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Detection method for biological efficacy of umbilical cord mesenchymal stem cell

A stem cell biology, stem cell technology, applied in the field of detection of the biological efficacy of umbilical cord mesenchymal stem cells, can solve problems such as the lack of defined cell quality cell treatment effects

Inactive Publication Date: 2013-03-20
天津和泽干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the mesenchymal stem cell products used in preclinical or clinical use are based on the three criteria for defining mesenchymal stem cells proposed by the International Cell Therapy Association in 2006 [2], but lack the definable cell quality or preliminary judgment for a certain type of disease Methods of Cell Therapy Effects

Method used

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  • Detection method for biological efficacy of umbilical cord mesenchymal stem cell
  • Detection method for biological efficacy of umbilical cord mesenchymal stem cell
  • Detection method for biological efficacy of umbilical cord mesenchymal stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 mRNA level detection of galectin-3 expression

[0023] Three batches of human umbilical cord mesenchymal stem cells (hUC-MSCs) were continuously cultured. After cultured to the P4 generation, they were digested and centrifuged separately, and the total RNA of the cells was extracted with an RNA extraction kit (product of Invitrogen Company). After reversed to cDNA, galectin-3 The primers were subjected to conventional PCR amplification (forward primer: 5′-CCAAAGAGGGAATGATGTTGCC-3′, reverse primer: 5′-TGATTGTACTGCAACAAGTGAGC-3′), and agarose electrophoresis analysis (result Figure 1A ); select the same batch of P2-P4 generation cells to extract RNA and reverse it to cDNA, use real-time qPCR (real-time quantitative PCR) analysis, the primer sequence is the same as PCR, and the real-time qPCR results are analyzed for the target product 2 -ΔCt Analysis (results see Figure 1B ). The results showed that hUC-MSCs expressed galectin-3 at the mRNA level, and there w...

Embodiment 2

[0024] Example 2 Western blot detection of galectin-3 protein expression

[0025] Human umbilical cord mesenchymal stem cells were cultured with a culture system of 10 ml. After 48 hours of culture, the supernatant was collected for later use; the cells were digested and counted by centrifugation, and 1 ml of RIPA reagent (cell lysate, Sigma Company) was added to lyse the cells per 1 million cells. The cell supernatant and RIPA lysate were subjected to SDS-PAGE electrophoresis at the same time, and the electrophoresis band was transferred to PVDF membrane by semi-dry method for Western blot analysis. The primary antibody was goat anti-galectin-3 antibody (R&D Company), and the secondary antibody It is rabbit anti-goat IgG (Abcam Company), see the results figure 2 . It can be seen from the results that hUC-MSC can express galectin-3 protein, and this protein can be secreted into the cell culture medium.

Embodiment 3

[0026] Example 3 ELISA detection of galectin-3 protein expression

[0027] Continuously culture 3 batches of human umbilical cord mesenchymal stem cells, the culture system is 10ml, and the supernatant is collected after 48 hours of culture; the cells are digested at the end of 48 hours, and 1ml RIPA reagent is added for every 1 million cells to lyse the cells. Cell supernatant and RIPA lysate were tested by ELISA (galectin-3ELISA kit, Bender company) at the same time, and the results were as follows: image 3 . It is further proved that galectin-3 protein can be expressed in hUC-MSC cells and can be secreted extracellularly.

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Abstract

The invention provides a detection method for the biological efficacy of the umbilical cord mesenchymal stem cell. The method is characterized in that: the immunosuppression ability of the cell is judged by detecting the content of galectin-3 in the umbilical cord mesenchymal stem cell culture supernatant, the pertinence is high, and the quantification is accurate; and moreover, digestive cells are not required, thus the method is suitable for detecting the immunosuppression efficacy in large-scale continuous umbilical cord mesenchymal stem cell culture. In the method provided by the invention, a detection sample is easy to process, and automatic or semi-automatic detection can be performed by use of instrument.

Description

technical field [0001] The invention relates to stem cell detection technology, in particular to a detection method for the biological effectiveness of umbilical cord mesenchymal stem cells. Background technique [0002] Umbilical cord-mesenchymal stem cells (UC-MSCs) are a kind of stem cells isolated from umbilical cord tissue with multilineage differentiation potential. The umbilical cord is the tissue that connects the mother to the fetus during pregnancy and consists of two arteries, a vein and mucus connective tissue (Wharton’s Jelly). Umbilical cord jelly, umbilical cord vessel wall and perivascular are rich in mesenchymal stem cells (mesenchymal stem cells, MSCs) and MSCs-like cells (including stromal cells, perivascular cells, etc.). Since Mitchell et al[1] first successfully isolated stromal cells from umbilical cord and confirmed their neural differentiation potential in 2003, the research on umbilical cord tissue-derived MSCs has attracted widespread attention. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N33/68
Inventor 刘广洋朱德琳刘拥军徐燚聂艳波
Owner 天津和泽干细胞科技有限公司
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