In-vitro evaluation method and application of aging state of umbilical cord mesenchymal stem cells

Abstract

The invention provides an in-vitro evaluation method for the aging state of umbilical cord mesenchymal stem cells and application, and relates to the technical field of biochemical index detection. In the application provided by the invention, a target gene comprises at least one of BMP2, ADAMTSL4, IFIT2, RRAS2 and GADD45A. According to the present invention, by detecting the expression level of the target gene, the aging state of the umbilical cord mesenchymal stem cell can be stably and objectively evaluated on the molecular level, such that the influence caused by the insufficient subjectivity and the insufficient sensitivity of the traditional method can be reduced, and the early-stage identification of the aging trend can be easily achieved.

Description

Technical Field

[0001] This invention relates to the field of biochemical indicator detection technology, and more specifically, to an in vitro assessment method and application for the aging status of umbilical cord mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal and multi-lineage differentiation potential, showing great promise in tissue engineering and regenerative medicine. Among them, umbilical cord-derived mesenchymal stem cells (UCMSCs) have become highly promising seed cells in cell therapy due to their convenient sourcing, low immunogenicity, and strong proliferative capacity. However, when UCMSCs are expanded and cultured in vitro to obtain a sufficient number of cells for clinical use, cellular senescence is inevitable. Senescent cells not only have significantly reduced proliferative capacity, but their multi-lineage differentiation potential and key therapeutic functions such as immunomodulation are also weakened, thus seriously affecting their final clinical therapeutic effect. Therefore, accurate assessment of the senescence status of UCMSCs before application in clinical treatment is crucial to ensuring the quality of cell products and the safety of treatment.

[0003] Currently, the assessment of cellular senescence typically relies on a range of biological indicators. Commonly used detection techniques include analyzing cell proliferation capacity, such as calculating cell population doubling time; histochemical staining utilizing the high expression of senescence-associated β-galactosidase (SA-β-gal) in senescent cells; detecting the expression levels of key cell cycle repressor proteins (such as p16INK4a and p21CIP1); and analyzing various cytokines in the senescence-associated secretory phenotype (SASP). Furthermore, telomere shortening and the accumulation of DNA damage markers are also considered indicators of cellular senescence. These methods reflect the senescence process of cells from different dimensions.

[0004] However, existing aging detection technologies have many limitations in practical applications. First, the process of comprehensive evaluation relying on combinations of multiple biomarkers is complex, and the lack of unified standards between different detection methods makes it difficult to compare results across different methods. Second, single detection methods are insufficient in terms of specificity, sensitivity, and quantitative accuracy. For example, the results of cell proliferation capacity detection are easily affected by external conditions such as culture medium composition and serum batch; SA-β-gal staining is essentially a semi-quantitative method, and the interpretation of results is easily affected by subjective factors, making it difficult to achieve accurate quantitative analysis; while the expression levels of proteins such as p16 and p21 are dynamic and transient, and highly dependent on the specific causes of aging and cell type, the correlation between their expression levels and cellular functional aging is not absolute, resulting in insufficient sensitivity when used to assess the aging of UCMSCs.

[0005] In summary, existing methods for assessing the aging status of UCMSCs suffer from limitations due to complex procedures, difficulty in standardization, or the lack of specificity, sensitivity, and precise quantification of single indicators, failing to meet the urgent clinical needs for cell quality control. Specifically, current technologies lack a single molecular marker that can rapidly, accurately, sensitively, and universally determine the degree of aging in UCMSCs. Therefore, there is an urgent need in this field to develop new technologies to establish a more reliable and efficient aging diagnostic system, thereby ensuring the effectiveness and safety of cell therapy products.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an in vitro assessment method and application for the aging status of umbilical cord mesenchymal stem cells. This invention utilizes specific target genes as molecular markers to achieve a sensitive, accurate, and quantifiable assessment of the aging status of umbilical cord mesenchymal stem cells, thereby overcoming the limitations of traditional methods and improving the quality control level of cell products.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the use of a reagent for detecting the expression level of a target gene in the preparation of a detection product for assessing the aging status of umbilical cord mesenchymal stem cells, wherein the target gene includes at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

[0009] In an optional implementation, the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; Preferably, the detection product assesses the aging state by calculating an aging score; the aging score is calculated based on the following formula: ; Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

[0010] In an optional implementation, a higher aging score indicates a more severe degree of aging of the umbilical cord mesenchymal stem cells; and / or, The detection product is used to screen umbilical cord mesenchymal stem cells with aging scores below a preset threshold.

[0011] In an optional embodiment, the target gene includes BMP2; elevated BMP2 expression levels are used to indicate decreased hepatocyte growth factor expression levels and / or weakened anti-fibrotic ability of the umbilical cord mesenchymal stem cells; and / or, The reagent includes at least one of the following: primer pairs for specifically amplifying the target gene mRNA, probes for specific hybridization, and a detection chip; and / or, The testing product is a kit used for quality monitoring during the in vitro expansion and culture of umbilical cord mesenchymal stem cells.

[0012] In a second aspect, the present invention provides a kit for assessing the senescence status of umbilical cord mesenchymal stem cells, comprising reagents for detecting the expression level of at least one target gene in a gene combination including BMP2, ADAMTSL4, IFIT2, RRAS2 and GADD45A. The reagent includes at least one of the following: primer pairs for specifically amplifying the target gene, probes for specific hybridization, or detection chips coated with specific antibodies.

[0013] In optional embodiments, the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; and / or, The kit also includes an instruction manual; the instruction manual guides the user to calculate an aging score based on the detected target gene expression levels. Preferably, the instruction manual guides the user to calculate an aging score based on the following formula; ; Where S represents the aging score; E BMP2 Represents the relative expression level of the BMP2 gene; EIFIT2 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

[0014] Thirdly, the present invention provides an in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells, comprising: Receive relative expression level data of target genes from umbilical cord mesenchymal stem cell samples to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; An aging score is calculated based on the relative expression level data; wherein the aging score is calculated based on the following formula: ; Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 Represents the relative expression level of the RRAS2 gene; The aging status assessment result of the cell is output based on the aging score.

[0015] Fourthly, the present invention provides an in vitro assessment device for the aging status of umbilical cord mesenchymal stem cells, comprising: The receiving module is used to receive relative expression level data of target genes in the umbilical cord mesenchymal stem cell sample to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; The calculation module is used to calculate an aging score based on the relative expression level data; The output module is used to output the aging status assessment result of the cell based on the aging score; wherein the aging score is calculated based on the following formula: ; Where S represents the aging 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 ADAMTSL4Represents the relative expression level of the ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

[0016] Fifthly, the present invention provides an electronic device including a memory and a processor, wherein the memory stores a computer program, and the processor executes the program to implement the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells as described in the foregoing embodiments.

