Method for preparing composition, included within exosome obtained from nanog-introduced, fetus-derived mesenchymal stem cell in amniotic fluid, for hair growth
A technology of stem cells and exosomes, applied in the field of compositions for promoting hair growth, can solve the problems of recurring hair loss, no significant effect on hair growth, side effects and increased cost, etc.
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Embodiment 1
[0068] Embodiment 1: establish the amniotic fluid source MSCs that introduced Nanog
[0069] In order to obtain an amniotic fluid-derived MSC cell line that is easy for cell expansion, the Nanog gene was introduced into the amniotic fluid-derived MSCs using a retroviral vector system to induce the overexpression of Nanog, thereby establishing an amniotic fluid-derived MSC cell line that introduced Nanog. The detailed method is as follows.
[0070] Fetal-derived cells were isolated from amniotic fluid obtained from pregnant women and subcultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 1 % L-glutamine, 4 ng / ml basic fibroblast growth factor (bFGF), 5 ng / ml selenium and 50 μg / ml ascorbic acid to obtain fetal-derived MSCs in amniotic fluid.
[0071] The Nanog gene was introduced into the obtained amniotic fluid-derived MSCs by using a retrovirus vector system to induce the overexpression of...
Embodiment 2
[0073] Example 2: Verification of Nanog expression in amniotic fluid-derived MSCs introduced with Nanog
[0074] For RT-PCR, total RNA was isolated from Nanog-introduced amniotic fluid-derived MSCs using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. 500 μg of isolated total RNA was reverse transcribed to cDNA using a cDNA prep mix (Bioneer). The test tubes were allowed to stand at 45°C for 60 minutes, then at 95°C for 5 minutes, and then stored at -20°C until use. cDNA was treated with Taq polymerase (Promega) and Nanog mRNA-specific primers (Exo-Nanog forward: 5'-GCTTGGATACACGCCGC-3' (SEQ ID NO: 2); and Exo-Nanog reverse: 5'-GATTGTTCCAGGATTGGGTG-3' (SEQ ID NO: 3)) for amplification. In RT-PCR, a cycle of 94°C for 20 seconds, 60°C for 30 seconds and 72°C for 2 minutes was repeated 35 times, and then the final synthesis was performed at 72°C for 10 minutes. PCR products were electrophoresed and analyzed on 1% agarose gels.
[0075] For the W...
Embodiment 3
[0078] Example 3: Analysis of exosomes in conditioned medium derived from amniotic fluid-derived MSCs introduced with Nanog
[0079] Select a part of cells of each amniotic fluid-derived MSC cell line established in Examples 1 and 2, prepare their 100 ml conditioned medium, and then isolate exosomes therefrom.
[0080] Exosomes were extracted from conditioned medium using an Amicon Ultra-15 10K centrifugal filter device (Millipore), which is designed to filter proteins of certain molecular weights from conditioned medium produced by fetal-derived MSCs. 14.8 ml of the conditioned medium was placed in the upper cap of the device, and then centrifuged at 5,000 xg for 15 minutes to 40 minutes. Concentrate 100 ml of the conditioned medium in this way, then add the Exosome Isolation Kit (Invitrogen) to the concentrated medium in an amount of 1 / 2 the final volume, and then mix well. The completed samples were stored at 4°C for 12 hours. Thereafter, each sample was centrifuged at 13...
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