Compound and method for delaying aging of human umbilical cord mesenchymal stem cells

Abstract

The invention relates to a compound and a method for delaying senescence of human umbilical cord mesenchymal stem cells, belongs to the technical field of biological medicines, and aims to solve the technical problem of delaying senescence of human umbilical cord mesenchymal stem cells. The invention relates to a compound for delaying the aging of human umbilical cord mesenchymal stem cells, and the compound is pyrroloquinoline quinone (PQQ). The culture solution containing the PQQ is used for culturing the hUC-MSCs, so that the activity of cells can be effectively improved, cell proliferation can be promoted, the expression of senescence-related genes can be reduced, the dryness of the cells can be maintained, the replicative senescence of the human umbilical cord mesenchymal stem cells can be effectively delayed, and the amplification efficiency and the cell activity of in-vitro culture of the hUC-MSCs can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a compound and a method for delaying the aging of human umbilical cord mesenchymal stem cells. Background Art

[0002] With the increasing aging of the population, anti-aging research has gradually become one of the hot areas. Aging is a multifaceted and complex process, which is manifested by the decline of the normal physiological functions of tissues and organs, leading to obvious weakness, death and chronic diseases such as bone, cardiovascular and cognitive disorders. Stem cell aging is one of the main theories of biological aging. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are derived from the umbilical cord of newborns. They have the characteristics of self-renewal, multidirectional differentiation and immunomodulation, and can be used to treat a variety of diseases and injuries. hUC-MSCs can replace damaged cells by relying on their self-renewal and multi-lineage differentiation potential, regulate immune and inflammatory responses through paracrine effects and cell-to-cell interactions, reduce oxidative stress and DNA damage, and improve mitochondrial function, thereby delaying the aging process. At present, many clinical trials have confirmed the efficacy and safety of human umbilical cord mesenchymal stem cells in anti-aging, such as for the treatment of skin wrinkles, tendon injuries, premature ovarian failure, osteoporosis and other aging-related diseases.

[0003] However, the current therapeutic effects of hUC-MSCs are not ideal, and there are obstacles to their widespread clinical application. This is because the number of hUC-MSCs that can be expanded in vitro is limited, and they have immunogenicity and cell heterogeneity. In addition, the existing in vitro culture methods of hUC-MSCs greatly reduce their self-renewal ability. Continuous expansion in vitro is prone to replicative senescence, which affects their transplantation and clinical treatment applications. The aging of hUC-MSCs limits the speed and total amount of their large-scale expansion, and also limits the feasibility of large-scale market applications. Therefore, how to delay the replicative senescence of hUC-MSCs and obtain hUC-MSCs of sufficient "quantity" and "quality" has become a technical problem that needs to be solved urgently. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a compound and method for delaying the aging of human umbilical cord mesenchymal stem cells, so as to solve the technical problem of how to delay the aging of human umbilical cord mesenchymal stem cells.

[0005] The purpose of the present invention is mainly achieved through the following technical solutions:

[0006] In one aspect, the present invention provides a compound for delaying the aging of human umbilical cord mesenchymal stem cells, wherein the compound is pyrroloquinoline quinone.

[0007] On the other hand, the present invention also provides a method for delaying the aging of human umbilical cord mesenchymal stem cells, the method comprising culturing primary human umbilical cord mesenchymal stem cells in a complete culture medium, and during continuous passage, adding pyrroloquinoline quinone for treatment when the cell growth density reaches 60-70%.

[0008] Furthermore, the amount of pyrroloquinoline quinone added is such that the concentration of pyrroloquinoline quinone in the complete culture medium is 10 to 100 μmol / L, and the treatment time is 24 to 72 hours.

[0009] Furthermore, the amount of pyrroloquinoline quinone added was such that the concentration of pyrroloquinoline quinone in the complete culture medium was 70 μmol / L, and the treatment time was 48 h.

[0010] Furthermore, the method can upregulate the expression of stemness genes Klf4 and c-Myc in human umbilical cord mesenchymal stem cells of generations P3 to P6.

[0011] Furthermore, the method can upregulate the expression of stemness genes Oct4, Sox2, Klf4 and c-Myc in P10 human umbilical cord mesenchymal stem cells, and upregulate the protein expression of stemness genes Oct4 and Sox2.

[0012] Furthermore, the method can down-regulate the expression of aging-related genes p53 and p21 in P10 human umbilical cord mesenchymal stem cells, and down-regulate the protein expression of aging-related genes p16 and p53.

[0013] Furthermore, the method can reduce the activity of β-galactosidase and the level of reactive oxygen species (ROS) in human umbilical cord mesenchymal stem cells at the P10 generation.

[0014] Furthermore, the method can reduce the G0 / G1 phase ratio of the cell cycle of human umbilical cord mesenchymal stem cells at the P10 generation, while increasing the G2 / M phase and S phase ratios of the cell cycle.

[0015] Furthermore, the method can increase the relative length of telomeres of P10 human umbilical cord mesenchymal stem cells and reduce telomere loss.

