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Stem cell bioactive protein and its preparation and application

A technology of stem cell biology and active protein, which is applied in the field of stem cell bioactive protein and its preparation. It can solve the problems of low production efficiency, limited secretion of stem cell active protein, and high cost of cultivation, etc., and achieve high-efficiency preparation and improvement of stem cell biological activity. Protein expression, fast expression effect

Active Publication Date: 2020-12-08
GUANGZHOU ZHUNYOU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this prior art has improved the cell growth rate to a certain extent, only by increasing the cell growth rate, the secreted amount of stem cell active protein finally obtained is very limited, the culture cost is expensive, and the production efficiency is low.

Method used

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  • Stem cell bioactive protein and its preparation and application
  • Stem cell bioactive protein and its preparation and application
  • Stem cell bioactive protein and its preparation and application

Examples

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preparation example Construction

[0036]The invention relates to an efficient method for preparing stem cell biologically active protein, and a preparation process (seefigure 1 ) Mainly: pretreat mesenchymal stem cells with core induction reagents to induce a large number of biologically active proteins; then use a one-step ultrafiltration method to concentrate and purify the culture supernatant to effectively improve the purification of biologically active proteins Recovery efficiency. This technology relates to biologically active proteins expressed by eukaryotic systems such as cells. There are no post-translational folding modification differences and complex inclusion body denaturation and renaturation problems, and can ensure that the extracted protein maintains its natural conformation and function. The invention induces high-efficiency protein expression through the induction reagent and one-step ultrafiltration method, simplifies the purification process, has simple operation and high purification efficienc...

Embodiment 1

[0039]This embodiment provides a method for preparing an efficient stem cell biologically active protein. The preparation includes the following steps:

[0040](1) Select 100 mL of fresh and healthy sheep placenta tissue after screening;

[0041](2) Wash the placenta tissue thoroughly and cut it into small pieces, remove the blood vessel and mucosal tissue, and cut it into 5mm3;

[0042](3) Attach the tissue fragments to a 10 cm sterile petri dish, add DMF12 culture medium, and place it in a 5% carbon dioxide incubator until the cells can be subcultured, and routinely pass to the third generation, that is, mesenchymal stem cells;

[0043](4) Select the active mesenchymal stem cells of the third generation and culture them until their growth density is 60%, and then wash them with PBS solution and then change the medium; among them, the conventional culture in this step refers to the culture using DMF12 Basically, it is cultured at 37℃ and the culture system contains 5% carbon dioxide; the mediu...

Embodiment 2

[0050]This embodiment provides a method for preparing an efficient stem cell biologically active protein. The preparation includes the following steps:

[0051](1) Select fresh and healthy sheep umbilical cord tissue 10cm long after selection;

[0052](2) Fully clean and cut the umbilical cord tissue into small pieces, remove blood vessels and mucous membrane tissue, and cut into 5mm3;

[0053](3) Attach the tissue fragments to a 10 cm sterile petri dish, add DMF12 culture medium, and place it in a 5% carbon dioxide incubator until the cells can be subcultured, and routinely pass to the third generation, that is, mesenchymal stem cells;

[0054](4) Select the active mesenchymal stem cells of the third generation and culture them until their growth density is 60%, and then wash them with PBS solution and then change the medium; among them, the conventional culture in this step refers to the culture using DMF12 Basically, it is cultured at 37℃ and the culture system contains 5% carbon dioxide; th...

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Abstract

The invention provides a preparation method of stem cell bio-active protein. The preparation method comprises the following steps: acquiring mesenchymal stem cells; implementing induced culture on themesenchymal stem cell through a DMEM medium which is added with an inducing reagent, so that a culture is obtained, wherein the inducing reagent consists of IL-1[beta] and EGF; removing cell bodies and cell debris in the culture, so that a coarse extracting solution of the stem cell bio-active protein is obtained; and concentrating and purifying the coarse extracting solution of the stem cell bio-active protein. According to the preparation method provided by the invention, by adding the inducing reagent of proper variety to the cell medium, and especially by adding a proper amount of the inducing reagent for coordination, a specific inducing medium can be formed; and the inducing medium, when used for inducing the mesenchymal stem cells, can obviously improve expression of the stem cellbio-active protein.

Description

Technical field[0001]The invention relates to the field of biotechnology, in particular to a stem cell bioactive protein and its preparation and application.Background technique[0002]Stem cells have powerful paracrine functions, secreting up to 236 biologically active proteins, including a variety of growth factors, transcription factors, receptors, nutritional factors, etc.; participating in more than 14 biological functions, regulating cell differentiation and proliferation, and promoting cell synthesis and secretion , Chemotaxis induction, inhibition of inflammation, etc., start the metabolic process within the cell and the transfer of materials and information between cells. In particular, KGF and TGF-β can effectively promote wound healing, HGF and FGF can repair scars, and VEGF can promote angiogenesis, nourish nerve cells, and repair the nervous system. A variety of stem cell biologically active proteins have immunomodulatory activity in vivo and in vitro, and play an importa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12P21/00C07K1/34A61K8/64A61Q19/00
CPCA61K8/64A61K2800/10A61Q19/00C07K1/34C12N5/0662C12N2501/11C12N2501/2301C12P21/00
Inventor 吴道贫陶然赖小华李文禄
Owner GUANGZHOU ZHUNYOU BIOLOGICAL TECH CO LTD
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