
[0020] Chemical synthesis of anti-aging ultrashort peptide Mapin-WH, Tinin-WH, Orin-G-OT:
[0021] (1) Chemical synthesis method of anti-aging ultrashort peptide Mapin-WH, Tinin-WH, Orin-G-OT: According to the amino acid sequence of the ultrashort peptide, use an automatic peptide synthesizer (433A, Applied Biosystems) to synthesize its full sequence, and pass HPLC reverse phase column chromatography desalting.
[0022] (2) Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).
[0023] (3) Three kinds of purified anti-aging ultrashort peptides Mapin-WH, Tinin-WH, Orin-G-OT were identified by high-performance liquid chromatography (HPLC) for their purity, and the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI- TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was de...
Embodiment 2
[0028] Hemolytic Assay:
[0029] The collected healthy human blood was mixed with Alfred's solution for anticoagulation, washed twice with normal saline and resuspended into 10 7 -10 8 cell / ml suspension. The above-mentioned diluted erythrocyte suspension was mixed with the ultrashort peptide sample dissolved in normal saline, incubated at 37°C for 30 minutes, then centrifuged at 1000rpm for 5 minutes, and the supernatant was measured at 540nm for absorbance. Negative control uses physiological saline, positive control uses Triton X-100, and the hemolysis percentage is calculated according to the following formula: hemolysis percentage H%=(A sample-A negative control) / A positive control×100%.
[0030] Table 1. Hemolytic activity of three ultrashort peptides
[0031]
[0032] The results showed that the percentage of hemolysis of the three ultrashort peptides Mapin-WH, Tinin-WH and Orin-G-OT was less than 1% when the concentration was 160μg / ml. It shows that Mapin-WH, Ti...
Embodiment 3
[0034] Cytotoxicity Assay:
[0035] The toxicity of this group of ultrashort peptides to human skin fibroblast HFF-1 was detected by MTT method.
[0036] Human skin fibroblasts HFF-1 were purchased from Shanghai Cell Bank. First culture the fibroblasts in DMEM containing 15% fetal bovine serum and double antibodies (penicillin and streptomycin each 100 U / ml), after the cells are congested, digest them with 0.25% trypsin, and wash Twice, resuspend the cells, and after counting the cells, add 100 μl of the cell suspension to a 96-well cell culture plate, so that the number of cells per well reaches 10 5 indivual. Add the sample, add the same volume of sterilized ultrapure water to the control group, set at 37°C, 5% CO 2Cultivate in the incubator for 24h. After the culture was over, add 20 μl of 5 mg / ml MTT solution (prepared with cell culture PBS buffer) to each well of the 96-well cell culture plate, continue to cultivate for 5 hours, suck out the liquid in the wells with a...
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