A compound and method for delaying senescence of human umbilical cord mesenchymal stem cells

Abstract

The present application relates to a kind of compound and method for delaying human umbilical cord mesenchymal stem cell senescence, belong to biological medicine technical field, to solve the technical problem of delaying human umbilical cord mesenchymal stem cell senescence.A kind of compound for delaying human umbilical cord mesenchymal stem cell senescence, compound is pyrroloquinoline quineone (PQQ).By the present application, PQQ-containing culture solution is used to culture hUC-MSCs, not only can effectively improve the activity of cell, promote cell proliferation, but also can down-regulate the expression of its senescence-related gene, maintain cell stemness, and effectively delay the replicative senescence of human umbilical cord mesenchymal stem cell, significantly improve the expansion efficiency and cell activity of in vitro culture hUC-MSCs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a compound and method for delaying the aging of human umbilical cord mesenchymal stem cells. Background Technology

[0002] With the increasing aging of the population, anti-aging research has gradually become a hot topic. Aging is a multifaceted and complex process, characterized by a decline in the normal physiological functions of tissues and organs, leading to significant weakness, death, and chronic diseases such as bone, cardiovascular, and cognitive impairments. Stem cell aging is one of the main theories of aging in organisms. Human umbilical cord mesenchymal stem cells (hUC-MSCs), derived from the umbilical cord of newborns, possess characteristics such as self-renewal, multi-lineage differentiation, and immunomodulation, and can be used to treat various diseases and injuries. hUC-MSCs can replace damaged cells by relying on their potential for self-renewal and multi-lineage differentiation. Through paracrine effects and cell-cell interactions, they regulate immune and inflammatory responses, reduce oxidative stress and DNA damage, and improve mitochondrial function, thereby delaying the aging process. Currently, several clinical trials have confirmed the efficacy and safety of human umbilical cord mesenchymal stem cells in anti-aging, such as in the treatment of age-related diseases like wrinkles, tendon injuries, premature ovarian failure, and osteoporosis.

[0003] However, the therapeutic effects of these hUC-MSCs are currently not ideal, hindering their widespread clinical application. This is because the in vitro expansion of hUC-MSCs is limited, they possess immunogenicity and cellular heterogeneity, and existing in vitro culture methods significantly reduce their self-renewal capacity. Continuous in vitro expansion easily leads to replicative senescence, affecting their transplantation and clinical application. Furthermore, the senescence of hUC-MSCs limits the speed and quantity of large-scale expansion, thus limiting the feasibility of large-scale commercial application. Therefore, how to delay the replicative senescence of hUC-MSCs and obtain a sufficient quantity and quality of hUC-MSCs has become an urgent technical problem to be solved. Summary of the Invention

[0004] To address the above-mentioned technical problems, this invention provides a compound and method for delaying the aging of human umbilical cord mesenchymal stem cells, thereby solving the technical problem of how to delay the aging of human umbilical cord mesenchymal stem cells.

[0005] The objective of this invention is mainly achieved through the following technical solutions: On the one hand, the present invention provides a compound that delays the aging of human umbilical cord mesenchymal stem cells, the compound being pyrroloquinoline quinone.

[0006] On the other hand, the present invention also provides a method for delaying the aging of human umbilical cord mesenchymal stem cells. The method involves culturing primary human umbilical cord mesenchymal stem cells in a complete culture medium, and adding pyrroloquinoline quinone for treatment when the cell growth density reaches 60-70% during continuous passage.

[0007] Furthermore, the amount of pyrroloquinoline quinone added was 10-100 μmol / L in the complete culture medium, and the treatment time was 24-72 h.

[0008] Furthermore, the amount of pyrroloquinoline quinone added was 70 μmol / L in the complete culture medium, and the treatment time was 48 h.

[0009] Furthermore, the method was able to upregulate the expression of the stem genes Klf4 and c-Myc in human umbilical cord mesenchymal stem cells from generations P3 to P6.

[0010] Furthermore, the method can upregulate the expression of stem genes Oct4, Sox2, Klf4 and c-Myc in P10 generation human umbilical cord mesenchymal stem cells, and upregulate the protein expression of stem genes Oct4 and Sox2.

[0011] Furthermore, the method can downregulate the expression of aging-related genes p53 and p21 in P10 generation human umbilical cord mesenchymal stem cells, and downregulate the protein expression of aging-related genes p16 and p53.

[0012] Furthermore, the method can reduce the activity of β-galactosidase and the level of reactive oxygen species (ROS) in P10 generation human umbilical cord mesenchymal stem cells.

[0013] Furthermore, the method can reduce the proportion of cell cycle phase G0 / G1 in P10 generation human umbilical cord mesenchymal stem cells, while increasing the proportion of cell cycle phase G2 / M and S.

[0014] Furthermore, the method can increase the relative telomere length of P10 generation human umbilical cord mesenchymal stem cells and reduce telomere loss.