[0016] Compared with the prior art, the present invention can achieve at least one of the following technical effects:

[0017] (1) The present invention uses a 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 and maintain cell stemness, thereby effectively delaying the replicative aging of human umbilical cord mesenchymal stem cells and significantly enhancing the expansion efficiency and cell activity of hUC-MSCs cultured in vitro.

[0018] (2) The present invention improves the antioxidant capacity of hUC-MSCs under culture conditions by applying PQQ, reduces the accumulation of reactive oxygen species (ROS) in aged hUC-MSCs, promotes cell growth and proliferation and the operation of the cell cycle, reduces telomere loss, and better reverses the expression of β-galactosidase, a marker of hUC-MSCs aging.

[0019] (3) The present invention reduces the expression levels of aging-related factors p53 and p21 in hUC-MSCs and increases the expression levels of stemness genes Oct4, Sox2, Klf4 and c-Myc in hUC-MSCs by applying PQQ, thereby ultimately alleviating the aging process of hUC-MSCs.

[0020] (4) The present invention increases the expression levels of hUC-MSCs stem cell Oct4 and Sox2 proteins and reduces the expression levels of aging-related genes p16 and p53 proteins by applying PQQ, thereby alleviating the aging of hUC-MSCs.

[0021] (5) The present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference numerals refer to like components throughout the drawings.

[0023] Figure 1 This is a graph showing the morphological changes of hUC-MSCs at generations P2 and P10 over time;

[0024] Figure 2 It is a growth curve diagram of hUC-MSCs of P2 and P10 generations of the present invention;

[0025] Figure 3 To test the survival rate of hUC-MSCs at generation P10 treated with different concentrations of PQQ in Example 1;

[0026] Figure 4 This is the expression data analysis diagram of the hUC-MSCs gene Klf4 of the control group and the PQQ-administered group of Example 2 from P3 to P6;

[0027] Figure 5 This is the analysis diagram of the expression data of the c-Myc gene in the hUC-MSCs of the P3 to P6 generations in the control group and the PQQ-administered group in Test Example 2;

[0028] Figure 6 The microscopic images and data analysis diagrams of β-galactosidase detection of hUC-MSCs in the young group, control group and PQQ administration group in test case 3;

[0029] Figure 7 This is the cell cycle distribution and data analysis diagram of hUC-MSCs in the young group, control group and PQQ administration group in test case 4;

[0030] Figure 8 This is a data analysis diagram of the relative average intensity of ROS fluorescence of hUC-MSCs in the young group, control group and PQQ-administered group in test case 5;

[0031] Fig. 9 This is a data analysis chart of the relative telomere length of hUC-MSCs in the young group, control group and PQQ administration group in test case 6.

[0032] Fig.10 This is the analysis diagram of the expression data of some genes in hUC-MSCs of the young group, control group and PQQ administration group in test case 7;

[0033] Fig.11 These are some gene protein expression graphs and data analysis graphs of hUC-MSCs in the young group, control group and PQQ-administered group of test case 8. DETAILED DESCRIPTION

[0034] The following is a further detailed description of a compound and method for delaying the aging of human umbilical cord mesenchymal stem cells in conjunction with specific examples. These examples are only for comparison and explanation purposes, and the present invention is not limited to these examples.

[0035] Pyrroloquinoline quineone (PQQ), a compound with potential anti-aging properties, has attracted widespread attention from the scientific community. It has been found to have a strong antioxidant effect, capable of scavenging free radicals and protecting cells from oxidative damage. It is beneficial to the energy metabolism and mitochondrial function of cells, helping to improve cell vitality and anti-aging ability. PQQ has also been found to promote neuroprotection and neuroregeneration, which is important for preventing nervous system aging and neurodegenerative diseases. However, there is still a lot of room for development in the mechanism of action, effect, and quantitative research of PQQ on specific types of cells, and there is a gap in the specific role and effect of PQQ on human umbilical cord mesenchymal stem cells.

[0036] In view of this, the inventors proposed the use of pyrroloquinoline quinone to delay the aging of human umbilical cord mesenchymal stem cells. Specifically, the pyrroloquinoline quinone, namely PQQ, has a molecular formula of C 14 H6N2O8.

[0037] It should be noted that the inventors have found that during the continuous culture of human umbilical cord mesenchymal stem cells, hUC-MSCs, in vitro, a series of unique morphological and biological functional changes will occur with the increase in the number of passages. hUC-MSCs from P2 to P4 have normal cell morphology, but enter the senescence stage after P5, and P9 cells show obvious replicative senescence characteristics, and senescence is also accompanied by the loss of cell proliferation ability. Figure 1 The morphological changes of hUC-MSCs of generation P2 and P10 over time are shown in the figure. Comparison shows that the cells of generation P10 are flat in morphology, enlarged in volume, with more cell matrix and more particles or vacuoles. Figure 2 This is a growth curve diagram of hUC-MSCs of generation P2 and P10 of the present invention. Comparison shows that the proliferation rate of cells of generation P10 is significantly reduced on days 3 to 6.

[0038] In the specific application of PQQ, the present invention proposes a method for delaying 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 passaging, pyrroloquinoline quinone, i.e., PQQ, is added for treatment when the cell growth density reaches 60-70%.

