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Method for evaluating influence of GFP gene transfection on hPMSCs metabonomics based on CILLC-MS

A technology of metabolomics and gene transfection, which is applied in the field of evaluating the impact of green fluorescent protein gene transfection on the metabolomics of placenta-derived mesenchymal stem cells, which can solve the problems of poor extraction efficiency and avoid inconsistency and chance , improve the sensitivity and the effect of accurate quantitative analysis

Active Publication Date: 2018-02-16
ZHEJIANG UNIV
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Problems solved by technology

However, when used for the extraction of cell metabolites before and after GFP gene transfection, there is a problem of poor extraction efficiency when used alone

Method used

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Embodiment Construction

[0033] First of all, it is explained that the placental tissue used in the present invention is clinical waste and directly comes from the medical institution where the puerpera gave birth. The content of the present invention is based on the utilization of fresh placental tissue, but does not directly relate to the operation of removing or resecting the placental tissue.

[0034] The technical solutions of the present invention will be described in detail below in conjunction with specific implementation examples.

[0035] Comparing hPMSCs with GFP + Metabolomic differences between hPMSCs

[0036] (1) Isolation, culture and transfection of mesenchymal stem cells

[0037] Isolation and culture of hPMSCs:

[0038] Under aseptic conditions, the clinically discarded fresh placental tissue was fully washed with PBS to remove blood stains, and the membrane on the surface of the placental tissue was cut off. Mince the rest of the placental tissue, digest it in 0.2% type IV colla...

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Abstract

The present invention relates to a placenta-derived mesenchymal stem cell metabolite extraction and detection technology. A purpose of the present invention is to provide a method for evaluating the influence of GFP gene transfection on hPMSCs metabonomics based on CILLC-MS. According to the present invention, the differential metabolites and the differential metabolic pathways before and after transfection are further analyzed by extracting and by detecting and quantitating the intracellular metabolites of hPMSCs before and after GFP gene transfection, and the biological safety of green fluorescent protein in labeling and tracking of mesenchymal stem cells is evaluated from the perspective of metabonomics; the analysis process for comparing the metabonomics difference between hPMSCs and GFP-hPMSCs is firstly created, and the two sample preparation methods are combined for the extraction of the stem cell metabolites, such that the intracellular metabolites are completely released; by combining the CIL LC-MS in the detection of the stem cell intracellular metabolites, the precise quantitative analysis of intracellular metabolites is achieved, and the identification type of metabolites is substantially increased; and with the method, the sensitivity, the reliability and the repeatability of the results are substantially improved, and the operation-induced non-parallelism and theoperation-induced contingency are avoided.

Description

field of invention [0001] The present invention relates to the extraction and detection technology of placenta-derived mesenchymal stem cell metabolites, in particular to the evaluation of the effect of green fluorescent protein gene transfection on placenta-derived mesenchymal stem cells based on isotope-labeled liquid chromatography-mass spectrometry (CIL LC-MS). Methods of metabolomics impact. Background technique [0002] As an important member of the stem cell family, mesenchymal stem cells (MSCs) are fibroblast-like, highly plastic and self-renewing pluripotent stem cells that can differentiate into a variety of terminal cells. The placenta is a clinical waste, and its placenta-derived mesenchymal stem cells (hPMSCs) have the advantages of rich sources, simple isolation methods, strong proliferation ability, low immunogenicity, and no ethical restrictions, and have gradually become the main source of stem cells. Advantages of seeding cells for transplantation therapy....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 曹红翠李兰娟俞炯陈德莹
Owner ZHEJIANG UNIV
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