[0015] Compared with the prior art, the present invention can achieve at least one of the following technical effects: (1) The present invention uses culture medium containing PQQ to culture hUC-MSCs, which can not only effectively enhance cell activity and promote cell proliferation, but also downregulate the expression of aging-related genes, maintain cell stemness, and thus effectively delay the replicative aging of human umbilical cord mesenchymal stem cells, significantly improving the expansion efficiency and cell activity of in vitro cultured hUC-MSCs.

[0016] (2) By applying PQQ, this invention improves the antioxidant capacity of hUC-MSCs under culture conditions, reduces the accumulation of reactive oxygen species (ROS) in senescent hUC-MSCs, promotes cell growth and proliferation and cell cycle progression, reduces telomere loss, and better reverses the expression of β-galactosidase, a marker of senescence in hUC-MSCs.

[0017] (3) By applying PQQ, this invention reduces the expression levels of aging-related factors p53 and p21 in hUC-MSCs and increases the expression levels of stem genes Oct4, Sox2, Klf4 and c-Myc in hUC-MSCs, thereby ultimately alleviating the aging process of hUC-MSCs.

[0018] (4) By applying PQQ, this invention increases the expression levels of Oct4 and Sox2 proteins in hUC-MSCs stem cells and reduces the expression levels of aging-related genes p16 and p53 proteins, thereby alleviating the aging of hUC-MSCs.

[0019] (5) This invention provides an effective method for delaying the aging of hUC-MSCs and has the potential for wide application in tissue engineering and regenerative medicine. Attached Figure Description

[0020] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0021] Figure 1 This is a diagram showing the morphological changes of the P2 and P10 generation hUC-MSCs of this invention over time. Figure 2 This is a growth curve diagram of the inventor's P2 and P10 generation hUC-MSCs; Figure 3 To examine the survival rate of generation P10 hUC-MSCs under different concentrations of PQQ treatment in Example 1; Figure 4 The graph shows the expression data of the Klf4 gene in hUC-MSCs from generations P3 to P6 in Example 2, in the control group and the PQQ-treated group. Figure 5 This is a graph showing the expression data of the c-Myc gene in generation P3 to P6 hUC-MSCs of Example 2 control group and PQQ-treated group. Figure 6 Microscopic images and data analysis diagrams of β-galactosidase detection in hUC-MSCs of the young group, control group and PQQ-treated group in Example 3 were obtained. Figure 7To examine the cell cycle distribution and data analysis of hUC-MSCs in the young group, control group and PQQ-treated group in Example 4; Figure 8 This is a data analysis graph showing the relative average intensity of ROS fluorescence in hUC-MSCs from the young group, control group, and PQQ-treated group in Example 5. Figure 9 To examine the relative telomere length data of hUC-MSCs in the young group, control group and PQQ-treated group in Case 6.

[0022] Figure 10 To examine the expression data analysis of some genes of hUC-MSCs in the young group, control group and PQQ-treated group of Case 7; Figure 11 To examine the partial gene and protein expression maps and data analysis diagrams of hUC-MSCs in the young group, control group and PQQ-treated group of Case 8. Detailed Implementation

[0023] The following detailed description of a compound and method for delaying the aging of human umbilical cord mesenchymal stem cells, with reference to specific embodiments, is provided. These embodiments are for comparative and illustrative purposes only, and the present invention is not limited to these embodiments.

[0024] Pyrroloquinoline quineone (PQQ) is a compound with potential anti-aging properties that has attracted widespread attention in the scientific community. It has been found to possess potent antioxidant activity, capable of scavenging free radicals and protecting cells from oxidative damage. It is beneficial to cellular energy metabolism and mitochondrial function, contributing to improved cellular vitality and anti-aging capabilities. PQQ has also been found to promote neuroprotection and nerve regeneration, which is significant for preventing neurological aging and neurodegenerative diseases. However, there is still considerable room for development in the study of PQQ's mechanisms of action, effects, and quantitative aspects on specific cell types. The specific effects of PQQ on human umbilical cord mesenchymal stem cells remain unexplored.

[0025] In view of this, the inventors, through research, proposed the application of pyrroloquinoline quinone to delay the aging of human umbilical cord mesenchymal stem cells. Specifically, the pyrroloquinoline quinone, namely PQQ, has the molecular formula [missing information]. .

[0026] It should be noted that the inventors' research revealed that human umbilical cord mesenchymal stem cells (hUC-MSCs) undergo a series of unique morphological and biological functional changes during continuous in vitro culture with increasing passage number. hUC-MSCs from passages P2 to P4 exhibit normal cell morphology, while cells from passage P5 onwards enter a senescent phase. Passage P9 cells show obvious replicative senescence characteristics, accompanied by a loss of cell proliferation capacity. Figure 1 The diagram shows the morphological changes of P2 and P10 generation hUC-MSCs over time. It can be seen that the P10 generation cells are flattened, enlarged in size, have increased cell matrix, and contain more granules or vacuoles. Figure 2 The growth curves of P2 and P10 generation hUC-MSCs of this invention are shown in the figure. It can be seen that the proliferation rate of P10 generation cells is significantly reduced from day 3 to day 6.

