A class of ultrashort peptides for promoting collagen secretion and its application
A collagen secretion, ultra-short technology, applied in the field of biomedicine, can solve the problems that it cannot penetrate the stratum corneum of the skin and act on the fibroblasts of the dermis, the molecular weight of collagen is large, immune rejection, etc., and achieve no hemolytic activity, Low cytotoxicity, collagen secretion promoting effect
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Embodiment 1
[0018] Chemical synthesis of two ultrashort peptides, Magin-FL and Purin-WH3, which promote collagen secretion:
[0019] (μg1 / )ml Chemical synthesis of two ultrashort peptides, Magin-FL and Purin-WH3, which promote collagen secretion: According to the amino acid sequence of the ultrashort peptide, use an automatic peptide synthesizer (433A, Applied Biosystems) to synthesize its full sequence, and use HPLC Desalted by reverse phase column chromatography.
[0020] (2) Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).
[0021] (3) The purity of the purified two collagen-promoting ultrashort peptides Magin-FL and Purin-WH3 was identified by high-performance liquid chromatography (HPLC), 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 focusing electrophoresis, and the amino acid seque...
Embodiment 2
[0025] Hemolytic activity assay:
[0026] 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 for absorbance at 540nm. 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%.
[0027] Table 1. Hemolytic activity of two ultrashort peptides
[0028]
[0029] The results showed that the percentage of hemolysis was 0.41% and 1.62% when the concentration of the two collagen-promoting ultrashort peptides, Magin-FL and Purin-WH3, was 160 μg / ml. It sho...
Embodiment 3
[0031] Cytotoxicity Assay:
[0032] The toxicity of this group of ultrashort peptides to human skin fibroblast HFF-1 was detected by MTT method.
[0033] 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 2 Cultivate 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 ...
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