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Method for authenticating activity of bone marrow mesenchymal stem cells

A bone marrow mesenchymal and stem cell technology, applied in the direction of material inspection products, measuring devices, instruments, etc., can solve the problems of reduced cell number, low content of BMSCs, complicated operation process, etc., to achieve strong proliferation ability, reduce damage, and simple process Effect

Inactive Publication Date: 2014-12-17
WUHAN GAOHUA CELL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the complex operation process of BMSCs used in stem cell therapy, such as separation, concentration, purification, identification, detection, washing, and cryopreservation, and the low content of BMSCs, it is difficult to directly isolate and purify them, and the number of cells varies with the growth environment. Aging will decrease, which will reduce the effect of stem cell therapy to a certain extent
The decline in cell activity will not only affect the therapeutic effect of the disease, but also cause some side effects, such as fever, etc.

Method used

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  • Method for authenticating activity of bone marrow mesenchymal stem cells
  • Method for authenticating activity of bone marrow mesenchymal stem cells
  • Method for authenticating activity of bone marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Activity Identification of Bone Marrow Mesenchymal Stem Cells

[0023] (1) Immunofluorescence localization of the expression distribution of GAPDH in BMSCs

[0024] Bone marrow mesenchymal stem cells were sliced, soaked, fixed, membrane ruptured, and sealed, then incubated overnight with GAPDH primary antibody, then incubated with FITC-labeled fluorescent secondary antibody, and then observed and collected images under a fluorescent microscope. The specific operation is as follows:

[0025] first day:

[0026] ①In the culture plate, soak the slides with bone marrow mesenchymal stem cells on which the bone marrow mesenchymal stem cells have climbed up with PBS for 3 times, each time for 3 minutes;

[0027] ②Fix slides with 4% paraformaldehyde for 15 minutes, soak and wash slides with PBS 3 times, 3 minutes each time;

[0028] ③ 0.5% Triton X-100 (prepared in PBS) permeabilized at room temperature for 20 minutes;

[0029] ④ Soak slides in PBS for 3 times, 3 ...

Embodiment 2

[0036] Example 2 Effect of the expression distribution level of GAPDH protein in BMSCs on the therapeutic effect of pulmonary hypertension

[0037] 1. Establishment of animal model of pulmonary hypertension

[0038] Animal-related operations were performed in accordance with international standards, and 180 g male SD mice were selected to inject monocrotaline (MCT, 60 mg / kg) into the tail vein to establish a pulmonary hypertension model for a total of 30 days. From the fourth day of modeling, the cell therapy group was injected with H-GAPDH and L-GAPDH BMSCs (1×10 6 per 0.5mL PBS), and the blank control group was injected with 0.5mL PBS. Rats were sacrificed 30 days after modeling, and the muscularization of pulmonary artery and right ventricular hypertrophy were detected.

[0039] 2. Detection of right ventricular hypertrophy and pulmonary artery muscularization

[0040] The right ventricle and left ventricle were separated from the septum, weighed and recorded respectivel...

Embodiment 3

[0047] Example 3 The influence of the expression distribution level of GAPDH protein in BMSCs on the proliferation ability of BMSCs

[0048] 1. Detection of proliferation ability of BMSCs

[0049] BMSCs with different expression types were digested and made into single-cell suspensions. After counting, single-cell suspensions with a certain concentration were prepared for cell plating. Each sample was set up with 5 to 6 multiple wells, and sterile water or PBS was added to the edge wells. , 37 degrees, 5% CO 2 cultured in an incubator. After 72 hours, treat with CCK-8, detect with a microplate reader, and analyze according to the statistics of the data.

[0050] 2. Experimental results

[0051] Such as Figure 4 As shown, the normal 24h proliferation rate of cells in the high-expression GAPDH (H-GAPDH) group was 1 (1.005±0.046, n=7) for normalization, and the low-expression GAPDH (L-GAPDH) normal 24h proliferation was H-GAPDH (0.909±0.054, n=8); after H-GAPDH cells were s...

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Abstract

The invention discloses a method for detecting expression distribution level of GAPDH protein in bone marrow mesenchymal stem cells, and the application of the method in authenticating the activity of the bone marrow mesenchymal stem cells. The invention also discloses a method for authenticating the activity of the bone marrow mesenchymal stem cells. The method comprises the following steps: (1) detecting the expression distribution level of the GAPDH protein in the bone marrow mesenchymal stem cells by adopting a cell immunofluorescence staining method; (2) if over 60 percent of the GAPDH protein is distributed in cell nuclei, determining that the GAPDH protein is of a highly expressed type and the activity of the bone marrow mesenchymal stem cells is high; if less than 40 percent of the GAPDH protein is distributed in cell nuclei, determining that the GAPDH protein is of a lowly expressed type and the activity of the bone marrow mesenchymal stem cells is low. According to the method, the relationship between the expression distribution level of the GAPDH protein in the bone marrow mesenchymal stem cells and the activity of the bone marrow mesenchymal stem cells is discovered for the first time; by the provided method, the bone marrow mesenchymal stem cells which have a good treatment effect on diseases and high multiplication capacity can be authenticated quickly, so that the method can be used for high-flux screening of the bone marrow mesenchymal stem cells.

Description

technical field [0001] The invention relates to a method for detecting the expression and distribution level of GAPDH protein in bone marrow mesenchymal stem cells and its application in identifying the activity of bone marrow mesenchymal stem cells. The invention also relates to a method for identifying the activity of bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are a group of non-hematopoietic stem cells derived from bone marrow tissue and belong to pluripotent stem cells. Because BMSCs have multi-directional differentiation ability, they can differentiate into various connective tissues and some tissues derived from ectoderm after in vitro culture to form adipocytes, muscle cells, chondrocytes, nerve cells, endothelial cells and osteoblasts, etc. It has broad application prospects in the medical field. Compared with embryonic stem cells, it has obvious advantages, and there are no problems such as immune re...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N21/64
CPCG01N21/64G01N33/56966G01N33/577G01N33/68
Inventor 胡清华叶平陈凤容
Owner WUHAN GAOHUA CELL TECH
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