[0027] In the specific application of PQQ, this invention proposes a method to delay the aging of human umbilical cord mesenchymal stem cells: primary human umbilical cord mesenchymal stem cells are cultured in a complete culture medium, and during continuous passage, when the cell growth density reaches 60-70%, pyrroloquinoline quinone, i.e., PQQ, is added for treatment.

[0028] Specifically, the hUC-MSCs primary cells were commercially available products. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium.

[0029] The cultivation conditions include: at 2×10 4 live cells / cm 2 Cells were seeded at a density of 60% to 70%, cultured in a complete culture medium, and placed in a 37°C incubator with 5% CO2. Once the cells adhered to the incubator and were in good growth condition, and the cell density reached 60% to 70%, they were treated with PQQ.

[0030] It should be noted that hUC-MSCs that have undergone continuous in vitro expansion to generation P10 and beyond exhibit a series of obvious aging characteristics. Therefore, the inventors first conducted preliminary exploratory experiments by treating generation P10 hUC-MSCs with PQQ, as shown in Test Example 1. Through comparison of different PQQ concentrations and treatment times, it was found that the effective amount of PQQ added was 10~100 μmol / L in complete culture medium, with a treatment time of 24~72 h, and the preferred concentration was 70 μmol / L with a treatment time of 48 h.

[0031] Furthermore, to verify that PQQ also plays a role in maintaining stemness in young hUC-MSCs, the inventors, based on the aforementioned effective PQQ dosage, administered PQQ to each generation of hUC-MSCs from P3 to P6 (see Test Case 2). The results showed that PQQ intervention significantly increased the expression levels of stemness genes Klf4 and c-Myc in hUC-MSCs from P3 to P6. Notably, compared with the control group of hUC-MSCs of the same generation, PQQ increased Klf4 gene expression in P4 to P6 generation hUC-MSCs by approximately 1.6 to 9.0 times, while PQQ increased c-MYC gene expression in P3 to P5 generation hUC-MSCs by approximately 1.8 to 3.2 times.

[0032] This invention, through the application of PQQ, enhances the antioxidant capacity of hUC-MSCs under culture conditions, reduces the accumulation of reactive oxygen species (ROS) in senescent hUC-MSCs, promotes cell growth and proliferation and cell cycle progression, reduces telomere loss, and better reverses the expression of β-galactosidase, a marker of senescence in hUC-MSCs. Simultaneously, PQQ reduces the expression levels of senescence-related factors p53 and p21, decreases the protein expression levels of senescence-related genes p16 and p53, and increases the expression levels of stem cell genes Oct4, Sox2, Klf4, and c-Myc in hUC-MSCs, upregulating the expression of stem cell genes Oct4 and Sox2, thereby ultimately alleviating the senescence process of hUC-MSCs.

[0033] Test Example 1 The cell viability of P10 generation hUC-MSCs was detected by CCK-8 assay.

[0034] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged to the P10 generation for detection of CCK-8 cell proliferation toxicity assay.

[0035] Phase 1: P10 generation hUC-MSC cells were introduced at a rate of 5 × 10⁶ cells / year. 3Cells were seeded per well in 96-well plates, with 100 μL of complete culture medium added to each well. The plates were then incubated at 37 °C with 5% CO2. Blank, control, and PQQ-treated groups were established. In the PQQ-treated groups, after cell adhesion and when cell growth was good and the cell density reached 60%–70%, cells were treated with 10 μmol / L, 35 μmol / L, 70 μmol / L, 100 μmol / L, and 150 μmol / L PQQ for 24 h, respectively. Each PQQ concentration group, control group, and blank group had six replicates. The complete culture medium was then replaced with medium containing 10 μL of CCK-8 reagent, and the plates were incubated for 2.5 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated for each group. Cell viability (%) = (Experimental group A450 nm - Blank group A450 nm) / (Control group A450 nm - Blank group A450 nm) × 100%.

[0036] Phase 2: The same experiments were conducted in the blank group, control group, and PQQ-treated group. Compared with Phase 1, the only difference was that the PQQ treatment time was changed to 48 hours.

[0037] Phase 3: The same experiments were conducted in the blank group, control group, and PQQ-treated group. Compared with Phase 1, the only difference was that the PQQ treatment time was changed to 72 hours.

[0038] Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s represents the within-group mean, and s represents the within-group standard deviation. One-way ANOVA was used for comparisons between groups within each stage. A p-value < 0.05 was considered statistically significant. Cell viability statistics are shown in Table 1 and [Table data missing]. Figure 3 .