[0017] In a sixth aspect, the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the in vitro assessment method for the aging state of umbilical cord mesenchymal stem cells as described in the foregoing embodiments.

[0018] In a seventh aspect, the present invention provides a quality control method for the preparation process of umbilical cord mesenchymal stem cells, comprising: Obtain lysates or nucleic acid extracts of the umbilical cord mesenchymal stem cells to be tested in vitro; The relative expression levels of target genes in the extract were determined, including BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A. An aging score is calculated based on the relative expression level; wherein the aging score is calculated based on the following formula: ; Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 Represents the relative expression level of the RRAS2 gene; Cells with an aging score below a preset threshold are classified as qualified stem cell products.

[0019] Compared with conventional techniques, this application, by employing the specific target gene as a molecular marker, can effectively address the multiple challenges currently faced in the assessment of aging in umbilical cord mesenchymal stem cells. Existing methods for assessing cellular aging often rely on a combination of multiple indicators, such as cell proliferation capacity, β-galactosidase staining, or p16 / p21 protein expression. This process is not only complex and time-consuming but also lacks standardized criteria. More importantly, these single traditional detection indicators have significant shortcomings in specificity, sensitivity, and quantitative accuracy. For example, β-galactosidase staining results are easily influenced by subjective judgment, while the expression of proteins such as p16 / p21 is unstable and transient, making it difficult to sensitively reflect the aging process.

[0020] This newly discovered set of target genes provides a more precise and reliable molecular basis for aging assessment. The expression levels of these specific genes show a stable and significant correlation with the aging degree of umbilical cord mesenchymal stem cells. Therefore, by detecting the expression levels of these genes, cellular aging trends can be identified earlier and more sensitively. Furthermore, since the detection of gene expression levels is based on molecular biology techniques such as reverse transcription polymerase chain reaction (RT-PCR), the results are objective numerical values, fundamentally overcoming the drawbacks of semi-quantitative or subjective judgment in traditional methods. This makes the assessment results not only accurate but also highly reproducible and comparable, laying the foundation for establishing a standardized quality control system.

[0021] The target genes were not obtained through empirical screening or single literature reports, but rather based on transcriptome data from different generations of umbilical cord mesenchymal stem cells, combined with differential expression analysis, functional pathway enrichment analysis, and expression trend consistency screening, among other criteria. The target genes identified through this screening pathway exhibited stable expression changes across different cell sources and aging models, thus ensuring their reliability and reproducibility as aging biomarkers.

[0022] Therefore, the establishment of this technical approach makes it possible to rapidly and accurately screen the quality of umbilical cord mesenchymal stem cells used in clinical practice. By accurately assessing the aging status of cell batches, it is possible to screen out cell populations with greater potential and superior function for treatment, effectively avoiding poor treatment effects or potential safety risks caused by the use of senescent cells. Ultimately, this significantly improves the overall quality, efficacy, and safety of cell therapy products, meeting the urgent needs of clinical applications. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells in Embodiment 4 of this application. Figure 2 This is a schematic diagram of the functional modules of the in vitro assessment device for the aging status of umbilical cord mesenchymal stem cells in an embodiment of this application; Figure 3 The results of the detection of doubling time of umbilical cord mesenchymal stem cells at different passages in Experiment Example 1 of this application are shown in the figure. Figure 4 The results of detecting the ratio of SA-β-gal (β-galactosidase) positive / senescent cells in different generations of umbilical cord mesenchymal stem cells in Experiment Example 1 of this application; Figure 5 The qRT-PCR results of the relative expression levels of the p21 gene in different passages of umbilical cord mesenchymal stem cells in Experiment Example 1 of this application (UCMSCs-1 and UCMSCs-2). Figure 6 The qRT-PCR results of the relative expression levels of the p16 gene in different passages of umbilical cord mesenchymal stem cells in Experiment Example 1 of this application (UCMSCs-1 and UCMSCs-2). Figure 7 The image shows the qRT-PCR verification results of the candidate aging marker gene in Experiment Example 3 of this application at different passages of umbilical cord mesenchymal stem cells UCMSCs-1, UCMSCs-2 and UCMSCs-3 (BMP2 gene). Figure 8 This image shows the qRT-PCR validation results of the candidate aging marker gene in Experiment Example 3 of this application at different passages of umbilical cord mesenchymal stem cells UCMSCs-1, UCMSCs-2 and UCMSCs-3 (GADD45A gene). Figure 9 The image shows the qRT-PCR verification results of the candidate aging marker gene in Experiment Example 3 of this application at different passages of umbilical cord mesenchymal stem cells UCMSCs-1, UCMSCs-2 and UCMSCs-3 (ADAMTSL4 gene). Figure 10This image shows the qRT-PCR verification results of the candidate aging marker gene in Experiment Example 3 of this application at different passages of umbilical cord mesenchymal stem cells UCMSCs-1, UCMSCs-2 and UCMSCs-3 (RRAS2 gene). Figure 11 The image shows the qRT-PCR verification results of the candidate aging marker gene in Experiment Example 3 of this application at different passages of umbilical cord mesenchymal stem cells UCMSCs-1, UCMSCs-2 and UCMSCs-3 (IFIT2 gene). Figure 12 This is the ROC curve plot of the correlation between gene markers and aging of umbilical cord mesenchymal stem cells in Experiment Example 4 of this application. Figure 13 This is a graph showing the results of gene expression detection in the hydrogen peroxide-induced umbilical cord mesenchymal stem cell aging model in Experiment Example 5 of this application (BMP2 gene). Figure 14 This is a graph showing the results of gene expression detection in the hydrogen peroxide-induced umbilical cord mesenchymal stem cell aging model in Experiment Example 5 of this application (ADAMTSL4 gene). Figure 15 This is a graph showing the results of gene expression detection in the hydrogen peroxide-induced umbilical cord mesenchymal stem cell aging model in Experiment Example 5 of this application (IFIT2 gene). Figure 16 This is a graph showing the results of gene expression level detection in the hydrogen peroxide-induced umbilical cord mesenchymal stem cell aging model in Experiment Example 5 of this application (RRAS2 gene). Figure 17 This is a graph showing the results of gene expression detection in the hydrogen peroxide-induced umbilical cord mesenchymal stem cell aging model in Experiment Example 5 of this application (GADD45A). Figure 18 The experimental results (UCMSCs-1) show the relationship between BMP2 and HGF expression levels in Experiment Example 6 of this application. Figure 19 The figure shows the experimental results of the relationship between BMP2 and HGF expression levels in Experiment Example 6 of this application (UCMSCs-2). Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] Example 1 This application provides the use of a reagent for detecting the expression level of a target gene in the preparation of a detection product for assessing the aging status of umbilical cord mesenchymal stem cells, wherein the target gene includes at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

[0027] The aforementioned "reagents" refer to chemical or biological substances that can specifically identify and quantify the activity level of specific genes in molecular biology experiments. They are not general-purpose chemicals, but rather substances specifically designed to measure the "expression level" of a particular gene, that is, the degree to which the gene is activated and transcribed into messenger RNA (mRNA).

