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Application of periostin recombinant protein in maintaining the function of tendon stem cells

A technology of tendon stem cells and recombinant proteins, which is applied in the application field of Periostin recombinant proteins in maintaining the function of tendon stem cells, can solve the problems of low ratio of tendon stem cells, affecting the use effect of tendon stem cells, proliferation of tendon stem cells, decline in self-renewal and differentiation ability, etc. Achieve the effect of maintaining proliferation, promoting self-renewal ability, and good proliferation

Active Publication Date: 2021-12-31
PEKING UNIV SCHOOL OF STOMATOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the proportion of tendon stem cells in the body is low, and after isolation from patients, long-term expansion in vitro is required to obtain sufficient numbers for the repair and regeneration of tendon tissue defects
The same problem as normal adult stem cells is that the proliferation, self-renewal and differentiation ability of tendon stem cells after long-term expansion will seriously affect the application effect of tendon stem cells in tendon tissue engineering.
[0005] At present, there is no report about the long-term expansion and culture of tendon stem cells in vitro using Periostin recombinant protein to promote the self-renewal and differentiation capabilities of stem cells

Method used

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  • Application of periostin recombinant protein in maintaining the function of tendon stem cells
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  • Application of periostin recombinant protein in maintaining the function of tendon stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Achilles tendons from 6-8 week old rats were obtained, minced thoroughly, digested with collagenase and dispase, resuspended in DMEM medium, placed in 5% CO 2 The culture medium was cultured in a 37°C incubator with a high concentration, and the fresh medium was replaced every 2-3 days. The cell morphology was observed under an optical microscope, and the ratio of positive mesenchymal stem cell surface markers (CD105, CD90) and negative hematopoietic stem cell surface markers (CD34, CD45) in tendon stem cells was detected by flow cytometry Distribution.

[0034] The results show:

[0035] (1) TSPCs showed fibroblast-like morphology under the microscope, see Figure 1a ;

[0036](2) Flow cytometry analysis showed that TSPCs highly expressed mesenchymal markers CD105 (94.57%) and CD90 (92.12%), and lowly expressed hematopoietic stem cell markers CD45 (1.76%) and CD34 (0.17), see Figure 1b ;

[0037] The above results indicated that TPSCs cultured in vitro belonged to...

Embodiment 2

[0039] In order to verify the effect of early generations of Periostin recombinant protein on the self-renewal ability and proliferation ability of tendon stem cells, we verified by Ki67 immunofluorescence staining and cell clone formation experiments.

[0040] Add PBS solution to TSPCs in the control group, add the same volume of Periostin recombinant protein to TSPCs in the experimental group, and perform the following treatments:

[0041] ① After continuous culture for 48-72 hours under the same conditions, perform Ki67 immunofluorescence staining, randomly select and take pictures under the microscope and count the number of Ki67-positive cells;

[0042] ② After continuous culture for 14 days under the same conditions, carry out crystal violet staining, take pictures under the microscope and count the number of clone populations with a diameter greater than 40 μm.

[0043] The results show:

[0044] (1) The number of Ki67-positive cells in the experimental group was large...

Embodiment 3

[0047] In order to verify the effect of early generations of Periostin recombinant protein on the tendon differentiation ability of tendon stem cells, we verified by Sirius red, Masson staining and cell immunofluorescence staining experiments.

[0048] The blank group did not add any treatment, the TSPCs of the control group were added with PBS solution, the TSPCs of the experimental group were added with the same volume of Periostin recombinant protein, the control group and the experimental group were added and replaced with fresh tendon-forming medium every 2-3 days, and the following treatments were performed:

[0049] ①After 14 days of continuous culture, carry out Sirius red and Masson staining, randomly take photos under the microscope and count the proportion of stained positive areas;

[0050] ② After continuous culture for 14 days under the same conditions, use cell immunofluorescence staining technology, randomly take pictures under the microscope and count the posit...

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Abstract

The invention provides the application of Periostin recombinant protein in maintaining the function of tendon stem cells, including the in vitro culture method of tendon stem cells, which adopts culture medium containing Periostin recombinant protein; the in vitro culture medium of tendon stem cells, wherein the culture medium contains Periostin recombinant protein. The present invention utilizes the Periostin recombinant protein to successfully establish a method for culturing and mass-expanding tendon stem cells in vitro, and can obtain a sufficient number of tendon stem cells. The ability of tendon differentiation makes it possible for tendon stem cells to be used in tendon tissue engineering, repair and regeneration therapy.

Description

technical field [0001] The invention belongs to the field of biomedicine, and particularly relates to the application of Periostin recombinant protein in maintaining the function of tendon stem cells. Background technique [0002] Tendon injury is a common clinical disease. Epidemiological surveys have found that the current incidence of sports injuries accounts for 10-20%, often directly caused by trauma, strenuous exercise, or secondary to chronic tendon inflammation or tumors. Adult tendon is a tissue with low cell density and low blood vessels, and its self-repair and regeneration ability is poor. However, there are still many problems in the current traditional treatment methods for tendon repair and regeneration, such as structural specificity and difficult restoration of integrity, low degree of functional recovery, secondary The recurrence rate of secondary injury is high. In recent years, the development of stem cell biology and tissue engineering technology has br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/077A61K38/17A61P19/04A61P39/06
CPCC12N5/0668C12N5/066A61K38/1709A61P19/04A61P39/06C12N2501/998
Inventor 刘燕王禹金姗姗朱丽莎
Owner PEKING UNIV SCHOOL OF STOMATOLOGY
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