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Mesenchymal stem cell affinity peptide screening and use

A kind of mesenchymal stem cell and application technology, applied in library screening, medical preparations containing active ingredients, peptides, etc., can solve the problems of receptor molecules losing their natural conformation and function, and difficulties in natural receptor molecules

Active Publication Date: 2007-06-13
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the past, phage random peptide library technology was used to study the interaction between receptors and ligands. Most of the purified receptor molecules screened the peptide library to obtain peptides that bind to the receptor molecule. However, when the structural information of a certain receptor molecule is known There are not many, and in the case of unknown receptors on the cell membrane surface, it will be very difficult to obtain purified natural receptor molecules, and the purified receptor molecules may lose their natural conformation and function. Cells are an easy choice as a screening target

Method used

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  • Mesenchymal stem cell affinity peptide screening and use
  • Mesenchymal stem cell affinity peptide screening and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, taking rat as an example to illustrate the isolation and culture of MSC

[0055] The experimental steps of this embodiment are as follows:

[0056] Select 6-week-old healthy wistar rats (about 150 grams, male or female), separate the femur and tibia under aseptic conditions, wash out the bone marrow, blow and beat into a cell suspension, and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, resuspend the cells in PBS, gently add the cell suspension to the centrifuge tube pre-prepared with an equal volume of lymphocyte separation medium (specific gravity 1.083) to suspend in the upper layer, centrifuge at 1800rpm for 25min. Collect the mononuclear cells in the cloudy buffy coat in another centrifuge tube, wash once with PBS (centrifuge at 1200 rpm for 5 min), discard the supernatant, and resuspend with L-DMEM containing 10% newborn calf serum. Take 1×10 5 / ml seeded cells in a culture flask, cultured in 5% CO under the conditions of saturatio...

Embodiment 2

[0057] Example 2, identification of rat bone marrow MSC surface markers and purity

[0058] Rat bone marrow mesenchymal stem cells cultured and expanded at passage 4 were collected, digested with 0.25% trypsin to collect cells, and centrifuged at 1200 rpm for 5 min. After washing the cells twice with PBS, resuspend the cells with 1ml PBS and count. Adjust the cell concentration to 1 x 10 with PBS 6 pcs / ml, according to 5×10 5 Each cell / tube was divided into several EP tubes equally, and PE or FITC-labeled multiple antibodies CD90, CD45, CD44 and CD31 (products of Peprotech) were added to each tube in 5 μl, incubated at room temperature for 30 min, and then used Wash with PBS twice, add 0.5ml PBS to resuspend the cells, and use a flow cytometer to detect and identify the surface markers of the cultured and amplified cells. The results showed that the cultured and expanded rat bone marrow mesenchymal stem cells expressed positively CD90, CD45, CD44 and CD31 (see Figure 3).

Embodiment 3

[0059] Embodiment 3, the screening of MSC affinity peptide

[0060] The experimental steps of this embodiment are as follows:

[0061] Inoculate the isolated and purified MSCs of the 2nd or 3rd passage in a 6-well culture plate supplemented with L-DMEM medium (containing 10% fetal bovine serum) and culture for 2 days, until the cells grow well until the monolayer is adherent and confluent At the bottom of the plate, absorb the culture solution, wash it twice with L-DMEM medium, add 1ml stock solution containing 10μL Ph.D.-C7C phage peptide library (titer is 6.5×10 12 pfu / mL) L-DMEM culture based on incubation at 37°C for 1h. Wash 4 times with PBS solution containing 0.1% Tween20, 1 min each time. Phage non-specifically bound to MSCs were removed, and then 1 mL of glycine buffer at pH 2.2 was added to each well, placed on ice for 8 min, and shaken occasionally to elute phage bound to the cell surface. The eluted phages were sucked out and quickly neutralized with Tris (pH9.8...

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Abstract

This invention relates to biotechnology field of cell and polypeptide. The aim of this invention is screening a small peptide that can strongly combine with MSC, and using it to separate and purify MSC and using it as carrying agent to prepare targeted therapeutic agent. The stated binding peptide sequence of MSC is ringed seven peptide sequence that abundantly contains Ser, Thr, Asn, Lys. Finally this invention uses phage display technique to screen small peptide that can strongly combine with MSC in random libraries of ringed seven peptides by affinity screening with MSC. MSC can link to solid phase sustentaculum through small peptide screened by using this invention. It can be used for screening, separating and purifying MSC. It can be used to prepare tracer agent of MSC after linking with fluorescent tumor marker. It can be used as carrying agent of MSC and medicine to prepare praeparatum about targeted therapy.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to the screening of affinity peptides on the surface of mesenchymal stem cells by using a phage random peptide library and the application of the screened affinity peptides. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells derived from mesoderm with high self-renewal ability and multilineage differentiation potential. Widely present in connective tissue and interstitial organs of the whole body, the content is most abundant in bone marrow tissue. In recent years, MSCs have been isolated from umbilical cord blood, umbilical vein subendothelium, periosteum, tendon, blood vessels, and adipose tissue of premature fetuses. MSC has high differentiation potential, among which bone marrow mesenchymal stem cells can differentiate into mature mesenchymal cells under different induction conditions, such as osteoblasts, chondrocytes, adipocytes, tenocytes, ret...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07H21/04C07K19/00C40B30/04A61K38/16A61P43/00G01N33/532
Inventor 裴雪涛赵敬湘杨丽平王韫芳闫舫师伟秦立蓬管利东
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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