[0039] Table 1. hUC-MSC cell survival rates under different PQQ concentrations and treatment times From Table 1 and Figure 3 As can be seen, the cell viability after treatment with 10 μmol / L, 70 μmol / L, and 100 μmol / L PQQ for 24–72 h was higher than that of the control group; the cell viability after treatment with 35 μmol / L PQQ for 24 h and 72 h was higher than that of the control group, while the cell viability after treatment with 35 μmol / L PQQ for 48 h was slightly lower than that of the control group; the cell viability after treatment with 150 μmol / L PQQ for 24 h was higher than that of the control group, while the cell viability after treatment for 48 h and 72 h was significantly lower than that of the control group. From the above data, the following general trends can be observed: (1) When the concentration of PQQ is below 10~70 μmol / L, the cell survival rate increases with the increase of treatment time within the range of 24~72h; (2) When the concentration of PQQ is 70 μmol / L, the cell survival rate increases with the increase of treatment time in the range of 24 to 48 h, but in the range of 48 to 72 h, a reversal occurs, and the cell survival rate decreases with the increase of treatment time. (3) When the concentration of PQQ exceeds 70 μmol / L, such as 100 μmol / L or 150 μmol / L, the cell survival rate decreases with the increase of treatment time in the range of 24 to 72 h. In particular, the cell survival rate after treatment with 150 μmol / L PQQ for 48 h and 72 h is significantly lower than that of the control group, and is lower than that of the cell survival rate when the PQQ concentration is 70 μmol / L under the same treatment time.

[0040] Therefore, based on the above trends, it can be deduced that PQQ concentrations of 10–70 μmol / L and treatment times of 24–72 h can improve the survival rate of hUC-MSCs. Cell survival rate generally increases with increasing concentration or treatment time, reaching a peak at a PQQ concentration of 70 μmol / L and a treatment time of 48 h. PQQ concentrations of 70–100 μmol / L and treatment times of 24–72 h also improve hUC-MSC survival rate, but after the peak, cell survival rate gradually decreases with increasing concentration or treatment time. The slightly lower cell survival rate after 48 h of treatment with 35 μmol / L PQQ compared to the control group does not conform to the overall trend derived from numerous experimental data, and can be clearly attributed to experimental error.

[0041] Notably, compared with the control group, after treatment with 70 μmol / L PQQ for 24 h, 48 h, and 72 h, the cell viability of hUC-MSCs was (109.3±2.59)%, (112.4±4.27)%, and (109.5±0.47)%, respectively. The significance levels of these results, as determined by one-way ANOVA, were [significantly high]. , and Treatment with 150 μmol / L PQQ for 72 h significantly inhibited cell proliferation, reducing the survival rate of hUC-MSCs to (76.70 ± 1.129)%, with a significance level of [missing data]. Based on the above experimental results, the effective PQQ concentration range is 10~100μmol / L, and the treatment time is 24~72h, with a preferred concentration of 70μmol / L and a treatment time of 48h.

[0042] Test Example 2 The total amount of polymerase chain reaction products after cycling was determined by quantitative real-time PCR (qPCR), i.e., the expression levels of stem genes Klf4 and c-Myc in each generation of P3-P6 hUC-MSCs were detected by qPCR.

[0043] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged to P3-P6. Each generation of hUC-MSCs from P3 to P6 was then subjected to qPCR detection.

[0044] Specifically, the qPCR detection steps and process parameters for P3 generation hUC-MSCs are as follows: The P3 generation hUC-MSCs were 1.0 × 10 5 Cells were seeded per well in six-well plates, with 2.5 ml of complete culture medium added to each well. The plates were then incubated at 37 °C with 5% CO2. A control group and a PQQ-treated group were set up. In the PQQ-treated group, after cell adhesion and good cell growth, when the cell density reached 60%–70%, the cells were treated with 70 μmol / L PQQ for 48 h. Both the PQQ-treated group and the control group had three replicates.

[0045] After PQQ treatment, total RNA was extracted using a kit and reverse transcribed into cDNA. Real-time quantitative PCR was performed using a two-step method. The reaction conditions were: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 10 s, and annealing / extension at 60℃ for 30 s. β-actin was used as an internal reference gene. The relative expression level of the target gene was calculated using the method. The experiment was repeated three times, and the primer sequences are shown in Table 4.

[0046] The steps and process parameters for qPCR detection of hUC-MSCs in each of the remaining P4 to P6 generations are exactly the same as those for qPCR detection of the P3 generation hUC-MSCs.

[0047] Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s represents the within-group mean, and s represents the within-group standard deviation. One-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant. The test results are shown in Table 2 and... Figure 4 , Figure 5 .

[0048] Table 2. qPCR data on the stem genes Klf4 and c-Myc in P3–P6 generation hUC-MSCs. From Table 2 and Figure 4 , Figure 5 It is evident that PQQ treatment significantly increased the expression levels of the stem genes Klf4 and c-Myc in hUC-MSCs from generations P3 to P6. Notably, compared to the control group of the same generation of hUC-MSCs, PQQ increased Klf4 gene expression in hUC-MSCs from generations P4 to P6 by approximately 1.6 to 9.0 times. One-way ANOVA showed that the significance level between the P4 and P5 generations was [missing data]. The significance level between the P6 generation groups was [missing information]. In P3-P5 generation hUC-MSCs, c-MYC gene expression was increased by approximately 1.8-3.2-fold by PQQ. One-way ANOVA showed that the significance levels among the P3-P5 generation groups were all within the range of [missing data]. .

