Preparation method and product of sericin-agarose composite gel with activity of promoting cell proliferation

A cell proliferation and composite gel technology, applied in prosthesis, medical science and other directions, can solve the problems of rarely obtaining clinical approvals for finalized products, restricting the marketization of silk biomaterials, weak cell adhesion, etc., and achieve good mechanical properties. , The effect of good water absorption performance and simple method

Active Publication Date: 2020-06-30
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the complex preparation process of traditional silk biomaterials, single functionality, and its own defects such as weak cell adhesion, even if it can be improved by adding functional substances later, the source and efficacy of functional substances The risk factors brought about, as well as the increase in production costs, have limited the marketization of silk biomaterials, and so far few clinical approvals and finalized products have been obtained.

Method used

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  • Preparation method and product of sericin-agarose composite gel with activity of promoting cell proliferation
  • Preparation method and product of sericin-agarose composite gel with activity of promoting cell proliferation
  • Preparation method and product of sericin-agarose composite gel with activity of promoting cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a preparation method and a product of a sericin-agarose composite gel having the activity of promoting cell proliferation and a product, sericin of recombinant FGF1 protein and FGF2 protein can be extracted from transgenic human FGF1 gene and human FGF2 gene silk, a sericin solution containing the recombinant FGF1 protein and the FGF2 protein is obtained, the sericin solution containing the recombinant FGF1 protein and the FGF2 protein is renatured to obtain a renatured sericin solution containing the recombinant FGF1 protein and the FGF2 protein, the renatured sericinsolution and an agarose solution are mixed for injection molding and molding to obtain the sericin-agarose composite gel having the activity of promoting cell proliferation, and the prepared composite gel has good stability, can better support the proliferation and growth of NIH3T3 cells, and has low cytotoxicity, and has broad application prospects in tissue engineering.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of a sericin-agarose composite gel with the activity of promoting cell proliferation, and also relates to a product prepared by the method. Background technique [0002] Silk is mainly composed of silk fibroin in the inner layer and sericin in the outer layer, the content of which is 75% and 25% respectively. For a long time, a large amount of sericin protein produced by the silk reeling industry has been regarded as "waste" and discharged, resulting in serious environmental safety hazards. With the deepening of research, researchers found that sericin has the functions of anti-oxidation, moisturizing, promoting cell proliferation and accelerating wound healing, and it is a potential raw material for biomaterials. Sericin can be prepared such as sericin ointment, sericin-gelatin film, sericin-carboxymethyl cellulose, sericin poly(ethylene) after chemical crosslink...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/26A61L27/52A61L27/50
CPCA61L27/26A61L27/50A61L27/52A61L2300/412C08L89/00C08L5/12
Inventor 夏庆友王元成王峰田弛赵萍
Owner SOUTHWEST UNIV
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