[0028] This reagent can take many specific forms, including (but not limited to): Primer pairs are required for real-time quantitative PCR (qRT-PCR) detection. These pairs of short DNA fragments (primers) specifically bind to and amplify the reverse transcription product of the target gene's mRNA. Therefore, primer pairs designed for specific genes (such as BMP2) described below represent a concrete implementation of this reagent.

[0029] In techniques such as qRT-PCR or in situ hybridization, a probe is a fragment of DNA or RNA labeled with fluorescence or radioactivity that specifically binds to the target gene sequence. The intensity of the signal emitted by the probe is proportional to the amount of the target gene, thus achieving precise quantification.

[0030] Gene chips: This is a more advanced form of reagent, with thousands of different probes immobilized on a single chip, which can simultaneously detect the expression levels of multiple or even all target genes.

[0031] This embodiment provides the "purpose" and "application direction" of the above-mentioned reagents. The value of these reagents lies not in themselves, but in their role as key components in the manufacture of a specific "detection product." This final "detection product" is used to determine the degree of aging of umbilical cord mesenchymal stem cells (UCMSCs).

[0032] Compared to traditional methods such as observing cell morphology and detecting proliferation rate, this gene expression level-based detection product has core advantages: the detection results are specific numerical values, eliminating subjective errors from human observation and making the assessment results highly reproducible; changes in gene expression levels often occur before significant changes in cell morphology and function occur, thus enabling early warning of aging; and because the results are quantitative, it is easy to establish unified standards for "qualified" or "aged," facilitating industry supervision and comparison of results between different institutions.

[0033] In this embodiment, the specific objects to be measured as "molecular scales" are one or more of five specific genes (BMP2, ADAMTSL4, IFIT2, RRAS2, GADD45A). This means that the study found that the expression levels of these five genes are directly and stably associated with the aging state of UCMSCs, acting as "messengers" or "indicators" of aging, and as biomarkers.

[0034] Specifically, this can be achieved by detecting only one gene. For example, BMP2 can be chosen as the core indicator. The prepared detection product (such as a qRT-PCR kit) contains primers and probes that specifically amplify the BMP2 gene. For example, if a laboratory receives a batch of UCMSCs to be tested, it can extract their total RNA and use this product for detection. If the relative expression level of BMP2 is found to be very high (e.g., more than 10 times that of fresh, low-passage cells), it can be determined that these cells have entered a severe senescent state and are not recommended for clinical treatment.

[0035] While testing only one gene is feasible, testing multiple genes together can provide more comprehensive and robust assessment results, reducing the random bias that may be caused by a single indicator.

[0036] In summary, this embodiment provides an innovative technical approach by employing at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A as molecular markers for assessing the senescence status of umbilical cord mesenchymal stem cells. This approach leverages the stable and significant correlation between the expression levels of these specific genes and the degree of cellular senescence, overcoming numerous shortcomings of traditional detection methods (such as β-galactosidase staining, p16 / p21 protein detection, etc.), including complex procedures, low specificity, insufficient sensitivity, and difficulty in precise quantification. Since the detection of gene expression levels can produce objective and reproducible numerical results, this approach enables a more sensitive, accurate, and standardized assessment of cellular senescence status, thereby effectively screening high-quality, low-senescence cells for clinical use and significantly improving the quality control, efficacy, and safety of cell therapy products.

[0037] Example 2 Based on the foregoing embodiments, this application provides an example of a use whereby the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

[0038] In this embodiment, the final evaluation result is defined as being based on comprehensive information from a “gene combination” or “gene panel” consisting of these five genes, rather than relying on the performance of a single gene.

[0039] It's important to note that biological systems are complex, and the expression of individual genes can sometimes fluctuate due to random factors. By simultaneously detecting five aging-related genes, the risks associated with such individual differences or random fluctuations can be effectively mitigated. If all five indicators consistently point to aging, the accuracy and reliability of the assessment will be far greater than relying on only one indicator. This is similar to a doctor diagnosing a disease; a comprehensive assessment of multiple laboratory indicators (such as complete blood count, C-reactive protein, and imaging) is more reliable than relying solely on body temperature. These five genes may be associated with different aspects or pathways of the aging process. A comprehensive evaluation of them provides a more complete and multi-dimensional picture of the cellular aging state.

[0040] Furthermore, the detection product assesses the aging state by calculating an aging score; the aging score is calculated based on the following formula (Formula 1): ; Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

[0041] This embodiment provides a specific algorithm for converting the detection data of five genes into a single, quantified score. It no longer simply checks whether each gene is "high" or "low," but rather integrates them into a final result called an "aging score" using the aforementioned mathematical formula.

[0042] Formula 1 simplifies complex biological information into a single, intuitive number, achieving a leap from qualitative / semi-quantitative to precise quantification. Theoretically, any laboratory using the same method and formula to test the same sample will obtain the same aging score, completely eliminating subjective judgment and making it possible to establish industry standards.

[0043] For example, technicians use a qRT-PCR kit containing primers and probes for the five genes mentioned above to detect the relative expression levels (E values) of these five genes in UCMSCs samples. Assume a set of data is obtained: E... BMP2 =2.0,E IFIT2 =3.0,E GADD45A =1.5,E ADAMTSL4 =2.2,E RRAS2 =1.8. Therefore, using Formula 1, we have: S = (1.572 × 2.0) + (2.477 × 3.0) + (1.118 × 1.5) + (1.355 × 2.2) + (0.642 × 1.8) ≈ 3.144 + 7.431 + 1.677 + 2.981 + 1.156 = 16.389. Therefore, we can conclude that the aging score of this sample is 16.389.

