Methods for promoting differentiation and growth of epidermal stem cells
A technology of epidermal stem cells and neuron cells, applied in the field of stem cells, can solve the problem that epidermal stem cells are not induced to become neuron cells, and achieve the effect of good proliferation effect and good application prospect.
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Embodiment 1
[0028] Example 1 Preparation of Epidermal Stem Cells
[0029] Take the skin specimens, rinse the skin specimens 3 times with D-hank's solution containing gentamicin 800u / ml under aseptic conditions. Use ophthalmic scissors to cut the skin slices into 1cm*1cm size skin slices, put them into a culture bottle, add 0.25% Trypsin-0.02% EDTA dropwise, the volume ratio of the two is 1:6, and digest them at 4°C for 10h. Take out the skin slices, add a little fetal bovine serum dropwise to stop digestion, and rinse with D-hank's solution twice. The epidermis and dermis were separated, and the epidermis was gently uncovered with ophthalmic forceps; K-SFM was added, and the epidermis was carefully blown repeatedly for 15 minutes with a straw. Filter through a 200-mesh sieve, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add K-SFM to make a cell suspension. The cells were inoculated on the pretreated type IV collagen culture plate, and cultured in a constant tempera...
Embodiment 2
[0033] Example 2 Preparation of Transgenic Epidermal Stem Cells
[0034] 1. Gene acquisition and plasmid construction
[0035]The CLL gene shown in SEQ ID NO: 1 was synthesized by the method of whole gene sequence synthesis, and enzyme cleavage sites: HindIII and EcoRI were respectively added at the two ends of the gene. The pcDNA3.1 plasmid and the CLL gene were double digested with HindIII and EcoRI, respectively. The product was identified by 1% agarose gel electrophoresis, and the DNA was recovered. The recovered product after double digestion was subjected to the action of T4 DNA ligase to obtain the CLL expression plasmid pcDNA3.1-CLL. Take an appropriate amount of DH5α competent cells, add 9 μl of the CLL expression plasmid pcDNA3.1-CLL after the ligation reaction, mix well, and put it in an ice bath for 30 minutes; put it in a 42°C water bath, heat shock for 2 minutes; immediately transfer to an ice water bath to cool for 1 minute ; After cooling, add 400 μl LB liqu...
Embodiment 3
[0039] Example 3 Compounds Induce Differentiation of Epidermal Stem Cells to Neurons
[0040] The compound of formula (1) is synthesized and prepared according to the method of CN101437785B.
[0041] The epidermal stem cells prepared in Example 1 and the transgenic epidermal stem cells prepared in Example 2 of the fourth generation were respectively taken, digested with 0.25% trypsin-EDTA and inoculated in a 24-well plate coated with poly-lysine, and the cells adhered to the wall Afterwards, the culture medium was changed to the induction medium (experimental group): DMEM / F12+1% penicillin+20 μg / L bFGF+20 μg / L compound of formula (1), half of the medium was changed every other day. Detection was carried out 7 days after induction. In addition, the epidermal stem cell group prepared in Example 1 with the addition of DMEM / F12+1% penicillin+20 μg / L bFGF as the induction solution at the fourth generation was used as control 1, and the epidermal stem cell group prepared in Example...
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