[0049] Test Example 3 SA-β-Gal is a commonly used biomarker of cellular senescence. SA-β-Gal (Senescence-Associated β-Galactosidase) is a β-galactosidase whose activity increases significantly during cellular replicative senescence. By detecting the activity level of SA-β-Gal in cells, the degree of cellular senescence can be assessed. Therefore, this test case compares the detection rate of SA-β-Gal positive cells in hUC-MSCs.

[0050] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged, and hUC-MSCs from generations P2 and P10 were used for β-galactosidase assay.

[0051] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶.5 Cells were seeded per well in 12-well plates, with 2 ml of complete culture medium added to each well. The plates were then incubated at 37 °C in a 5% CO2 incubator. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and good cell growth, when the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 72 h. All three groups (young cell group, control group, and PQQ-treated group) had three replicates. Subsequent experiments were performed using a β-galactosidase assay kit. The complete culture medium was replaced with β-galactosidase fixative for 15 min, followed by washing three times with PBS buffer. β-galactosidase staining working solution was prepared according to the kit instructions, with 1 ml added to each well. Cells were incubated at 37 °C, CO2-free, pH 6.0 in the β-galactosidase staining working solution for 24 h.

[0052] The number of SA-β-gal positive cells was observed in 6 random fields of view under an optical microscope. The number of positive cells was expressed as the positive cell rate. The SA-β-gal positive cell rate (%) = number of SA-β-gal positive cells / total number of cells in the same field of view × 100%.

[0053] Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s is the mean of each group and s is the standard deviation of each group. One-way ANOVA was used for comparisons between groups. A statistically significant difference was considered when the significance level was P < 0.05. Figure 6 Microscopic images and data analysis figures of β-galactosidase detection in hUC-MSCs from the young group, control group, and PQQ-treated group.

[0054] from Figure 6 Microscopic images showed that, compared with the P2 generation young group, the hUC-MSCs in the P10 generation control group had increased β-galactosidase activity and significantly deeper staining. Data analysis showed that the SA-β-gal positive cell rate in the P2 generation young group was (21.98±2.56)%, while the SA-β-gal positive cell rate in the P10 generation control group was (59.8±2.49)%, with a significant difference compared to the P2 generation young group. This indicates that the activity level of SA-β-Gal in hUC-MSCs is significantly increased, which can indeed be used to assess the degree of cellular senescence.

[0055] Compared with the control group of generation P10, the PQQ-treated group of generation P10 showed significantly reduced staining. The percentage of SA-β-gal positive cells in the PQQ-treated group of generation P10 was (39.4±1.50)%, which was significantly different from that of the control group of generation P10. The microscopic images and data analysis graphs showed that PQQ could reduce the activity of β-galactosidase in hUC-MSCs and delay the aging of hUC-MSCs.

[0056] Test Example 4 The cell cycle regulates cellular senescence, and permanent cell cycle arrest in the G1 / G0 phase is a characteristic feature of cellular senescence. This arrest is associated with intracellular DNA replication and damage repair. Compared to young cells, senescent cells show a further decrease in the number of cells in the S and G2 / M phases, while the proportion of cells in the G1 / G0 phase increases. Therefore, detecting the distribution of different phases of the cell cycle can also be used to monitor senescence. Cell cycle analysis was performed using flow cytometry.

[0057] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged, and the P2 and P10 generations of hUC-MSCs were analyzed by flow cytometry.

[0058] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶. 5 Cells were seeded at 2.5 ml of complete culture medium per well in six-well plates and incubated at 37 °C with 5% CO2. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and good cell growth, when the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 48 h. Three replicates were used for each of the young cell group, control group, and PQQ-treated group. Cells were then digested using Tryple, and the cell pellet was collected and transferred to 1.5 ml centrifuge tubes. Subsequent experiments were performed using a cell cycle assay kit. Cells were resuspended in 1 ml of PBS buffer and centrifuged at 1000 g for 5 min, and the supernatant was discarded. Cells were then fixed with 1 ml of 70% ethanol for 24 h. After resuspending in 1 ml of PBS buffer and centrifuging, propidium iodide staining solution was prepared according to the instructions of the cell cycle assay kit. 500 μl of this solution was added to each sample, and the cells were incubated at 37 °C in the dark for 30 min. Flow cytometry was used for analysis, and data were collected and analyzed using ModFit LT 5.0 software. GraphPad Prism 9.5 software was used for data processing. Results are presented as follows: express, s represents the within-group mean, and s represents the within-group standard deviation. One-way ANOVA was used for comparisons between groups, and a p-value < 0.05 was considered statistically significant. Results are shown below. Figure 7 .

[0059] from Figure 7 As can be seen, compared with the young group, the proportion of G0 / G1 phase in hUC-MSCs in the control group increased from 65.93% to 91.56%, and one-way ANOVA showed a significant difference. The proportion of the G2 / M phase decreased from 14.58% to 6.14%, and the proportion of the S phase decreased from 19.48% to 2.29%. One-way ANOVA showed that the significance levels for both the G2 / M and S phases were [not specified]. This indicates that the replicative senescence of hUC-MSCs involves cell cycle arrest.