[0044] In some implementations, a higher aging score indicates a more severe degree of aging of the umbilical cord mesenchymal stem cells.

[0045] This provides a clear "directional" interpretation of the aging score. It tells users that the number is not random, but rather "positively correlated" with the degree of cellular aging. The higher the score, the "older" the cells, and the lower their biological activity and therapeutic potential may be, making the test results easily understandable even to non-professionals. A score of "16.4" is clearly worse than a score of "3.2".

[0046] In some implementations, the detection product is used to screen umbilical cord mesenchymal stem cells with aging scores below a preset threshold.

[0047] In this embodiment, the provided "preset threshold" is a key concept, serving as the dividing line between "acceptable" and "unacceptable." This threshold needs to be determined through extensive preclinical or clinical studies to ensure that cells below this score have ideal therapeutic effects and safety.

[0048] In the application presented in this embodiment, a complex biological assessment problem is transformed into a simple "yes / no" decision, greatly improving production efficiency and the objectivity of quality control. By establishing such a clear checkpoint, it is possible to systematically prevent functionally degraded or potentially harmful senescent cells from entering clinical applications, thereby ensuring the safety and efficacy of the final cell therapy product.

[0049] In some embodiments, the target gene includes BMP2; the elevated expression level of BMP2 is used to indicate a decrease in hepatocyte growth factor expression and / or a weakened anti-fibrotic ability of the umbilical cord mesenchymal stem cells.

[0050] In this embodiment, a molecular marker (high expression of BMP2) is directly linked to a key cellular "therapeutic function" (anti-fibrotic ability). Hepatocyte growth factor (HGF) is an important secretory factor for UCMSCs to exert their anti-fibrotic and other repair functions. This means that BMP2 is not only a "marker" of aging, but also an "early warning signal" of cellular function loss. It transcends the description of "aging" as a state and directly addresses the core issue of "effectiveness" in clinical treatment.

[0051] In some embodiments, the reagent includes at least one of primer pairs for specifically amplifying the target gene mRNA, a probe for specific hybridization, and a detection chip.

[0052] This embodiment provides the physical items constituting the "detection reagent". Primer pairs and probes are key components required for implementing qRT-PCR technology. The detection chip is a core component required for implementing gene chip technology. This demonstrates that this application can be achieved using currently mature and standardized molecular biology techniques (such as qRT-PCR), and is highly feasible.

[0053] In some embodiments, the detection product is a kit for quality monitoring during the in vitro expansion culture of umbilical cord mesenchymal stem cells.

[0054] The aforementioned kit contains all necessary reagents (such as primers, probes, standards, reaction buffers, etc.) and (optionally) a detailed instruction manual. This kit is used during the "in vitro amplification and culture process," falling under "process control" or "quality control" (QC) before final release. The kit format significantly lowers the barrier to entry, enabling any laboratory with basic molecular biology skills to easily and repeatedly perform the assay, facilitating the promotion and commercialization of the technology.

[0055] Example 3 This application provides a kit for assessing the aging status of umbilical cord mesenchymal stem cells, comprising reagents for detecting the expression level of at least one target gene in a gene combination including BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; the reagents include at least one of primer pairs for specifically amplifying the target gene, probes for specific hybridization, or detection chips coated with specific antibodies.

[0056] The aforementioned "kit" is a pre-packaged set of components designed to perform a specific experimental task. Its function is to "assess the aging status of umbilical cord mesenchymal stem cells." The core contents of the kit are "reagents." These reagents are used to detect the "expression level" of one or more specific target genes (selected from a list of five genes), i.e., the activity level of the genes.

[0057] The primer pairs described above can be one or more sets of two short, synthetically produced single-stranded DNA molecules. Their sequences are carefully designed to bind, and only to, the two ends of the DNA strand reverse transcribed from the target gene (such as BMP2) mRNA. In the PCR reaction, they act as "start markers," guiding DNA polymerase to begin replication from these points, thereby achieving the amplification of the target gene fragment in large quantities.

[0058] The probe used for specific hybridization described above is a short DNA strand that can also specifically bind to the target gene sequence, but it additionally carries a "signaling device" (such as a fluorescent group). When it successfully binds to the target gene, it emits a signal that can be detected by instruments.

[0059] The aforementioned detection chip coated with specific antibodies represents a more integrated technology. It refers to the immobilization of antibodies (antigens) capable of specifically recognizing and capturing proteins (antigens) expressed by a target gene on a solid-phase carrier (chip). By detecting antibody-protein binding events, the gene expression level can be indirectly reflected.

[0060] The core principle behind this kit is molecular-specific recognition. Primers, probes, and antibodies are all designed according to strict molecular complementary pairing principles (A to T, G to C; or antigen-antibody binding), ensuring that in the complex cellular environment, they can precisely find and bind to their unique target molecule, like a cruise missile, ignoring all other irrelevant molecules. By detecting the intensity of this binding event (e.g., the intensity of the fluorescence signal), the expression level of the target gene can be quantitatively reflected.

[0061] This kit pre-packages all necessary, optimized components into a single box, greatly simplifying the experimental process. Users do not need to design, synthesize, or optimize various reagents themselves; it is ready to use right out of the box, ensuring ease of use and consistency of results among different operators. Because the reagents are "tailor-made" for specific target genes, it ensures that the test results only reflect the true situation of the target gene, avoiding false positives and false negatives, thus guaranteeing the accuracy of the assessment. By limiting testing to "at least one," the product line can be highly flexible. A "basic" kit that detects only core genes (such as BMP2) is available, as well as an "advanced" kit that detects all five genes, meeting the needs of different users.

[0062] In some implementations, the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

[0063] In this embodiment, five genes are detected simultaneously, which is equivalent to observing the aging state of cells from five different dimensions. These five dimensions corroborate each other, forming a more comprehensive "aging characteristic spectrum," whose reliability far surpasses that of any single dimension.

[0064] In some embodiments, the kit also includes instructions for use; the instructions instruct the user to calculate an aging score based on the detected target gene expression levels; Furthermore, the instruction manual guides users to calculate an aging score based on the following formula; ; Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

[0065] The weighting coefficients in the formula (1.572, 2.477, etc.) reflect the different contributions of different genes to aging. This formula achieves intelligent and differentiated integration of multidimensional data by assigning high weights to genes with high contributions (such as IFIT2) and low weights to genes with low contributions (such as RRAS2). This ensures that the final "aging score" can most accurately reflect the overall degree of cellular aging.

