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Production method for promoting yield of stem cell cytokines

A cytokine and stem cell technology, applied in the field of promoting the production of stem cell cytokines, can solve the problems of not verifying the adipogenic differentiation of bone marrow mesenchymal stem cells, and not involving cytokine production and enrichment.

Active Publication Date: 2021-06-08
上海天安智谷干细胞科技集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the prior art, it has not been verified to promote the adipogenic differentiation of bone marrow mesenchymal stem cells after rapid proliferation, detect the changes of corresponding cytokines, and there is no research involving the production and enrichment of corresponding cytokines.

Method used

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  • Production method for promoting yield of stem cell cytokines
  • Production method for promoting yield of stem cell cytokines
  • Production method for promoting yield of stem cell cytokines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 is specific to the preparation of the monoclonal antibody of PTN

[0020] The optimized PTN highly immunogenic epitope peptide sequence rtgaeckqtmktqrckipcnwkkqfgaeckyqfqawgecdlntalktrtg was synthesized for later use. Animal immunization: 4 7-week-old female Balb / c mice were taken, and the antigen was fully emulsified with an equal volume of Freund's adjuvant at a dose of 50 μg / mouse, and then subcutaneously injected into the abdomen at multiple points, and the immunization was boosted once every 2 weeks. Seven days after the third immunization, the serum of the immunized mice was collected by picking the eyeball method, which was the immune serum (positive serum). In addition, serum from unimmunized mice was collected as negative serum. On the 7th day after the 3 times of immunization, blood was collected from the tail vein, and the titer of mouse serum was determined by ELISA detection method. 10μg / mL, coated at 4°C for 12h; 50mg / L skimmed milk powder, ...

Embodiment 25

[0024] Example 2 Identification of 5H2 monoclonal antibody subtypes

[0025]The Mouse Monoelonal Antibody Isotyping Kit from Sigma was used for identification. Take hybridoma cells and mouse ascites, and make 1:10 dilution with PBS of pH 7.2; take 200 μL of the diluted solution and add it to the test tube, let it stand at room temperature for 30 seconds, insert the Isotrip colloidal gold test paper into the bottom of the tube, take it out after 10 minutes, and observe the result. The results showed that the antibody subtype of 5H2 monoclonal antibody was IgG1, and the light chain type was κ.

Embodiment 3

[0026] Example 3 Affinity Identification and Sequence Identification of 5H2 Monoclonal Antibody

[0027] The affinity was determined by Surface Plasmon Resonance (SPR). Immobilization of the capture molecule (anti-mouse Fc fragment capture antibody), the channel with the anti-mouse Fc fragment capture antibody immobilized as the detection channel, and the channel with the unfixed anti-mouse Fc fragment capture antibody as the control channel, the process is as follows: (1) surface balance: HBS-EP buffer solution, equilibrate the chip surface at a flow rate of 15 μl / min for 5 min; (2) surface activation: inject a 1:1 mixture of 'NHS+EDC', activate the chip surface at a flow rate of 15 μl / min for 7 min; (3) couple Coupled antibody: inject goat anti-mouse Fc fragment capture antibody (diluted in 10mM sodium acetate (pH 4.5) buffer), couple at a flow rate of 15μl / min for 6min; control channel omit this step; (4) surface blocking: inject ethanolamine , the surface was blocked for ...

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Abstract

The invention relates to a production method for promoting the yield of stem cell cytokines. According to the method, a monoclonal antibody capable of specifically aiming at PTN is used; the antibody can effectively induce adipogenic differentiation of bone marrow mesenchymal stem cells; and furthermore, the antibody can effectively promote high secretion of SCF and VEGF cytokines after differentiation. The high-yield cytokines can be used for subsequent research through separation and purification.

Description

technical field [0001] The present application relates to the field of biology, and more specifically, relates to a production method for promoting the production of stem cell cytokines. Background technique [0002] Mesenchymal stem cells (MSCs) are important members of the stem cell family, not only in bone marrow, fat, umbilical cord blood and peripheral blood, but also in perichondrium, periosteum, muscle and trabecular bone. At present, bone marrow mesenchymal stem cells (BMSCs) are the most studied. Its basic characteristics are: self-renewal ability, can proliferate to form tissue and maintain its own quantity in vivo, and can grow clones in vitro. Proliferation and differentiation ability, under the induction of certain environment and specific factors, can differentiate into tissues such as muscle, nerve, cartilage, bone, fat, tendon and ligament. BMSCs have become the most commonly used seed cells for bone tissue engineering research due to their advantages of mu...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/077C07K16/22C12P21/02C07K14/475C07K14/515
CPCC12N5/0663C12N5/0653C07K14/475C07K14/515C12P21/02C07K16/22C12N2506/1353C12N2501/33C12N2500/40C12N2501/999C12N2501/10C07K2317/56C07K2317/92
Inventor 张海涛李娜
Owner 上海天安智谷干细胞科技集团有限公司
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