Preparation method and product of sericin-agarose composite gel having activity of promoting cell proliferation and product
A technology of cell proliferation and composite gel, applied in medical science, prostheses, etc., can solve the problems of few products that have obtained clinical approval, limit the marketization of silk biomaterials, and increase production costs, and achieve good mechanical properties and water absorption. Good performance and good stability
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Embodiment 1
[0032] Example 1, the extraction of sericin from silk with human FGF1 gene and human FGF2 gene
[0033] The transgenic human FGF1 gene and human FGF2 gene silk cocoon shells were ground into powder by liquid nitrogen and set aside. At a concentration of 30-50mg / ml, use an extraction buffer containing 8M urea (50mM Tris-HCl, 8M urea, pH 7.0) to extract at 80°C for 2h, and at 4°C, centrifuge at 18,000rpm for 10min. The supernatant is the sericin solution containing recombinant FGF1 protein and FGF2 protein.
Embodiment 2
[0034] Example 2, the preparation method of sericin-agarose composite gel (AH(FGF1+FGF2))
[0035] First, the sericin solution containing recombinant FGF1 protein and FGF2 protein obtained in Example 1 was refolded using the dithiothreitol / glutathione (DTT / GSSH) redox system to refold the FGF1 protein in the supernatant and FGF2 protein. Refolding process: Under the condition of 4°C, the supernatant was fully dialyzed for 12 hours with a dialysis bag (MWCO 1000Da, Spectrum Laboratory, Inc, USA), and replaced with a dialysate (8M urea, 1mM dithiothreitol (DTT), 50mM Tris-Cl ( pH7.0), and 250mM NaCl); using half-dilution method using refolding solution (2.0mM reduced glutathione (GSH), 0.2mM oxidized glutathione (GSSG), 1mM DTT, 50mM Tris–Cl (pH 7.0), and 250mMNaCl) to replace the dialysate, dialysis for 12h each time, repeat 4 times; dialyze with ultrapure water 6 times, remove the refolding solution, 12h each time. After renaturation is completed, the sericin aqueous solutio...
Embodiment 3
[0039] Example 3, performance detection of sericin-agarose composite gel
[0040] a. Electron microscope observation (SEM)
[0041] AH (FGF1+FGF2) gels with a length and width of 1 cm were freeze-dried, and the inner layer was sprayed with gold, and observed and photographed with a scanning electron microscope (Supra 55sapphire, Zeiss). All experiments were performed at room temperature with a voltage of 3.0 kV. The number of samples in each experiment is 5 independent samples, and each sample is taken 3 times, and the results are as follows image 3 shown. The results showed that the interior of the AH(FGF1+FGF2) composite gel was a porous loose structure; compared with the pore size (the transverse diameter was about 48 μm and the longitudinal diameter was about 137 μm) in the sericin gel material not mixed with agarose, the AH The void diameter in the (FGF1+FGF2) composite gel was significantly reduced, about 23 μm, indicating that the AH(FGF1+FGF2) composite gel had bet...
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