[0066] Example 4 refer to Figure 1 This application provides an in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells, comprising: Step S1: Receive the relative expression level data of target genes from the umbilical cord mesenchymal stem cell sample to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

[0067] This step is the starting point of the entire method, namely the data input stage. It means that when this method begins to work, a specific set of raw data needs to be obtained. This set of data is not arbitrary; it must come from the "umbilical cord mesenchymal stem cell sample to be tested".

[0068] The test provides the "relative expression levels" of five specific target genes (BMP2, ADAMTSL4, IFIT2, RRAS2, GADD45A). The "relative expression level" is a standardized value obtained through experimental techniques such as qRT-PCR, which reflects the fold relationship between the activity of the target gene in the test cells and a reference gene (such as an internal control gene).

[0069] The aforementioned "received" data can be achieved in several ways: it can be manually entered, meaning the operator manually inputs the E values ​​of the five genes obtained after analysis by the qRT-PCR instrument into a software interface; it can also be imported from a file or directly connected to the instrument. The fundamental principle of this step is that these five specific genes have been proven to be effective biomarkers of aging in umbilical cord mesenchymal stem cells. Therefore, their expression level data is the basis for all subsequent analyses and judgments, and is the "raw material" of the entire evaluation method.

[0070] This step clearly separates the biological experiments (wet experiments) from the data analysis (dry experiments). Regardless of the brand of qRT-PCR instrument used at the front end, as long as the expression data of these five genes can be provided in the correct format, the subsequent analysis process is completely consistent, which greatly promotes the standardization of evaluation methods.

[0071] Step S2: Calculate the aging score based on the relative expression level data.

[0072] This step is the core of the method, namely the data processing and computation stage. It receives the five discrete gene expression data from the previous step and integrates and calculates them into a single, comprehensive indicator, the "aging score," through a pre-defined mathematical model. This step performs a purely mathematical operation. Its core algorithm is defined by Equation 1 above.

[0073] The mechanism of this step is to integrate and optimize information through multivariate weighted summation. The expression of a single gene may fluctuate occasionally, but by weighting and combining the information of five related genes according to their importance, the "noise" of individual indicators can be effectively smoothed out, resulting in a more robust and reliable comprehensive score that better reflects the true aging state of the cells as a whole.

[0074] The calculation method transforms multiple ambiguous biological indicators into a precise and objective mathematical score, eliminating any subjective judgment. The weighted algorithm is not a simple summation; it reflects a deep understanding of the molecular mechanisms of aging, making the scoring results more scientific and predictive.

[0075] For example, after receiving the five E values ​​from the previous step, this method performs the calculation: S = (1.572 × 2.0) + (2.477 × 3.0) + (1.118 × 1.5) + (1.355 × 2.2) + (0.642 × 1.8) ≈ 16.389. This step completes, and the result is 16.389.

[0076] Step S3: Output the aging status assessment result of the cell based on the aging score.

[0077] This step marks the end of the method, namely the result output and interpretation stage. It transforms the purely mathematical score "S" calculated in the previous step into an "assessment result" that is meaningful and understandable to the user. This "output" can take several forms: for example, it can directly display the score, such as showing "Aging Score: 16.389" on the screen. Alternatively, it can be a qualitative conclusion, comparing the score with one or more preset thresholds and outputting simple textual conclusions, such as "Status: Severe Aging," "Grade: III," or "Recommendation: Not Clinically Applicable." It can also be a visual presentation, displaying the results graphically, such as a color bar from green to red, with the pointer moving towards the red area as the score increases. Finally, it can generate a report, automatically producing a PDF or printable report containing sample information, raw data, aging score, and final conclusions.

[0078] This method transforms an abstract number into an intuitive and actionable conclusion, directly providing a basis for users' (such as quality control personnel) decisions (e.g., whether this batch of cells is qualified). This allows users without a strong bioinformatics background to easily understand the meaning of the evaluation results.

[0079] This method can be extended to include a threshold setting module. Users or administrators can set their own scoring ranges for "qualified," "critical," and "senescent" based on different application needs (e.g., research vs. clinical) or regulatory requirements. For a long-term cultured cell batch, continuous evaluation can be performed, and each senescence score can be recorded to form a senescence trend curve. The output can not only show the current status but also display its historical change trajectory, providing data support for optimizing the culture process.

[0080] refer to Figure 2 This application also provides an in vitro assessment device for the aging status of umbilical cord mesenchymal stem cells, comprising: The receiving module 10 is used to receive the relative expression level data of target genes in the umbilical cord mesenchymal stem cell sample to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2 and GADD45A; Calculation module 20 is used to calculate an aging score based on the relative expression level data; Output module 30 is used to output the aging status assessment result of the cell based on the aging score; wherein the aging score is calculated based on formula 1.

[0081] It is understood that the device in this embodiment corresponds to the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells in the above embodiment, and the options in the above embodiment are also applicable to this embodiment, so they will not be described again here.

[0082] This application also provides an electronic device, including a memory and a processor. The memory stores a computer program, and when the processor executes the program, it implements the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells as described in the foregoing embodiments.

[0083] The processor can be an integrated circuit chip with signal processing capabilities. The processor can be a general-purpose processor, including at least one of a Central Processing Unit (CPU), Graphics Processing Unit (GPU), Network Processor (NP), Digital Signal Processor (DSP), Application-Specific Integrated Circuit (ASIC), Field-Programmable Gate Array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components. The general-purpose processor can be a microprocessor or any conventional processor, capable of implementing or executing the methods, steps, and logic block diagrams disclosed in the embodiments of this application.

[0084] The memory can be, but is not limited to, Random Access Memory (RAM), Read Only Memory (ROM), Programmable Read-Only Memory (PROM), Erasable Programmable Read-Only Memory (EPROM), Electrically Erasable Programmable Read-Only Memory (EEPROM), etc. The memory is used to store computer programs, and the processor can execute the computer programs accordingly after receiving execution instructions.

[0085] This application also provides a computer-readable storage medium storing a computer program thereon, which, when executed by a processor, implements the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells as described in the foregoing embodiments.

