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Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application

A technology of bone marrow mesenchyme and screening method, which is applied in the field of amino acid sequence and screening of bone marrow mesenchymal stem cell affinity polypeptide to achieve good repair effect

Active Publication Date: 2011-11-02
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In the process of realizing the present invention, the inventors found that the prior art has at least the following problems: in the prior art, there is no bone marrow mesenchymal stem cell affinity that can improve the specific affinity of biological materials for bone marrow mesenchymal stem cells. and peptide screening methods

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  • Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application
  • Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application
  • Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application

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Embodiment 1

[0055] Phage display technology screening bone marrow mesenchymal stem cell affinity peptide sequence:

[0056] (1) Primary culture of human bone marrow mesenchymal stem cells and human fibroblasts:

[0057] a. Primary culture and passage of human bone marrow mesenchymal stem cells

[0058] Obtain human bone marrow tissue from patients undergoing total knee arthroplasty (TKA), put it into a blood collection tube containing K2EDTA anticoagulant (BD Company), mix well, wash twice with PBS (0.01M, pH 7.4), and place Spread the bone marrow tissue evenly on the bottom of the culture dish, add DMEM (LG) complete medium containing 10% FBS, observe for 24-48 hours, change the medium after seeing adherent cells, continue to culture, and change the medium every 2-3 days , and passed on to one generation. as positive selection cells (see figure 1 ). Among them, DMEM is a medium containing various amino acids and glucose.

[0059] b. Primary culture and passage of human fibroblasts ...

Embodiment 2

[0114] Affinity identification of bone marrow mesenchymal stem cell affinity peptides for bone marrow mesenchymal stem cells derived from human, rat and rabbit Cell level affinity identification:

[0115] (1) Flow cytometry analysis

[0116] Method: Take a plate of bone marrow mesenchymal stem cells, digest with 0.05% trypsin-0.02% EDTA for 5 minutes, until the cells are completely detached, collect the suspension, centrifuge at 1000rpm for 5 minutes, discard the supernatant, and wash with PBS (0.01 M, pH 7.4) to suspend the cells, repeat 3 times, filter the cells with a 400-mesh filter, centrifuge again (2000rpm, 5min), re-suspend the cells in 0.1mlPBS (0.01M, pH 7.4), add 1nM SMSC affinity polypeptide ( FITC-EPLQLKMC) and mismatch peptide (FITC-MLKPLEQC), incubate at room temperature for 1h, centrifuge (2000rpm, 5min), wash with PBS (0.01M, pH 7.4) 3 times, resuspend cells in 0.5mlPBS (0.01M, pH 7.4) Transfer to a flow cytometry tube for flow cytometry analysis.

[0117] R...

Embodiment 3

[0123] Bone marrow mesenchymal stem cell-specific polypeptide modified PCL nano-electrospun fiber membrane:

[0124] (1) Polypeptide synthesis: the bone marrow mesenchymal stem cell affinity peptide EPLQLKM and the mismatched peptide MLKPLEQC (control) (Scilight-peptide Inc, China) were synthesized and screened separately, and the peptides were labeled with FITC fluorescent dye. The surface is covalently modified, and a cysteine ​​residue (C) is linked to the 3' of the polypeptide to facilitate the imino group (-NH 2 ) covalently bonded;

[0125] (2) Trim the PCL nano-electrospun fiber membrane into a circle equal to the hole area of ​​the 24-well plate, then put it into the hole of the 24-well plate, add 500 μl of 10% w / v 1 to each hole, In 6-hexamethylenediamine / isopropanol solution, 37°C, place for 1h;

[0126] (3) Rinse with deionized water for 3 to 5 times, double distilled water for 3 to 5 times, and PBS (0.01M, pH 7.4) for 3 times;

[0127] (4) Add 400ul of cross-lin...

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Abstract

The invention discloses an amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, a screening method and an application and belongs to the field of bio-pharmaceuticals. The screening method comprises the following steps: utilizing a phage display process to respectively perform negative screening of human fibroblasts and positive screening of a bone marrow mesenchymal stem cell; after screening, extracting the bone marrow mesenchymal stem cell, and then decomposing the cell; extracting a phage segment which has specific affinity to the bone marrow mesenchymal stem cell; and sequencing the phage segment, thereby acquiring a peptide segment which has high affinity to the bone marrow mesenchymal stem cell. The amino acid sequence of bone marrow mesenchymal stem cell affinity peptide can be used for promoting the specific affinity of biological material to the bone marrow mesenchymal stem cell and has profound significance and wide application prospect in the field of repairing tissue engineered cartilages.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an amino acid sequence, screening method and application of a bone marrow mesenchymal stem cell affinity polypeptide. Background technique [0002] After nearly 20 years of development, tissue engineering (Tissue Engineering, TE) has been widely used in various clinical fields, including regeneration and repair of bone tissue, cartilage, nerves, blood vessels, skin, gastrointestinal tract and genitourinary system. In the development process of tissue engineering, the continuous improvement and updating of scaffolds for various tissues is the key, including updating of materials, improvement of fabrication methods and continuous innovation of construction concepts. Another obvious conceptual change in recent tissue engineering is "simplification", that is, instead of imitating the composition of normal tissues as much as possible through various complex means and techniques ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/00A61L27/18A61L27/54
CPCA61L27/227A61L2430/06C07K7/06
Inventor 敖英芳邵振兴皮彦斌张辛周春燕
Owner PEKING UNIV THIRD HOSPITAL
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