[0039] Specifically, hUC-MSCs primary cells were purchased commercially available products, and the complete culture medium consisted of 475 ml serum-free basal culture medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture, which was then filtered with a 0.22 μm needle filter to obtain the complete culture medium.

[0040] The culture conditions include: 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured with complete medium in a 37°C incubator with 5% CO2. After the cells adhered to the wall and grew well, when the density reached 60% to 70%, PQQ was administered.

[0041] It should be noted that after continuous in vitro expansion, hUC-MSCs of generation P10 and beyond have a series of obvious aging characteristics. Therefore, the inventors first treated hUC-MSCs of generation P10 with PQQ and conducted preliminary exploratory experiments, see Test Example 1. After comparing different PQQ concentrations and treatment times, it was found that the effective amount of PQQ added was a concentration of 10 to 100 μmol / L of PQQ in complete culture medium and a treatment time of 24 to 72 h, with a preferred concentration of 70 μmol / L and a treatment time of 48 h.

[0042] Further, in order to verify that PQQ also has an effect on maintaining the stemness of young hUC-MSCs cells, the inventors, based on the above-mentioned effective PQQ addition amount, conducted PQQ administration treatment for each generation of hUC-MSCs from P3 to P6, as shown in Test Example 2. The results showed that PQQ intervention can significantly increase the expression levels of hUC-MSC stemness genes Klf4 and c-Myc in P3 to P6 generations. It is noteworthy that compared with the hU-MSCs control group of the same generation, the expression of Klf4 gene in P4 to P6 generations of hUC-MSCs was increased by about 1.6 to 9.0 times by PQQ, while the expression of c-MYC gene in P3 to P5 generations of hUC-MSCs was increased by about 1.8 to 3.2 times by PQQ.

[0043] The present invention improves the antioxidant capacity of hUC-MSCs under culture conditions by applying PQQ, reduces the accumulation of reactive oxygen species ROS in aged hUC-MSCs, promotes cell growth and proliferation and the operation of cell cycle process, reduces telomere loss, and better reverses the expression of β-galactosidase, a senescence marker of hUC-MSCs. At the same time, PQQ reduces the expression levels of senescence-related factors p53 and p21 in cells, reduces the protein expression levels of senescence-related genes p16 and p53, increases the expression levels of stemness genes Oct4, Sox2, Klf4 and c-Myc of hUC-MSCs, and upregulates the expression of cell stemness Oct4 and Sox2 proteins, thereby ultimately alleviating the senescence process of hUC-MSCs.

[0044] Test Example 1

[0045] The cell viability of hUC-MSCs at generation P10 was detected by CCK-8 method.

[0046] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 1.5 and cultured with complete medium in a 37°C incubator with 5% CO2. They were cultured continuously to P10 for detection of CCK-8 cell proliferation toxicity assay.

[0047] The first stage: P10 hUC-MSC cells were cultured at 5×10 3Each well was inoculated in a 96-well plate, 100 μL of complete medium was added to each well, and the cells were cultured in a 37°C incubator with 5% CO2. A blank group, a control group, and a PQQ administration group were set up. After the cells adhered to the wall, the cells grew well and the density reached 60% to 70%. 10 μmol / L, 35 μmol / L, 70 μmol / L, 100 μmol / L, and 150 μmol / L of PQQ were respectively given for 24 hours. Six replicate wells were set for each PQQ administration group, control group, and blank group at each PQQ concentration. After that, the complete medium was replaced with a medium containing 10 μL of CCK-8 reagent, and the culture was incubated in the incubator for 2.5 hours. The absorbance value at 450 nm was detected by an enzyme marker to calculate the cell survival rate of each group. Cell survival rate (%) = (A450 nm of the experimental group-A450 nm of the blank group) / (A450 nm of the control group-A450 nm of the blank group) × 100%.

[0048] Phase II: The same blank group, control group and PQQ treatment group experiments were conducted. Compared with the first phase, only the PQQ treatment time was changed to 48h, and the rest was exactly the same.

[0049] Phase III: The same blank group, control group and PQQ treatment group experiments were conducted. Compared with the first phase, only the PQQ treatment time was changed to 72h, and the rest was exactly the same.

[0050] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and the inter-group comparison within each stage was performed using one-way analysis of variance. When the significance level P<0.05, the difference was considered statistically significant. The statistical results of cell survival rate are shown in Tables 1 and Figure 3 .

[0051] Table 1 hUC-MSC cell survival rate at different PQQ concentrations and treatment times

[0052]

[0053] From Table 1 and Figure 3 It can be seen that the cell survival rates after 24-72 hours of treatment with 10μmol / L, 70μmol / L, and 100μmol / L PQQ were higher than those of the control group; the cell survival rates after 24 and 72 hours of treatment with 35μmol / L PQQ were higher than those of the control group, and the cell survival rate after 48 hours of treatment with 35μmol / L PQQ was slightly lower than that of the control group; the cell survival rate after 24 hours of treatment with 150μmol / L PQQ was higher than that of the control group, and the cell survival rates after 48 hours and 72 hours of treatment were significantly lower than those of the control group. The following general trends can be seen from the above data:

[0054] (1) When the concentration of PQQ was lower than 10-70 μmol / L, the cell survival rate increased with the increase of treatment time in the range of 24-72 h;

[0055] (2) When the concentration of PQQ was equal to 70 μmol / L, within the range of 24 to 48 h, the cell survival rate increased with the increase of treatment time, but within 48 to 72 h, a reversal occurred, and the cell survival rate decreased with the increase of treatment time;

[0056] (3) When the concentration of PQQ exceeded 70 μmol / L, such as 100 μmol / L and 150 μmol / L, the cell survival rate decreased with the increase of treatment time within the range of 24 to 72 h. In particular, the cell survival rate after 48 h and 72 h of treatment with 150 μmol / L PQQ was significantly lower than that of the control group, and was lower than that of the cell survival rate when the PQQ concentration was 70 μmol / L and 100 μmol / L at the same treatment time.