[0060] Compared with the control group, the proportion of hUC-MSCs in the PQQ-treated group decreased from 91.56% to 82.97%, the proportion in the G0 / G1 phase increased from 6.14% to 11.62%, and the proportion in the S phase increased from 2.29% to 5.40%. One-way ANOVA showed that the significance levels for the G0 / G1, G2 / M, and S phases were all [not specified in the original text]. The difference was statistically significant; PQQ treatment could delay the senescence of hUC-MSCs and promote cell cycle function.

[0061] Test Example 5 Existing technologies indicate that intracellular reactive oxygen species (ROS) are closely related to aging, and excessive ROS accumulation leads to cellular senescence. Therefore, this study aimed to determine the effect of PQQ treatment on hUC-MSCs by detecting changes in ROS levels. The fluorescent probe H2DCFH-DA was used to detect ROS levels.

[0062] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged, and the reactive oxygen species levels of hUC-MSCs from passages P2 and P10 were measured.

[0063] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶.5 Cells were seeded at 2.5 ml of complete culture medium per well in six-well plates and incubated at 37 °C in a 5% CO2 incubator. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and good growth, when the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 48 h. All three groups (young cell group, control group, and PQQ-treated group) had three replicates. The complete culture medium was then discarded, and the cells were washed once with PBS buffer. H2DCFH-DA was diluted to a final concentration of 2 μM with PBS buffer (10 mM), and the probe was loaded. The cells were incubated at 37 °C in the dark for 30 min. Cells were washed twice with PBS buffer to remove unbound probes, and the cells were photographed under a fluorescence microscope. The mean fluorescence intensity was measured using ImageJ software.

[0064] Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s is the mean of each group and s is the standard deviation of each group. One-way ANOVA was used for comparisons between groups. A statistically significant difference was considered when the significance level was P < 0.05. Figure 8 The graph shows the relative average intensity of ROS fluorescence in hUC-MSCs from the young group, control group, and PQQ-treated group.

[0065] from Figure 8 Microscopic images showed that, compared with the younger group, the control group had significantly increased ROS accumulation in hUC-MSCs. Data analysis plots showed that ROS increased from 1.00±0.218 to 6.22±0.42. One-way ANOVA showed a significance level of [missing data]. This indicates a significant increase in ROS levels in senescent cells. Compared to the control group, ROS accumulation in hUC-MSCs treated with PQQ was significantly reduced. Data analysis showed that ROS decreased from 6.22±0.42 to 3.94±0.34, and one-way ANOVA showed a significance level of [missing data]. This indicates that PQQ can reduce ROS levels in hUC-MSCs and delay the aging of hUC-MSCs.

[0066] Test Example 6 Telomeres are short segments of DNA located at the ends of chromosomes, maintaining the stability of genetic information. Current technology considers telomeres to be one of the most important indicators for measuring the degree of cellular aging. Telomere length is determined by genetic and environmental factors and decreases with age. Telomere length is closely related to DNA repair, aging, apoptosis, and tumorigenesis. Therefore, measuring the relative length of telomeres in cells is used to assess the degree of cellular aging.

[0067] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg and cultured in complete medium at 37°C in a 5% CO2 incubator. The cells were continuously passaged and the average telomere length of hUC-MSCs from generation P2 and P10 was directly compared using relative quantitative qPCR. The ratio of the copy number of telomere repeat sequences (Tel) to the copy number of single-copy genes (SCR) in the genome (T / S) was used as the relative telomere length.

[0068] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶. 5 Cells were seeded at 2.5 ml of complete culture medium per well in six-well plates and incubated at 37 °C with 5% CO2. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and good cell growth, when the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 48 h. All three groups (young cell group, control group, and PQQ-treated group) had three replicates. Genomic DNA was then extracted, and its concentration and purity were determined using a UV spectrophotometer.

[0069] For PCR reaction setup, approximately 50–100 ng of template DNA was added to a PCR system consisting of the telomere repeat sequence pair Tel1 (T-reaction primer) and the control single-copy gene 36b4 primer pair (S-reaction primer). The PCR conditions were as follows: PCR activation reaction at 95 °C for 5 min to activate Hot StarTaq DNA polymerase in the system, followed by a two-step cycling method. The T-reaction conditions were: 95 °C denaturation for 15 s, 62 °C annealing / extension for 30 s, for a total of 40 cycles. The S-reaction conditions were: 95 °C denaturation for 15 s, 60 °C annealing / extension mixing for 30 s, for a total of 40 cycles.

[0070] Data analysis: Based on the Ct values ​​from real-time quantitative PCR results, the average of three replicates was taken, and data from wells with large deviations were discarded. The relative telomere length (T / S) value was calculated. The relative telomere length is the difference between the sample T / S value and the mean T / S value of all samples. Table 3 shows the qPCR primer sequences for telomere length.

[0071] Table 3. qPCR primer sequences for telomere length Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s is the mean of each group and s is the standard deviation of each group. One-way ANOVA was used for comparisons between groups. A statistically significant difference was considered when the significance level was P < 0.05. Figure 9 This is a graph showing the data analysis of the relative telomere length of hUC-MSCs in the young group, control group, and PQQ-treated group.