[0086] The computer storage medium can be a readable storage medium, a non-volatile storage medium, or a volatile storage medium. For example, the computer storage medium may include, but is not limited to, various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0087] In the several embodiments provided in this application, it should be understood that the disclosed apparatus and methods can also be implemented in other ways. The apparatus embodiments described above are merely illustrative. For example, the flowcharts and block diagrams in the accompanying drawings show the architecture, functionality, and operation of possible implementations of apparatus, methods, and computer program products according to various embodiments of this application. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of code containing one or more executable instructions for implementing a specified logical function. It should also be noted that, in alternative implementations, the functions marked in the blocks may occur in a different order than those marked in the drawings. For example, two consecutive blocks may actually be executed substantially in parallel, and they may sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each block in the block diagram and / or flowchart, and combinations of blocks in the block diagram and / or flowchart, can be implemented using a dedicated hardware-based system that performs the specified function or action, or using a combination of dedicated hardware and computer instructions.

[0088] In addition, the functional modules or units in the various embodiments of this application can be integrated together to form an independent part, or each module can exist independently, or two or more modules can be integrated to form an independent part.

[0089] If the aforementioned functions are implemented as software functional modules and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a smartphone, personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this application.

[0090] This application also provides a quality control method for the preparation process of umbilical cord mesenchymal stem cells, including: Step S100: Obtain the lysate or nucleic acid extract of the umbilical cord mesenchymal stem cells to be tested in vitro; Step S200: Determine the relative expression level of the target genes in the extract, the target genes including BMP2, ADAMTSL4, IFIT2, RRAS2 and GADD45A; Step S300: Calculate the aging score based on the relative expression level; wherein the aging score is calculated based on Formula 1.

[0091] In step S400, cells with an aging score below a preset threshold are identified as qualified stem cell products.

[0092] It should be noted that the aging assessment method and detection products provided in this embodiment are particularly suitable for quality monitoring during the in vitro expansion and culture of umbilical cord mesenchymal stem cells. By detecting the expression of target genes in cells at different passage stages and under different aging induction conditions, and combining this with an aging scoring model, the aging trend of cells can be dynamically monitored, thereby providing early warning before significant functional decline occurs in cells and providing objective basis for batch screening and quality release in the cell preparation process.

[0093] The present invention will be further illustrated below with specific experimental examples. However, it should be understood that these experimental examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0094] Experimental Example 1: Evaluation of the aging degree of UCMSCs using existing technologies / common indicators (control system) 1. Detection of cell doubling time at different passages: In this experiment, the aging trend of UCMSCs at different generations was assessed using proliferation capacity (doubling time).

[0095] (1) Samples: Cells from three umbilical cords (UCMSCs-1, UCMSCs-2, UCMSCs-3), different passages (see figure for specific passages).

[0096] (2) Experimental method: Record the cell doubling time at different generations.

[0097] (3) Experimental results and analysis: Reference Figure 3 , representing the cell doubling time at different generations. Figure 3 In the middle, the proliferation capacity of UCMSCs-1 decreased significantly in the P23 generation; the trends of UCMSCs-2 and UCMSCs-3 were consistent, with both showing a significant decrease in proliferation capacity in the P17 generation.

[0098] Experimental results showed that the doubling time of umbilical cord mesenchymal stem cells from different umbilical cord sources differed significantly during in vitro expansion. Specifically, for example... Figure 3 As shown, the proliferation capacity of UCMSCs-1 decreased significantly at passage P23, while that of UCMSCs-2 and UCMSCs-3 decreased significantly at passage P17. This demonstrates that even umbilical cord mesenchymal stem cells of the same origin exhibit significant differences in the inflection point of their doubling time with passage passage.

[0099] Furthermore, before cells enter a significant senescence stage, the doubling time varies little between generations, making it difficult to distinguish between early and mid-stage senescence. Therefore, relying solely on doubling time as an indicator of senescence assessment not only has low sensitivity and lacks the ability to predict early cellular senescence, but is also easily affected by differences in cell source, culture conditions, and experimental procedures, making it difficult to establish a unified and stable standard for senescence evaluation.

[0100] 2. SA-β-gal (β-galactosidase) staining to detect the proportion of senescent cells: In this experiment, the degree of aging was assessed by the positive rate of SA-β-gal.

[0101] (1) Samples: UCMSCs-1 and UCMSCs-2 from different generations.

[0102] (2) Experimental method: β-gal staining, and statistical analysis of the proportion of β-gal positive cells.

[0103] (3) Experimental results and analysis: Reference Figure 4 The proportion of senescent cells at different generations (β-galactosidase expression / staining level). Figure 4 In the study, UCMSCs-1 showed a significant increase in the proportion of β-gal positivity at passage P20; UCMSCs-2 showed a significant increase in the proportion of β-gal positivity at passage P15.

[0104] Experimental results show that, Figure 4 As shown, the proportion of SA-β-gal positive cells in UCMSCs-1 increased significantly at passage P20, while that in UCMSCs-2 increased significantly at passage P15. The differences in the passages at which the proportion of SA-β-gal positivity increased significantly among cells from different origins suggest that this indicator has some inconsistency in reflecting the cellular senescence process.

[0105] Meanwhile, in some intermediate passages, SA-β-gal staining results showed weak positivity or uneven distribution, with unclear boundaries between positive and negative cells, introducing a degree of subjectivity into the interpretation of results. Furthermore, SA-β-gal staining is a semi-quantitative detection method, making it difficult to finely distinguish between different degrees of aging and carrying the risk of false positives. Therefore, this method usually needs to be used in conjunction with other molecular markers or detection methods; using it alone as an indicator of aging has certain limitations.

[0106] 3. qRT-PCR detection of the recognized aging genes p21 / p16 (sensitivity control): This experiment aims to evaluate the ability of traditional molecular markers (p21, p16) to distinguish different generations of aging.

[0107] (1) Samples: UCMSCs-1 and UCMSCs-2, different generations.

[0108] (2) Experimental method: In vitro qRT-PCR was used to detect the relative expression levels of p21 and p16.

[0109] (3) Experimental results and analysis: Reference Figures 5-6 The figures show the expression results of p21 and p16 at different passages. Specifically, for UCMSCs-1: p21 was low from P1 to P5; p21 increased after P10, but the aging difference between P10 and P20 could not be significantly distinguished; p16 significantly increased at P25. For UCMSCs-2: p21 was low from P1 to P5 (lowest at P1, no significant difference between P3 and P5); p21 increased after P10; p16 increased with each passage but fluctuated.

[0110] Experimental results show that, Figures 5-6 As shown, the expression changes of p21 and p16 in different generations of umbilical cord mesenchymal stem cells exhibit certain stage-specific characteristics. Specifically, in UCMSCs-1, p21 remained at a low level from generation P1 to P5, and increased after P10, but its expression level did not show a significant difference between generation P10 and P20, making it difficult to reflect the further deepening of cell senescence; p16, on the other hand, showed a significant increase mainly in generation P25, and its ability to distinguish between early and mid-stage senescence was limited.

