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Detection method for in-vitro inhibition of Th1 and Th17 by mesenchymal stem cells

A mesenchymal stem cell and detection method technology, applied in the field of stem cell biology, can solve the problems of not having a good mesenchymal stem cell immune regulation ability, ineffective effect, and poor repeatability, so as to improve repeatability, effect, and The effect of expression

Inactive Publication Date: 2021-04-30
华夏源(上海)生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are various methods for preparing mesenchymal stem cells, such as tissue block adherent culture method, umbilical cord homogenate collagenase digestion method and improved collagenase digestion method, etc., but most of the current research focuses on the clinical application of mesenchymal stem cells, and There is no good evaluation mechanism to identify the immunomodulatory ability of mesenchymal stem cells prepared by many preparation methods
[0004] CN201811507077.2 provides a detection method for the immunoregulatory ability of mesenchymal stem cells. The original sample is co-cultured with mesenchymal stem cells to be evaluated, and the inhibitory effect of mesenchymal stem cells is evaluated by detecting the change in the proportion of lymphocyte subsets , so as to evaluate the immunoregulatory ability of mesenchymal stem cells, but in the detection of mesenchymal stem cells inhibiting T lymphocyte subsets Th1 and Th17 in vitro, there are characteristics such as low inhibition rate, insignificant effect, and poor repeatability.

Method used

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  • Detection method for in-vitro inhibition of Th1 and Th17 by mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0074] This example provides a detection method for mesenchymal stem cells to inhibit Th1 and Th17 in vitro, including:

[0075] (1) MSC cell inoculation: take the MSC cell suspension, centrifuge at 400g for 5min, remove the supernatant, add complete medium to resuspend, sample and count; according to the counting results, adjust the cell concentration to 2×10 5 / mL; inoculate the adjusted cell suspension in 24-well culture plate, 500 μl per well, and inoculate 2 wells in total. Place the inoculated cell plates in a carbon dioxide incubator (CO 2 Concentration setting: 5.0%, temperature setting: 37.0°C), culture for 20-24 hours;

[0076] (2) MSC deproliferation treatment: After MSC cells were cultured for 20-24 hours, observe the cell adhesion under the microscope; discard the medium in each well, add 500 μl of complete medium containing 10 μg / mL mitomycin C to each well, placed in a carbon dioxide incubator (CO 2 Concentration setting: 5.0%, temperature setting: 37.0°C), i...

Embodiment 2

[0084] This example provides a detection method for mesenchymal stem cells to inhibit Th1 and Th17 in vitro. Centrifuge to get the cell pellet, add medium containing 1μg / mL PHA to stimulate the cells for 1h, then add 10μg / mL BFA, put in CO 2 Continue to incubate in the incubator for 5 hours; blow and disperse the aggregated cells evenly every 2 hours; centrifuge at 500g for 5 minutes, wash the obtained cell pellet twice with 2 mL of PBS, and then add 100 μL of PBS to resuspend to obtain a resuspension solution.

Embodiment 3

[0086]This example provides a detection method for mesenchymal stem cells inhibiting Th1 and Th17 in vitro. The specific implementation method is the same as in Example 1, except that in the step (5), 100 μL of fixative and 100 μL of fixative and After vortexing 100 μL of membrane disrupting agent for 5 seconds and fixing for 15 minutes, add staining antibody, incubate in the dark for 20 minutes, add 3 mL of balanced salt solution, centrifuge at 400 g for 5 minutes, and add 300 μL of resuspension solution to the obtained cell pellet for detection.

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Abstract

The invention relates to the technical field of stem cell biology, in particular to a detection method for in-vitro Th1 and Th17 inhibition of mesenchymal stem cells. The method comprises the following steps: 1, co-culturing mesenchymal stem cells and peripheral blood mononuclear cells; 2, carrying out fixed membrane rupture dyeing and detection. According to the detection method provided by the invention, lymphocyte proliferation and differentiation are promoted through co-culture of mesenchymal stem cells and peripheral mononuclear cells, so that the expression of the inhibition effect of the mesenchymal stem cells is promoted, and the inhibition effect is improved. According to the method, the PBMC cells obtained through co-culture are stimulated and blocked, so that factor secretion is promoted, meanwhile, inhibition and factor blocking are added for detection, so that expression of Th1 and Th17 in the PBMC cells is further promoted, expression of Th1 and Th17 obtained through detection is improved, and the inhibition effect is further improved.

Description

technical field [0001] The present invention relates to the technical field of stem cell biology, and more specifically, the present invention relates to a detection method for mesenchymal stem cells inhibiting Th1 and Th17 in vitro. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells, derived from the mesoderm and ectoderm in the early stages of development, and can be isolated from various tissues such as bone marrow, fat, synovium, bone, muscle, and umbilical cord. It has the potential to differentiate into osteoblasts, chondrocytes, adipocytes, bone marrow stroma, nerve cells, liver cells, islet cells and cardiomyocytes. The source is convenient, easy to separate, cultivate, expand and purify, and still has the characteristics of stem cells after multiple passages and expansions, without the characteristics of immune rejection. [0003] Mesenchymal stem cells can not only promote the regeneration and repair of damaged tissues...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12Q1/02
CPCC12N5/0636C12N2502/1352G01N33/5005
Inventor 朱灏
Owner 华夏源(上海)生命科技有限公司
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