CN-122012738-A - In-vitro evaluation method and application of umbilical cord mesenchymal stem cell aging state

CN122012738ACN 122012738 ACN122012738 ACN 122012738ACN-122012738-A

Abstract

The invention provides an in-vitro evaluation method and application of an umbilical mesenchymal stem cell aging state, and relates to the technical field of biochemical index detection. In the use provided by the present invention, the target gene comprises at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD 45A. According to the invention, the aging state of the umbilical mesenchymal stem cells can be evaluated stably and objectively at a molecular level by detecting the expression level of the target gene, so that the influence caused by subjectivity and insufficient sensitivity of the traditional method is reduced, and the early identification of the aging trend is facilitated.

Inventors

HE SHANGWEN
SUN XIAOYAN
LI HAO
ZHANG LI
ZHANG MIAO
Kou Dalian

Assignees

成都康景生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260305

Claims (10)

1. Use of an agent for detecting expression levels of a target gene, wherein the target gene comprises at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A, in the manufacture of a test product for assessing the senescence status of umbilical mesenchymal stem cells.
2. The use according to claim 1, wherein the genes of interest comprise BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; Preferably, the test product evaluates the aging state by calculating an aging score, which is calculated based on the following formula: ; Wherein S represents the senescence score, E BMP2 represents the relative expression level of the BMP2 gene, E IFIT2 represents the relative expression level of the IFIT2 gene, E GADD45A represents the relative expression level of the GADD45A gene, E ADAMTSL4 represents the relative expression level of the ADAMTS L4 gene, and E RRAS2 represents the relative expression level of the RRAS2 gene.
3. The use of claim 2, wherein a higher senescence score indicates a more severe senescence of the umbilical mesenchymal stem cells, and/or, The detection product is used for screening umbilical cord mesenchymal stem cells with senescence scores lower than a preset threshold value.
4. The use according to claim 1, wherein the target gene comprises BMP2, wherein an increase in the expression level of said BMP2 is indicative of a decrease in the expression level of hepatocyte growth factor and/or an impaired anti-fibrosis ability of said umbilical cord mesenchymal stem cells, and/or, The reagent comprises at least one of a primer pair for specifically amplifying the mRNA of the target gene, a probe for specific hybridization and a detection chip, and/or, The detection product is a kit for quality monitoring in the in-vitro amplification culture process of umbilical cord mesenchymal stem cells.
5. A kit for assessing the senescence state of umbilical cord mesenchymal stem cells, comprising reagents for detecting the expression level of at least one gene of interest in a gene combination comprising BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD 45A; the reagent comprises at least one of a primer pair for specifically amplifying the target gene, a probe for specifically hybridizing or a detection chip coated with a specific antibody.
6. The kit according to claim 5, wherein the target genes include BMP2, ADAMTS L4, IFIT2, RRAS, and GADD45A, and/or, The kit also comprises a specification for use, wherein the specification directs a user to calculate a senescence score based on the detected expression level of the target gene; Preferably, the instructions instruct the user to calculate the senescence score based on the following formula; ; Wherein S represents the senescence score, E BMP2 represents the relative expression level of the BMP2 gene, E IFIT2 represents the relative expression level of the IFIT2 gene, E GADD45A represents the relative expression level of the GADD45A gene, E ADAMTSL4 represents the relative expression level of the ADAMTS L4 gene, and E RRAS2 represents the relative expression level of the RRAS2 gene.
7. An in vitro method for assessing the senescence state of umbilical cord mesenchymal stem cells, comprising: receiving relative expression level data of target genes of an umbilical cord mesenchymal stem cell sample to be detected, wherein the target genes comprise BMP2, ADAMTS 4, IFIT2, RRAS2 and GADD45A; Calculating a senescence score from the relative expression level data, wherein the senescence score is calculated based on the following formula: ; Wherein S represents the senescence score, E BMP2 represents the relative expression level of the BMP2 gene, E IFIT2 represents the relative expression level of the IFIT2 gene, E GADD45A represents the relative expression level of the GADD45A gene, E ADAMTSL4 represents the relative expression level of the ADAMTS L4 gene, and E RRAS2 represents the relative expression level of the RRAS2 gene; Outputting the aging state evaluation result of the cells according to the aging score.
8. An in vitro assessment device for the aging state of umbilical mesenchymal stem cells, comprising: the receiving module is used for receiving the relative expression level data of target genes of the umbilical cord mesenchymal stem cell sample to be detected, wherein the target genes comprise BMP2, ADAMTS 4, IFIT2, RRAS2 and GADD45A; a calculation module for calculating a senescence score from the relative expression level data; The output module is used for outputting the aging state evaluation result of the cells according to the aging score, wherein the aging score is calculated based on the following formula: ; Wherein S represents the senescence score, E BMP2 represents the relative expression level of the BMP2 gene, E IFIT2 represents the relative expression level of the IFIT2 gene, E GADD45A represents the relative expression level of the GADD45A gene, E ADAMTSL4 represents the relative expression level of the ADAMTS L4 gene, and E RRAS2 represents the relative expression level of the RRAS2 gene.
9. An electronic device comprising a memory and a processor, wherein the memory has stored thereon a computer program, and wherein the processor, when executing the program, implements the method for in vitro assessment of the aging status of umbilical mesenchymal stem cells according to claim 8.
10. A computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, implements the in vitro method for assessing the senescence state of umbilical mesenchymal stem cells according to claim 8.