[0111] In UCMSCs-2, p21 expression remained low from generation P1 to P5, with no significant difference between P3 and P5, only showing a significant increase after generation P10. While p16 expression generally increased with generation, its changes fluctuated and lacked a stable, continuous increasing pattern. These results indicate that the expression changes of traditional aging-related genes p21 and p16 in different cell sources and generation levels are highly dynamic and fluctuating, making it difficult to finely distinguish different aging stages. Therefore, their sensitivity as single molecular markers for assessing the aging status of umbilical cord mesenchymal stem cells is limited.

[0112] Experimental Example 2: RNA-seq screening for senescence candidate genes in UCMSCs (discovery pathway) In this embodiment, gene markers that change consistently with the aging process are screened from the transcriptome of UCMSCs of different generations.

[0113] (1) Samples and groups: P1, P5, P15, P25 UCMSCs.

[0114] (2) Experimental method: 1) Transcriptome sequencing was performed using the DNBSEQ platform; differential analysis was performed between P1 and P25 (P<0.05 and |logFC|≥1); 2) GO / KEGG enrichment was performed on differentially upregulated genes from P25 relative to P1 to screen for aging-related pathways; 3) Statistical analysis of the expression trends of pathway genes at P1, P5, P15, and P25, and screening for genes whose expression increases with each generation.

[0115] (3) Experimental results and analysis: By comparing and analyzing the transcriptome sequencing results of umbilical cord mesenchymal stem cells from different generations, with P1 generation cells as the control group and P25 generation cells as the senescent group, differentially expressed genes were screened under the condition of meeting the significance threshold (P<0.05 and |logFC|≥1). Further GO and KEGG pathway enrichment analysis was performed on the differentially expressed genes upregulated in P25 generation compared to P1 generation. The results showed that most differentially expressed genes were enriched in functional pathways related to cellular senescence, stress response, cell cycle regulation, and extracellular matrix remodeling.

[0116] Based on this, a comprehensive analysis was conducted on the expression trends of candidate genes in the aforementioned aging-related pathways at each generation (P1, P5, P15, and P25). Genes exhibiting a continuous upregulation trend during cell passage and whose expression changes were consistent with the aging process were screened out. Ultimately, GADD45A, RRAS2, IFIT2, ADAMTSL4, and BMP2 were identified as potential aging markers for umbilical cord mesenchymal stem cells, providing candidate targets for subsequent molecular-level assessment of cellular senescence.

[0117] Experiment Example 3: Screening of 5 genes in multiple umbilical cord-derived, multi-generational UCMSCs: qRT-PCR validation + establishment of an aging scoring model 1. Validation by multi-source / multi-generation qRT-PCR: In this experiment, we verified whether candidate genes selected by RNA-seq are stable and increase with aging in different umbilical cord sources and generations.

[0118] (1) Samples: UCMSCs-1, UCMSCs-2 and UCMSCs-3, three umbilical cord-derived cells, from different passages.

[0119] (2) Experimental method: The expression levels of five candidate genes were detected by in vitro qRT-PCR.

[0120] (3) Experimental results (corresponding figures): refer to Figures 7-11 This study presents the expression results of five candidate genes from UCMSCs-1 / 2 / 3 at different generations.

[0121] Experimental results show that, Figures 7-11As shown, in umbilical cord mesenchymal stem cells from three different umbilical cord sources, GADD45A, RRAS2, IFIT2, ADAMTSL4, and BMP2 all exhibited a gradually upregulated expression trend with increasing passage number during different passages. The expression changes of these genes in UCMSCs-1, UCMSCs-2, and UCMSCs-3 were generally consistent, indicating that their expression levels were not significantly correlated with cell origin and possessed good stability and reproducibility.

[0122] Compared to traditional aging biomarkers, the five candidate genes exhibited continuous and distinguishable expression changes across different generations. BMP2 showed particularly significant expression differences across generations, demonstrating high resolution for different aging stages. These results indicate that this five-gene combination can stably reflect the aging changes of umbilical cord mesenchymal stem cells during in vitro expansion at the molecular level, making it suitable as a molecular biomarker system for assessing the aging status of umbilical cord mesenchymal stem cells.

[0123] 2. Cox regression is used to establish a aging tendency score / prediction formula: In this experiment, the expression levels of 5 genes were integrated into an aging score that can be used for sample grading.

[0124] (1) Analysis method: The expression levels of five genes were assessed for aging tendency using the Cox regression model, and the aging prediction formula was obtained based on the model risk ratio.

[0125] The formula is as follows: Please refer to Formula 1 in the aforementioned implementation method for details.

[0126] (2) Analysis conclusion: Based on the stable expression characteristics of the five candidate aging biomarkers in umbilical cord mesenchymal stem cells from different sources and at different passages, a Cox regression model was further used to comprehensively analyze the relationship between gene expression levels and cellular senescence tendency. By weighted integration of the risk coefficients of each gene, an aging scoring model based on multi-gene expression levels was constructed.

[0127] Compared to single-gene indicators, this aging scoring model can comprehensively reflect changes in multiple aging-related molecular signals, thereby reducing the impact of fluctuations in the expression of individual genes on the assessment results. Experimental results show that the scoring model can be used to quantitatively assess the degree of aging in different samples and identify umbilical cord mesenchymal stem cell samples with a higher tendency to aging; the higher the score, the more severe the corresponding cellular aging.

[0128] Experiment Example 4: Screening for the correlation between genes and aging / diagnostic efficacy analysis (Logistic regression + ROC / AUC) In this embodiment, the ability of the selected genes to act as markers of aging (correlation efficacy) is evaluated.

[0129] (1) Analysis method: SPSS 27.0 was used; Logistic regression analysis was performed to analyze the association between gene expression and cell senescence; ROC curves were plotted and AUC was calculated (AUC>0.7 represents high accuracy).

[0130] (2) Analysis results: Reference Figure 12 We screened gene markers and verified the ROC curves to confirm their correlation with aging in UCMSCs.

[0131] Based on the aforementioned detection results of umbilical cord mesenchymal stem cells from multiple sources and generations, further statistical analysis was performed on the screened aging marker genes. For example... Figure 12 As shown, the correlation between target gene expression levels and aging status of umbilical cord mesenchymal stem cells was assessed by logistic regression analysis, and receiver operating characteristic (ROC) curves were plotted based on this.