[0072] from Figure 9 As can be seen, compared with the young group, the telomere length of hUC-MSCs in the control group was significantly shortened from (3.41±0.07) to (1.44±0.10). One-way ANOVA showed a significance level of [missing data]. This indicates that telomere length is significantly shortened in senescent cells. Compared with the control group, the telomere length of hUC-MSCs in the PQQ-treated group increased from (1.44±0.10) to 1.88±0.02, and one-way ANOVA showed a significance level of [missing value]. The difference was statistically significant; PQQ treatment could delay the aging of hUC-MSCs and reduce telomere loss.

[0073] Test Example 7 Existing technologies suggest that cellular senescence involves the regulation of multiple genes and pathways, affecting cell proliferation and growth. As hUC-MSCs senescent, the expression of stem cell genes, such as Oct4, Sox2, Klf4, and c-Myc, is downregulated; conversely, the expression of senescence-related genes, such as p53, p16, and p21, is upregulated. Therefore, this invention determines the degree of senescence of hUC-MSCs by detecting the expression levels of stem cell genes and senescence-related genes within them.

[0074] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg, cultured in complete culture medium, and placed in a 37°C incubator with 5% CO2. The cells were continuously passaged, and the expression levels of relevant genes in the P2 and P10 generations of hUC-MSCs were detected by real-time quantitative qPCR.

[0075] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶. 5 Cells were seeded per well in six-well plates, with 2.5 ml of complete culture medium added to each well. The plates were then incubated at 37 °C in a 5% CO2 incubator. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and when cell growth was good and the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 48 h. All three groups (young cell group, control group, and PQQ-treated group) had three replicates. Total RNA was then extracted using a kit and reverse transcribed into cDNA. A two-step PCR quantitative assay was performed for detection. The reaction conditions were: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, and annealing / extension at 60 °C for 30 s. β-actin was used as an internal control gene. The relative expression level of the target gene was calculated using the method. The experiment was repeated three times, and the primer sequences are shown in Table 4.

[0076] Table 4. Primer sequences for qPCR detection of genes Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s represents the within-group mean, and s represents the within-group standard deviation. One-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant. Results are shown below. Figure 10 .

[0077] from Figure 10 It was observed that, compared with the young group, the expression of the stem genes Oct4, Sox2, Klf4, and c-Myc in hUC-MSCs of the control group was significantly downregulated. One-way ANOVA showed that the significance levels for Oct4, Klf4, and c-Myc were all [missing data]. The significance level of Sox2 is The expression of aging-related genes p53, p16, and p21 was significantly upregulated. One-way ANOVA showed that the significance levels for p53, p16, and p21 were all [missing data]. This indicates that senescence of hUC-MSCs is negatively correlated with the expression of stem genes Oct4, Sox2, Klf4, and c-Myc, and positively correlated with the expression of p53, p16, and p21.

[0078] Compared with the control group, the expression of Oct4, Sox2, Klf4 and c-Myc in hUC-MSCs of the PQQ-treated group was significantly upregulated. One-way ANOVA showed that the significance levels of Oct4, Sox2 and Klf4 were all [missing data]. The significance level of c-Myc is The expression of aging-related genes p53 and p21 was downregulated, and one-way ANOVA showed that the significance level of p53 was [not specified]. The significance level of p21 is qPCR results confirmed that during the senescence of hUC-MSCs cells, cell stemness gradually diminishes and senescence-related genes are activated, while PQQ can delay cell senescence and maintain cell stemness to some extent.

[0079] Test Example 8 As shown in Example 7, with the senescence of hUC-MSCs, the expression of stem genes Oct4, Sox2, Klf4, and c-Myc was downregulated, while the expression of senescence-related genes p53, p16, and p21 was upregulated. Gene expression regulation can occur at multiple levels, including the transcriptional and translational levels. Translational regulation is a mechanism that rapidly activates or inhibits mRNA translation in response to endogenous and exogenous signals, and can quickly and flexibly regulate intracellular protein expression to achieve physiological changes and maintain homeostasis. Therefore, by detecting the protein expression levels of these stem genes and senescence-related genes in hUC-MSCs, the role of PQQ in the senescence process of hUC-MSCs was further determined. Protein expression was detected using Western blot.

[0080] Although protein expression and gene expression are closely related, the temporal and spatial intervals between transcription and translation in eukaryotic gene expression, as well as the influences at various levels such as post-transcriptional processing, transcription product degradation, translation, post-translational processing, and modification, mean that results from the transcriptional and translational levels are not entirely consistent. Therefore, it is necessary to combine the results of both assays to comprehensively evaluate the anti-aging effect of PQQ on hUC-MSCs.

[0081] The primary hUC-MSCs cells were purchased commercially available. The complete culture medium consisted of 475 ml of serum-free basal medium, 25 ml of human platelet lysis buffer, and 5 ml of a penicillin-streptomycin-amphoteric B mixture, which was then filtered through a 0.22 μm needle filter to obtain the complete culture medium. The primary hUC-MSCs cells were cultured at a density of 2 × 10⁻⁶ cells / year. 4 live cells / cm 2 Cells were seeded at a density of 1000 mcg and cultured in complete culture medium at 37°C in a 5% CO2 incubator. The cells were continuously passaged, and the protein expression levels of Oct4, Sox2, p16 and p53 genes in P2 and P10 generations were detected by Western blot.