Description

In-vitro evaluation method and application of umbilical cord mesenchymal stem cell aging state Technical Field The invention relates to the technical field of biochemical index detection, in particular to an in-vitro evaluation method and application of an umbilical mesenchymal stem cell aging state. Background The mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCS) are adult stem cells with self-renewal and multidirectional differentiation potential, and have great application prospects in the fields of tissue engineering and regenerative medicine. Wherein, the mesenchymal stem cells (Umbilical Cord MESENCHYMAL STEM CELLS, UCMSCS) from umbilical cord become seed cells with great potential in cell therapy due to the advantages of convenient material taking, low immunogenicity, strong proliferation capability and the like. However, when UCMSCs are expanded and cultured in vitro to obtain a sufficient number of cells for clinical use, cell senescence inevitably occurs. The proliferation capacity of the aged cells is obviously reduced, and the key therapeutic functions such as multidirectional differentiation potential, immunoregulation and the like of the aged cells are weakened, so that the final clinical therapeutic effect of the aged cells is seriously influenced. Therefore, accurate aging state assessment of UCMSCs for clinical treatment prior to application is critical to ensuring quality of cell products and therapeutic safety. Currently, the industry's assessment of the state of cellular aging generally depends on a range of biological indicators. Common detection techniques include analysis of the proliferative capacity of cells, e.g., calculation of cell population doubling time, histochemical staining with the property of high expression of senescence-associated beta-galactosidase (SA-beta-gal) in senescent cells, detection of the expression levels of cell cycle critical inhibitory proteins, such as p16.sup.INK4a and p21.sup.CIP 1, and analysis of various cytokines in senescence-associated secretory phenotypes (SASPs). Furthermore, shortening of telomere length and accumulation of DNA damage markers are also considered indicators of cellular senescence. These methods reflect the aging process of cells from different dimensions. However, the existing aging detection technology has a plurality of limitations in practical application. Firstly, the comprehensive evaluation process by relying on various marker combinations is complex, and the lack of uniform standards between different detection methods leads to difficulty in transverse comparison of results. Secondly, single detection methods have shortcomings in terms of specificity, sensitivity and quantitative accuracy. For example, the detection result of the proliferation capacity of cells is extremely easy to be influenced by external conditions such as culture medium components, serum batches and the like, SA-beta-gal staining is a semi-quantitative method in nature, the interpretation of the results is easy to be influenced by subjective factors, accurate quantitative analysis is difficult to realize, and the expression levels of proteins such as p16, p21 and the like are dynamic and transient and highly dependent on specific causes and cell types for inducing senescence, the association of the expression levels with cell functional senescence is not absolute, and the sensitivity is insufficient when the UCMSCs are evaluated for senescence. In summary, the methods for evaluating the aging state of UCMSCs in the prior art are difficult to meet the urgent requirements of clinical application on cell quality control due to the problems of complex flow, difficult standardization, or weak single index specificity, insufficient sensitivity, and difficult accurate quantification. In particular, the prior art lacks a single molecular marker which can rapidly, accurately, sensitively and universally judge the aging degree of UCMSCs. Therefore, there is an urgent need in the art to develop new technical means to establish a more reliable and efficient aging diagnosis system, thereby guaranteeing the effectiveness and safety of cell therapy products. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide an in-vitro evaluation method and application of an umbilical mesenchymal stem cell aging state. The invention uses the specific target gene as a molecular marker, and can sensitively, accurately and quantitatively evaluate the aging state of the umbilical mesenchymal stem cells, thereby overcoming the limitation of the traditional method and improving the quality control level of cell products. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: in a first aspect, the present invention provides a use of a reagent for detecting an expression level of a target gene including at least one of BMP2, ADAMTSL4, IFIT2, RRAS, and GADD45A in