[0132] The analysis results show that the selected gene has a good ability to distinguish between umbilical cord mesenchymal stem cells in different aging states, and its area under the ROC curve (AUC) reaches a high level, indicating that the determination of cell aging state based on the gene expression information has good accuracy and reliability. These results further validate the effectiveness of the selected gene as a biomarker for aging of umbilical cord mesenchymal stem cells.

[0133] Experimental Example 5: Validation of marker genes in a hydrogen peroxide-induced aging model of UCMSCs (extrapolation validation) In this experimental example, it was verified that gene 5 not only increases with "passage aging", but also increases in the "oxidative stress-induced aging model", which enhances the persuasiveness of specificity / universality.

[0134] (1) Samples and grouping: Resuscitated UCMSCs P1 cells were divided into a normal environment group and a hydrogen peroxide induced group.

[0135] (2) Experimental method: 7000 cells / cm 2 High-density inoculation with T75; Normal group: 37℃, 5% CO2 incubator; Hydrogen peroxide group: Senescence induced in a 6% hydrogen peroxide environment; cultured for 3 days ± 0.5 days; Harvest cells, lyse them to extract RNA, and then detect and screen gene expression.

[0136] (3) Experimental results: refer to Figures 13-17 The results show the gene expression levels screened in the hydrogen peroxide-induced model.

[0137] Experimental results show that, Figures 13-17 As shown, under hydrogen peroxide-induced oxidative stress, the expression levels of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A in umbilical cord mesenchymal stem cells were significantly higher than those in the control group under normal culture conditions. These genes exhibited a consistent upregulation trend in the induced aging model, indicating that their expression changes are not only related to increased cell passage number but also closely associated with oxidative stress-induced aging.

[0138] These results demonstrate that the screened aging biomarkers do not only reflect replicative aging caused by passage during in vitro expansion, but also exhibit consistent response characteristics in aging models induced by different methods, thus demonstrating good specificity and universality. The above experiments further validate the reliability of the screened genes as biomarkers for assessing aging in umbilical cord mesenchymal stem cells.

[0139] Experiment Example 6: Correlation analysis of BMP2 and HGF expression levels (functional association / application implications) This experimental case investigates the relationship between aging biomarkers (taking BMP2 as an example) and UCMSCs functional factors (HGF, a key anti-fibrotic factor), providing a basis for its use as a "quality monitoring / efficacy indicator".

[0140] (1) Experimental / Analytical Methods: Expression levels of BMP2 and HGF in UCMSCs were obtained; Spearman correlation analysis was performed using SPSS 27.0, and the correlation coefficient r was used to represent the strength; P < 0.05 was considered significant.

[0141] (2) Experimental results and analysis: Experimental results show that, Figure 18 and Figure 19 As shown, in the tested umbilical cord mesenchymal stem cell samples, cells with higher BMP2 expression levels had relatively lower hepatocyte growth factor (HGF) expression levels. Further Spearman correlation analysis was used to statistically evaluate the expression levels of BMP2 and HGF, revealing a significant negative correlation between the two, with a correlation coefficient r of [value missing]. The value was 0.738, with a p-value of 0.037, indicating statistical significance.

[0142] The above results indicate that elevated BMP2 expression levels not only reflect the aging state of umbilical cord mesenchymal stem cells (UCMSCs) but are also associated with decreased expression of their key functional factor, HGF. Therefore, BMP2, as an aging marker, can characterize the degree of cellular senescence and, to some extent, indicate changes in cellular functional status, thus providing a reference for quality monitoring and efficacy evaluation of UCMSCs during in vitro expansion and culture.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a reagent for detecting the expression level of a target gene in the preparation of a detection product for assessing the aging status of umbilical cord mesenchymal stem cells, characterized in that, The target gene includes at least one of BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A.

2. The use as described in claim 1, characterized in that, The target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; Preferably, the detection product assesses the aging state by calculating an aging score; the aging score is calculated based on the following formula: ； Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

3. The use as described in claim 2, characterized in that, A higher aging score indicates a more severe degree of aging of the umbilical cord mesenchymal stem cells; and / or, The detection product is used to screen umbilical cord mesenchymal stem cells with aging scores below a preset threshold.

4. The use as described in claim 1, characterized in that, The target gene includes BMP2; elevated BMP2 expression levels are used to indicate decreased hepatocyte growth factor expression levels and / or weakened anti-fibrotic ability of the umbilical cord mesenchymal stem cells; and / or, The reagent includes at least one of the following: primer pairs for specifically amplifying the target gene mRNA, probes for specific hybridization, and a detection chip; and / or, The testing product is a kit used for quality monitoring during the in vitro expansion and culture of umbilical cord mesenchymal stem cells.

5. A kit for assessing the aging status of umbilical cord mesenchymal stem cells, characterized in that, Includes reagents for detecting the expression level of at least one target gene in a gene combination including BMP2, ADAMTSL4, IFIT2, RRAS2 and GADD45A; The reagent includes at least one of the following: primer pairs for specifically amplifying the target gene, probes for specific hybridization, or detection chips coated with specific antibodies.

6. The reagent kit as described in claim 5, characterized in that, The target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; and / or, The kit also includes an instruction manual; the instruction manual guides the user to calculate an aging score based on the detected target gene expression levels. Preferably, the instruction manual guides the user to calculate an aging score based on the following formula; ； Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

7. An in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells, characterized in that, include: Receive relative expression level data of target genes from umbilical cord mesenchymal stem cell samples to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; An aging score is calculated based on the relative expression level data; wherein the aging score is calculated based on the following formula: ； Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 Represents the relative expression level of the RRAS2 gene; The aging status assessment result of the cell is output based on the aging score.

8. An in vitro assessment device for the aging status of umbilical cord mesenchymal stem cells, characterized in that, include: The receiving module is used to receive relative expression level data of target genes in the umbilical cord mesenchymal stem cell sample to be tested; the target genes include BMP2, ADAMTSL4, IFIT2, RRAS2, and GADD45A; The calculation module is used to calculate an aging score based on the relative expression level data; The output module is used to output the aging status assessment result of the cell based on the aging score; wherein the aging score is calculated based on the following formula: ； Where S represents the aging 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 ADAMTSL4 gene; E RRAS2 This represents the relative expression level of the RRAS2 gene.

9. An electronic device, characterized in that, It includes a memory and a processor, wherein the memory stores a computer program, and the processor executes the program to implement the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells as described in claim 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the in vitro assessment method for the aging status of umbilical cord mesenchymal stem cells as described in claim 8.

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