The use of eIF6 in the regulation of melanin synthesis
A technology for the synthesis and application of melanin, applied in the field of whitening and hairdressing, to achieve the effect of improving whitening, treating pigmentation, and broad application prospects
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Embodiment 1eI
[0025] Example 1 Observation of coat color and hair-plucking experiment of eIF6 knockout mice and wild-type mice
[0026] (1) Observation of coat color of eIF6 knockout mice (gifted by Stefano Biffo, whose address is Molecular Histology and Cell Growth Laboratory, San Raffaele Science Institute, Via Olgettina58, 20132Milan, Italy) and wild-type mice: observation of eIF6 knockout at 28 days after birth Coat color of genetic mice and wild-type mice, genotype identification and photographing. see results figure 1 , where A is an eIF6 knockout mouse with brown hair; B is a wild-type mouse with dark black hair.
[0027] (2) Hair-plucking experiment of eIF6 knock-out mice (KO mice) and wild-type mice (WT mice): Take out 27 eIF6 knock-out mice and wild-type mice in the telogen phase at 49 days old, and pull out the mice with tweezers after conventional anesthesia Hair on the rear thighs on both sides of the back. The hair color was observed on the day, 1st, 3rd, 5th, 8th, 10th, 12...
Embodiment 2e
[0028] Example 2Melanin, tyrosinase, and MITF expression detection experiments in eIF6 knockout mice and wild-type mice
[0029] (1) Primary culture of mouse melanocytes: Take the back skin of the mouse, sterilize it with iodophor for 1 minute, wash it with PBS three times, and cut it into about 0.5cm 2 For large and small tissue pieces, add neutral protease II and stay overnight at 4°C; separate the epidermis and dermis the next day, cut the epidermis into pieces, digest them with 2.5g / L trypsin at 37°C for 10min, and use a volume fraction of 10% FCS (fetal calf serum). GIBCO) DMEM medium to terminate digestion; filter, centrifuge and collect cell counts. Adjust the cell concentration with K-SFM medium to (1~2)×10 6 cell / 25cm 2 Inoculate cells and place at 37°C with 5% CO 2Incubator for 3 days. Discard non-adherent cells, lightly wash twice with PBS, replace K-SFM (GIBCO) culture medium containing 10% FCS (fetal calf serum GIBCO) and add TPA10ng / mL (melanocyte growth fact...
Embodiment 3
[0032] Example 3 Inhibition of melanin synthesis by interfering with eIF6 in the simulated human base skin pigmentation experiment
[0033] Human primary melanocytes were infected with the stable lentiviral interference vector pFIV-H1 / U6-copGFP-eIF6RNAi and a negative control (both interference and control sequences were purchased from Chongqing Jinmai Biotechnology Co., Ltd.). The cells were co-cultured with rat tail collagen (Sigma Company) at a ratio of 1:7:3, that is, three-dimensional skin culture was carried out for one month. After being fixed with 4% paraformaldehyde, the sections were embedded in paraffin, and photographed under a microscope after being stained with silver ammonia solution staining solution. see results Figure 5 As shown, Figure A is the negative control group, and Figure B is the lentiviral interference vector pFIV-H1 / U6-copGFP-eIF6RNAi interference group. The results showed that the lentivirus interference vector pFIV-H1 / U6-copGFP-eIF6RNAi interf...
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