[0082] hUC-MSCs from generations P2 and P10 were sized at 1.0 × 10⁻⁶. 5Cells were seeded per well in six-well plates, with 2.5 ml of complete culture medium added to each well. The plates were then incubated at 37 °C with 5% CO2. Three groups were established: a young cell group (P2 generation), a control group (P10 generation), and a PQQ-treated group (P10 generation). In the PQQ-treated group, after cell adhesion and good growth, when the cell density reached 60%–70%, cells were treated with 70 μmol / L PQQ for 48 h. Three replicates were used for each of the young cell group, control group, and PQQ-treated group. Cells were then collected, and protein was extracted on ice using 1 mL of RIPA lysis buffer containing PMSF (phenylmethylsulfonyl fluoride). Protein concentration was determined using the BCA method, and the mixture was adjusted to the same concentration. The mixture was then boiled at 100 °C for 5 min to denature the protein and stored at -20 °C. The separated proteins were transferred to a PVDF membrane by SDS-PAGE gel electrophoresis. After washing, the membrane was blocked with 5% skim milk on a horizontal shaker at room temperature for 1 h. After washing, the membrane was stored at 4 °C with primary antibody for 24 h. After washing, the membrane was incubated with secondary antibody at room temperature on a shaker for 1 hour. The membrane was then washed again, stained with ECL chemiluminescence, exposed using a gel imaging system, and the protein grayscale values ​​were analyzed using ImageJ.

[0083] Data analysis was performed using GraphPad Prism 9.5 software, and the results are presented in... express, s represents the within-group mean, and s represents the within-group standard deviation. One-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant. Results are shown below. Figure 11 .

[0084] from Figure 11 ImageJ grayscale data analysis showed that, compared with the young group, the expression of aging genes p16 and p53-related proteins in the control group hUC-MSCs was significantly upregulated. One-way ANOVA showed that the significance levels for both p16 and p53-related proteins were [not specified in the original text]. The expression trends of the stem genes Oct4 and Sox2 in hUC-MSCs were opposite, with decreasing expression levels. One-way ANOVA showed that the significance level of Oct4 protein expression was [missing value]. The significance level of Sox2 protein was 100%. This indicates that senescence of hUC-MSCs is negatively correlated with the protein expression of stem genes Oct4 and Sox2, and positively correlated with the protein expression of p53 and p16.

[0085] Compared with the control group, the protein expression of p16 and p53 in hUC-MSCs of the PQQ-treated group was downregulated. One-way ANOVA showed that the significance levels of p16 and p53-related proteins were both [not specified]. The expression of the stem genes Oct4 and Sox2 was elevated in hUC-MSCs. One-way ANOVA showed that the significance level of Oct4 protein expression was [missing value]. The significance level of Sox2 protein expression was The results showed that PQQ treatment could increase the expression of Oct4 and Sox2 proteins in stem cells and decrease the protein expression levels of aging-related genes p16 and p53. This is consistent with the trend verified by qPCR, that is, PQQ can delay cell senescence and maintain cell stemness to a certain extent.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for delaying the aging of human umbilical cord mesenchymal stem cells during passage in a complete culture medium, characterized in that, The method involves culturing P10 generation human umbilical cord mesenchymal stem cells in a complete culture medium, and then adding pyrroloquinoline quinone for treatment when the cell growth density reaches 60-70%. The amount of pyrroloquinoline quinone added is 35~100 μmol / L in the complete culture medium, and the treatment time is 24~72 h.

2. The method according to claim 1, characterized in that, The amount of pyrroloquinoline quinone added was 70 μmol / L in the complete culture medium, and the treatment time was 48 h.

3. The method according to claim 1 or 2, characterized in that, The method can upregulate the expression of stem genes Oct4, Sox2, Klf4 and c-Myc in P10 generation human umbilical cord mesenchymal stem cells, and upregulate the protein expression of stem genes Oct4 and Sox2.

4. The method according to claim 1 or 2, characterized in that, The method can downregulate the expression of aging-related genes p53 and p21 in P10 generation human umbilical cord mesenchymal stem cells, and downregulate the protein expression of aging-related genes p16 and p53.

5. The method according to claim 1 or 2, characterized in that, The method can reduce the activity of β-galactosidase and the level of reactive oxygen species (ROS) in P10 generation human umbilical cord mesenchymal stem cells.

6. The method according to claim 1 or 2, characterized in that, The method can reduce the proportion of cell cycle phase G0 / G1 in P10 generation human umbilical cord mesenchymal stem cells, while increasing the proportion of cell cycle phase G2 / M and S.

7. The method according to claim 1 or 2, characterized in that, The method can increase the relative telomere length of P10 generation human umbilical cord mesenchymal stem cells and reduce telomere loss.

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