[0057] Therefore, from the above trend, it can be inferred that the survival rate of hUC-MSCs can be improved when the PQQ concentration is 10-70μmol / L and the treatment time is 24-72h, and the cell survival rate has a general rule of increasing with the increase of concentration or treatment time, reaching a peak when the PQQ concentration is 70μmol / L and the treatment time is 48h; the survival rate of hUC-MSCs can also be improved when the PQQ concentration is 70-100μmol / L and the treatment time is 24-72h, but after the peak, the cell survival rate has a rule of gradually decreasing with the increase of concentration or treatment time. As for the cell survival rate after 48h treatment with 35μmol / L PQQ, which is slightly lower than that of the control group, it does not conform to the overall rule obtained from many experimental data, and it can be obviously inferred that it is an experimental error.

[0058] It is noteworthy that compared with the control group, the cell survival rates of hUC-MSCs cells treated with 70 μmol / L PQQ for 24 h, 48 h and 72 h were (109.3±2.59)%, (112.4±4.27)% and (109.5±0.47)%, respectively. The significance levels of one-way analysis of variance were * P<0.05, ** P < 0.01 and * P<0.05; however, 150 μmol / L PQQ significantly inhibited cell proliferation for 72 h, and the survival rate of hUC-MSCs decreased to (76.70±1.129)%, with a significance level of **P<0.01. According to the above experimental results, it can be seen that the effective PQQ concentration range is 10-100 μmol / L, and the treatment time is 24-72 hours, preferably 70 μmol / L and 48 hours.

[0059] Test Example 2

[0060] The method of using real-time fluorescence quantitative PCR (qPCR) technology to determine the total amount of polymerase chain reaction products after cycling, that is, qPCR method to detect the expression levels of stemness genes Klf4 and c-Myc in each generation of hUC-MSCs from P3 to P6.

[0061] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously to P3 to P6 generations, and hUC-MSCs from each generation of P3 to P6 were taken for qPCR detection.

[0062] Specifically, the qPCR detection steps and process parameters of P3 generation hUC-MSCs are as follows:

[0063] hUC-MSCs at passage P3 were cultured at 1.0×10 5 Each well was inoculated in a six-well plate, 2.5 ml of complete medium was added to each well, and the plate was cultured in a 37°C incubator with 5% CO2. A control group and a PQQ-treated group were set up. After the cells adhered to the wall and the cells grew well, when the density reached 60% to 70%, 70 μmol / L PQQ was given for 48 hours. Three replicate wells were set for both the PQQ-treated group and the control group.

[0064] After PQQ treatment, total RNA was extracted using a kit and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was performed using a two-step method. 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 the internal reference gene. -ΔΔCt The relative expression of the target gene was calculated by the method. The experiment was repeated three times. The primer sequences are shown in Table 5.

[0065] The steps and process parameters for qPCR detection of hUC-MSCs of each generation from P4 to P6 are exactly the same as those for qPCR detection of hUC-MSCs of P3 generation.

[0066] GraphPad Prism 9.5 software was used for data analysis. express, The mean of each group, s is the standard deviation of each group, and the inter-group comparison was performed by one-way analysis of variance. When the significance level P<0.05, the difference was considered statistically significant. The test results are shown in Table 3 and Figure 4 , Figure 5 .

[0067] Table 3 qPCR detection of hUC-MSCs stemness genes Klf4 and c-Myc data from P3 to P6

[0068]

[0069] From Table 3 and Figure 4 , Figure 5 It can be seen that PQQ treatment can significantly increase the expression levels of hUC-MSC stemness genes Klf4 and c-Myc in P3 to P6 generations. It is noteworthy that compared with the hUC-MSC control group of the same generation, the expression of Klf4 gene in P4 to P6 generations of hUC-MSCs was increased by about 1.6 to 9.0 times by PQQ. The one-way ANOVA showed that the significance level between the P4 and P5 groups was **** P<0.0001, the significance level between the P6 generation groups is ** P<0.005; PQQ increased the expression of c-MYC gene in hUC-MSCs of P3 to P5 generations by about 1.8 to 3.2 times. The one-way ANOVA showed that the significance level between the P3 to P5 generations was **** P<0.0001.

[0070] Test Example 3

[0071] SA-β-Gal is a commonly used cell senescence marker. SA-β-Gal (Senescence-Associated β-Galactosidase) is a β-galactosidase, and its activity increases significantly during cell replicative senescence. By detecting the activity level of SA-β-Gal in cells, the degree of cell senescence can be evaluated. Therefore, this test case is used to compare the SA-β-Gal positive cell rate in hUC-MSCs.

