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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: 2012-08-22
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 Detection of galectin-3 expression at mRNA level

[0023] Three batches of human umbilical cord mesenchymal stem cells (hUC-MSC) were cultured in a row. After cultured to the P4 generation, they were digested and centrifuged separately. The total RNA of the cells was extracted with an RNA extraction kit (product of Invitrogen), and 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 (results) 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 result is the target product 2 -ΔCt Analysis (see results Figure 1B ). It can be seen from the results that hUC-MSC expresses galectin-3 at the mRNA level, and there is no significant...

Embodiment 2

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

[0025] Cultivate human umbilical cord mesenchymal stem cells with a culture system of 10ml, and collect the supernatant for use after culturing for 48 hours; after cell digestion, centrifuge and count, add 1ml of RIPA reagent (cell lysate, Sigma) for every 1 million cells to lyse the cells. The cell supernatant and RIPA lysate were simultaneously subjected to SDS-PAGE electrophoresis, and the electrophoretic bands were transferred to PVDF membrane by semi-dry method for Western blot analysis. The primary antibody was goat anti-galectin-3 antibody (R&D), and the secondary antibody Rabbit anti-goat IgG (Abcam), 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 cell culture medium.

Embodiment 3

[0026] Example 3 ELISA to detect galectin-3 protein expression

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

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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 a stem cell detection technology, in particular to a method for detecting the biological efficacy of umbilical cord mesenchymal stem cells. Background technique [0002] Umbilical cord-mesenchymal stem cells (UC-MSCs) are stem cells with multidirectional differentiation potential isolated from umbilical cord tissue. The umbilical cord is the tissue that connects the mother and the fetus during pregnancy. It is composed of two arteries, one vein and mucus connective tissue (Wharton’s Jelly). Umbilical cord glue, umbilical cord blood vessel walls and surrounding blood vessels are rich in stromal mesenchymal stem cells (MSCs) and MSCs-like cells (including stromal cells, perivascular cells, etc.). Since Mitchell et al. [1] successfully isolated stromal cells from the umbilical cord in 2003 and confirmed their neural differentiation potential, the research on MSCs derived from umbilical cord tissue has attracted widespread attention. ...

Claims

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Application Information

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Patent Type & Authority Applications(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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