[0072] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously and hUC-MSCs at P2 and P10 were taken for β-galactosidase detection.

[0073] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5 Cells were inoculated in 12-well plates, 2 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2, a control group of P10, and a PQQ-administered group of P10 were set up. After the cells attached to the wall, the cells grew well and the density reached 60% to 70% in the PQQ-administered group. 70 μmol / L PQQ was given for 72 hours. Three replicate wells were set up for the young group, the control group, and the PQQ-administered group. The β-galactosidase detection kit was used for subsequent experiments. The complete medium was replaced with β-galactosidase fixative for 15 minutes and washed 3 times with PBS buffer. The β-galactosidase staining working solution was prepared according to the ratio specified in the kit instructions, 1 ml was added to each well, and the cells were incubated in the β-galactosidase staining working solution at 37°C, no CO2, and pH 6.0 for 24 hours.

[0074] 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: SA-β-gal positive cell rate (%) = number of SA-β-gal positive cells / total number of cells in the same field of view × 100%.

[0075] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and one-way analysis of variance was used for comparison between groups. When the significance level P<0.05, the difference was considered statistically significant. Figure 6 Microscopic images and data analysis diagrams of β-galactosidase detection of hUC-MSCs in the young group, control group and PQQ-treated group.

[0076] from Figure 6The microscopic images show that compared with the young group of P2 generation, the β-galactosidase activity of hUC-MSCs in the control group of P10 generation increased, and the staining was significantly darker. The data analysis chart shows that the SA-β-gal positive cell rate of the young group of P2 generation was (21.98±2.56)%, and the SA-β-gal positive cell rate of the control group of P10 generation was (59.8±2.49)%, which was significant compared with the young group of P2 generation. ### P<0.001, indicating that the activity level of SA-β-Gal in hUC-MSCs increased significantly, which can indeed evaluate the degree of cell aging.

[0077] Compared with the control group of P10, the staining of the PQQ-treated group of P10 was significantly reduced. The SA-β-gal positive cell rate of the PQQ-treated group of P10 was (39.4±1.50)%, which was significantly lower than that of the control group of P10. ** P<0.01, the content of the microscopic image reaction is consistent with the data in the data analysis diagram, which also confirms that PQQ can reduce the activity of β-galactosidase in hUC-MSCs and delay the aging of hUC-MSCs.

[0078] Test Example 4

[0079] The cell cycle regulates cell aging. Permanent cell cycle arrest in the G1 / G0 phase is one of the characteristics of cell aging. This arrest is related to the replication and damage repair of intracellular DNA. Compared with young cells, the number of cells in the S phase and G2 / M phase in aging cells is further reduced, while the proportion of cells in the G1 / G0 phase increases. Therefore, the distribution of each phase of the cell cycle can also be used to monitor the aging status. The cell cycle is detected by flow cytometry.

[0080] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously and hUC-MSCs at P2 and P10 were analyzed by flow cytometry.

[0081] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5Cells were inoculated in six-well plates, 2.5 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2, a control group of P10, and a PQQ-treated group of P10 were set up. After the cells adhered to the wall, the cells grew well and the density reached 60% to 70%. 70 μmol / L PQQ was given for 48 hours. Three replicates were set up for the young group, the control group, and the PQQ-treated group. After that, Tryple was used to digest and collect the cell precipitate, which was transferred to a 1.5 ml centrifuge tube. Subsequent experiments were performed using a cell cycle detection kit. The cells were resuspended with 1 ml of PBS buffer and centrifuged at 1000 g for 5 minutes, and the supernatant was removed. 1 ml of 70% ethanol was added for fixation for 24 hours. After adding 1 ml of PBS buffer, resuspend and centrifuge, prepare propidium iodide staining solution according to the ratio specified in the instructions of the cell cycle detection kit, add 500 μl to each sample, incubate at 37°C in the dark for 30 minutes, detect using flow cytometer, collect data and analyze data using ModFit LT 5.0 software. GraphPad Prism 9.5 software was used for data processing, and the data results are shown in express, is the mean value within each group, and s is the standard deviation within each group; one-way analysis of variance was used for inter-group comparison, and the difference was considered statistically significant when the significance level P<0.05. Figure 7 .

[0082] from Figure 7 As can be seen, compared with the young group, the G0 / G1 ratio of hUC-MSCs in the control group increased from 65.93% to 91.56%, and the one-way ANOVA showed a significant level. ### P<0.001, the proportion of G2 / M phase decreased from 14.58% to 6.14%, and the proportion of S phase decreased from 19.48% to 2.29%. One-way ANOVA showed that the significance levels of G2 / M phase and S phase were ## P<0.01, indicating that the replicative senescence of hUC-MSCs resulted in the blockage of cell cycle progression.

[0083] Compared with the control group, the G0 / G1 phase ratio of hUC-MSCs in the PQQ-treated group decreased from 91.56% to 82.97%, the G2 / M phase ratio increased from 6.14% to 11.62%, and the S phase ratio increased from 2.29% to 5.40%. One-way ANOVA showed that the significance levels of G0 / G1 phase, G2 / M phase and S phase were all * P<0.05, the difference was statistically significant. PQQ treatment could delay the senescence of hUC-MSCs and promote the operation of cell cycle.

[0084] Test Example 5

[0085] Prior art shows that reactive oxygen species, or ROS, in cells are closely related to aging, and excessive ROS accumulation can lead to cell aging. Therefore, the effect of PQQ treatment on hUC-MSCs was determined by detecting changes in ROS levels in hUC-MSCs. The fluorescent probe H2DCFH-DA was used to detect the level of reactive oxygen species.

[0086] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously and the levels of reactive oxygen species were detected in hUC-MSCs at P2 and P10.

[0087] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5 Cells were inoculated in six-well plates, 2.5 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2, a control group of P10, and a PQQ-treated group of P10 were set up. After the cells attached to the wall, the cells grew well and the density reached 60% to 70%, and 70 μmol / L of PQQ was given for 48 hours. Three replicates were set up for the young group, the control group, and the PQQ-treated group. After that, the complete medium was discarded, the cells were washed once with PBS buffer, and H2DCFH-DA with a concentration of 10 mM was diluted with PBS buffer to a final concentration of 2 μM. The probe was loaded and incubated in a 37°C cell culture incubator in the dark for 30 minutes. The cells were washed twice with PBS buffer to remove the unbound probes, photographed under a fluorescence microscope, and the average fluorescence intensity was detected using ImageJ software.

[0088] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and one-way analysis of variance was used for comparison between groups. When the significance level P<0.05, the difference was considered statistically significant. Figure 8 This is a data analysis chart of the relative average intensity of ROS fluorescence of hUC-MSCs in the young group, control group and PQQ-treated group.

[0089] from Figure 8The microscopic images show that compared with the young group, the accumulation of ROS in hUC-MSCs in the control group increased significantly. The data analysis chart showed that ROS increased from 1.00±0.218 to 6.22±0.42. The one-way ANOVA showed a significant level of ## P<0.01, indicating that the level of ROS in aging cells increased significantly. Compared with the control group, the accumulation of ROS in hUC-MSCs in the PQQ-treated group was significantly reduced. The data analysis chart showed that ROS decreased from 6.22±0.42 to 3.94±0.34, and the one-way ANOVA showed a significant level of ** P<0.01, indicating that PQQ can reduce the level of ROS in hUC-MSCs and delay the aging of hUC-MSCs.

[0090] Test Example 6

[0091] Telomeres are a small section of DNA at the end of chromosomes that maintains the stability of genetic information. Existing technology believes that telomeres are one of the most important indicators for measuring the degree of cell aging. Telomere length is determined by genetic and environmental factors and decreases with age. Telomere length is closely related to DNA repair, aging, cell apoptosis, and tumorigenesis. Therefore, the relative length of telomeres in cells is detected to measure the degree of cell aging.

[0092] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / cm2 and cultured using complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously and hUC-MSCs of generations P2 and P10 were taken to directly compare the average telomere length of hUC-MSCs using relative quantitative qPCR, that is, the ratio of the copy number of the telomeric repeat sequence (Tel) to the copy number of the single copy gene (SCR) in the genome (T / S) was used as the relative telomere length.

[0093] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5Cells were inoculated in six-well plates, 2.5 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2, a control group of P10, and a PQQ-treated group of P10 were set up. After the cells adhered to the wall, the cells grew well and the density reached 60% to 70%, 70 μmol / L of PQQ was given for 48 hours. Three replicates were set up for the young group, the control group, and the PQQ-treated group. Genomic DNA was then extracted, and the concentration and purity of the DNA were detected using an ultraviolet spectrophotometer.

[0094] The PCR reaction was set up by taking about 50-100 ng of template DNA and adding it to the 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 reaction conditions were as follows: PCR activation reaction at 95°C for 5 min to activate the Hot Star Taq DNA polymerase in the system, and then a two-step cycle method was performed. The conditions for the T reaction were denaturation at 95°C for 15 s, annealing / extension at 62°C for 30 s, and a total of 40 cycles. The conditions for the S reaction were denaturation at 95°C for 15 s, annealing / extension mixing at 60°C for 30 s, and a total of 40 cycles.

[0095] Data analysis: Based on the Ct value of the real-time fluorescence quantitative PCR results, the three replicates were averaged, and the well data with large deviations were discarded to calculate the relative telomere length T / S value, T / S = [2Ct (telomeres) / 2Ct (36B4)] – 1 = 2 –ΔCt , the relative telomere length is the difference between the sample T / S value and the mean T / S value of all samples, 2 -(ΔCt1-ΔCt2) =2 –ΔΔCt Table 4 shows the qPCR primer sequences for telomere length.

[0096] Table 4 qPCR primer sequences for telomere length

[0097]

[0098]

[0099] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and one-way analysis of variance was used for comparison between groups. When the significance level P<0.05, the difference was considered statistically significant. Fig. 9 This is a data analysis chart of the relative length of hUC-MSCs telomeres in the young group, control group and PQQ-treated group.

[0100] from Fig. 9It can be seen that 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), and the one-way ANOVA showed a significant level of ## P<0.01, indicating that the telomere length in senescent cells was significantly shortened. 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 the one-way ANOVA showed a significant level of * P<0.05, the difference was statistically significant. PQQ treatment could delay the aging of hUC-MSCs and reduce the loss of telomeres.

[0101] Test Example 7

[0102] The prior art believes that cell senescence involves the regulation of multiple genes and pathways, which affects the proliferation and growth of cells. As hUC-MSCs age, the expression of their stemness genes is downregulated, such as Oct4, Sox2, Klf4, and c-Myc. On the contrary, the expression of genes associated with aging is upregulated, such as p53, p16, and p21. Therefore, the present invention determines the aging degree of hUC-MSCs by detecting the expression levels of stemness genes and aging-related genes in hUC-MSCs.

[0103] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 100 μg / mL and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were cultured continuously for generations. The expression levels of related genes in the cells were detected by real-time fluorescence quantitative qPCR technology.

[0104] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5Cells were inoculated in six-well plates, 2.5 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2 generation, a control group of P10 generation, and a PQQ-administered group of P10 generation were set up. After the cells adhered to the wall, the cells grew well and the density reached 60% to 70%, and 70 μmol / L of PQQ was given for 48 hours. Three replicate wells were set up for the young group, the control group, and the PQQ-administered group. Total RNA was then extracted using a kit and reversely transcribed into cDNA, and PCR fluorescence quantitative detection was performed using a two-step method. The reaction conditions were: pre-denaturation at 95°C for 30s, denaturation at 95°C for 10s, annealing / extension at 60°C for 30s, β-actin was used as the internal reference gene, and the relative expression of the target gene was calculated using the 2-ΔΔCt method. The experiment was repeated three times, and the primer sequences are shown in Table 5.

[0105] Table 5 qPCR detection gene primer sequences

[0106]

[0107]

[0108] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and one-way analysis of variance was used for inter-group comparison. When the significance level P<0.05, the difference was considered statistically significant. Fig.10 .

[0109] from Fig.10 It can be seen that compared with the young group, the expression of hUC-MSCs stemness genes Oct4, Sox2, Klf4 and c-Myc in the control group was significantly downregulated. One-way ANOVA showed that the significance levels of Oct4, Klf4 and c-Myc were ## P<0.01, the significance level of Sox2 was # P<0.05; the expression of aging-related genes p53, p16 and p21 was significantly upregulated, and one-way ANOVA showed that the significance levels of p53, p16 and p21 were ## P<0.01; indicating that the aging of hUC-MSCs was negatively correlated with the expression of stemness genes Oct4, Sox2, Klf4 and c-Myc, and positively correlated with the expression of p53, p16 and p21.

[0110] Compared with the control group, the expression of Oct4, Sox2, Klf4 and c-Myc in hUC-MSCs in the PQQ-treated group was significantly upregulated. One-way ANOVA showed that the significance levels of Oct4, Sox2 and Klf4 were** P<0.01, the significance level of c-Myc was * P<0.05; the expression of aging-related genes p53 and p21 genes was down-regulated, and the one-way ANOVA showed that the significance level of p53 was ** P<0.01, the significance level of p21 is * P<0.05. qPCR results confirmed that during the aging process of hUC-MSCs cells, cell stemness gradually disappeared and aging-related genes were activated, while PQQ could delay cell aging and maintain cell stemness to a certain extent.

[0111] Test Example 8

[0112] It can be seen from Test Example 7 that with the aging of hUC-MSCs, the expression of its Oct4, Sox2, Klf4 and c-Myc stemness genes is downregulated, and the expression of p53, p16, and p21 aging-related genes is upregulated. Gene expression regulation can be carried out at multiple levels, including transcription and translation levels. Translation regulation is a mechanism that quickly activates or inhibits mRNA translation in response to endogenous and exogenous signals, which can quickly and flexibly regulate intracellular protein expression, achieve physiological changes, and maintain homeostasis of the body environment. Therefore, the role of PQQ in the aging process of hUC-MSCs was further determined by detecting the protein expression levels of these stemness genes and aging-related genes in hUC-MSCs. Protein expression was detected by Western blot.

[0113] Although protein expression and gene expression are closely related, the results from the transcription level and translation level are not completely consistent due to the time and site of transcription and translation of eukaryotic gene expression, and the influence of post-transcription and post-transcription processing, transcript degradation, translation and post-translational processing and modification. Therefore, it is necessary to combine the results of the two tests to comprehensively evaluate the anti-aging effect of PQQ on hUC-MSCs.

[0114] hUC-MSCs primary cells were purchased from the market. The complete culture medium consisted of 475 ml serum-free basal medium, 25 ml human platelet lysate, and 5 ml penicillin-streptomycin-amphotericin B mixture. The complete culture medium was then filtered through a 0.22 μm needle filter. hUC-MSCs primary cells were cultured at 2 × 10 4 viable cells / cm 2 The cells were inoculated at a density of 1.54 μg / mL and cultured with complete medium in a 37°C incubator with 5% CO2. The cells were subcultured continuously and the protein expression levels of Oct4, Sox2, p16 and p53 genes in the cells were detected by Western blot at P2 and P10 hUC-MSCs.

[0115] hUC-MSCs at P2 and P10 were cultured at 1.0×10 5 Cells were inoculated in six-well plates, 2.5 ml of complete medium was added to each well, and the plates were cultured in a 37°C incubator with 5% CO2. A young group of P2, a control group of P10, and a PQQ-administered group of P10 were set up. After the cells attached to the wall, the cells grew well and the density reached 60% to 70%, and 70 μmol / L of PQQ was given for 48 hours. Three replicates were set up for the young group, the control group, and the PQQ-administered group. After that, the cells were collected and 1 mL of RIPA lysis buffer containing PMSF (phenylmethylsulfonyl fluoride) was added to extract proteins on ice. The protein concentration was determined by the BCA method, adjusted to the same protein concentration, mixed, boiled at 100°C for 5 minutes to denature the protein, and stored at -20°C. The separated protein blot was transferred to a PVDF membrane by SDS-PAGE gel electrophoresis. After washing the membrane, it was blocked with 5% skim milk powder on a horizontal shaker at room temperature for 1 hour. After washing the membrane, the primary antibody was placed in a 4°C refrigerator for 24 hours. After washing the membrane, the secondary antibody was incubated on a horizontal shaker at room temperature for 1 hour. The membrane was washed, stained with ECL chemiluminescence, exposed in a gel imaging system, and the protein grayscale value was analyzed using ImageJ.

[0116] GraphPad Prism 9.5 software was used for data analysis. express, is the mean value within each group, s is the standard deviation within each group, and one-way analysis of variance was used for inter-group comparison. When the significance level P<0.05, the difference was considered statistically significant. Fig.11 .

[0117] from Fig.11 The data analysis of the grayscale values ​​in ImageJ showed that the expression of aging genes p16 and p53-related proteins in the control group hUC-MSCs was significantly upregulated compared with the young group. One-way ANOVA showed that the significance levels of p16 and p53-related proteins were both ## P<0.01; the expression trends of stemness genes Oct4 and Sox2 in hUC-MSCs were opposite, and the expression levels decreased. One-way ANOVA showed that the significance level of Oct4 protein was ### P<0.001, the significance level of Sox2 protein was ## P<0.01; indicating that the aging of hUC-MSCs was negatively correlated with the protein expression of stemness genes Oct4 and Sox2, and positively correlated with the protein expression of p53 and p16.

[0118] Compared with the control group, the protein expressions of p16 and p53 in hUC-MSCs of the PQQ-treated group were downregulated. One-way ANOVA showed that the significance levels of p16 and p53-related proteins were both** P<0.01; the protein expressions of stemness genes Oct4 and Sox2 in hUC-MSCs were increased, and one-way ANOVA showed that the significance level of Oct4 protein expression was ** P<0.01, the significance level of Sox2 protein expression was * P<0.05. The results showed that PQQ treatment could increase the expression of Oct4 and Sox2 proteins in stem cells and reduce the protein expression levels of aging-related genes p16 and p53, which was consistent with the trend of qPCR verification, that is, PQQ could delay cell senescence and maintain cell stemness to a certain extent.

[0119] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A compound for delaying the aging of human umbilical cord mesenchymal stem cells, characterized in that: The compound is pyrroloquinoline quinone.

2. A method for delaying the aging of human umbilical cord mesenchymal stem cells, characterized in that: The method uses the pyrroloquinoline quinone as claimed in claim 1, and the method comprises culturing primary human umbilical cord mesenchymal stem cells in a complete medium, and during continuous passage, adding pyrroloquinoline quinone for treatment when the cell growth density reaches 60-70%.

3. The method according to claim 2, characterized in that The amount of pyrroloquinoline quinone added is such that the concentration of pyrroloquinoline quinone in the complete culture medium is 10 to 100 μmol / L, and the treatment time is 24 to 72 hours.

4. The method according to claim 3, characterized in that The amount of pyrroloquinoline quinone added is such that the concentration of pyrroloquinoline quinone in the complete culture medium is 70 μmol / L, and the treatment time is 48 hours.

5. The method according to any one of claims 2 to 4, characterized in that: The method can up-regulate the expression of stemness genes Klf4 and c-Myc of human umbilical cord mesenchymal stem cells of generations P3 to P6.

6. The method according to any one of claims 2 to 4, characterized in that: The method can up-regulate the expression of stemness genes Oct4, Sox2, Klf4 and c-Myc of P10 human umbilical cord mesenchymal stem cells, and up-regulate the protein expression of stemness genes Oct4 and Sox2.

7. The method according to any one of claims 2 to 4, characterized in that: The method can down-regulate the expression of aging-related genes p53 and p21 in P10 human umbilical cord mesenchymal stem cells, and down-regulate the protein expression of aging-related genes p16 and p53.

8. The method according to any one of claims 2 to 4, characterized in that: The method can reduce the activity of β-galactosidase and the level of reactive oxygen species (ROS) in human umbilical cord mesenchymal stem cells at the P10 generation.

9. The method according to any one of claims 2 to 4, characterized in that: The method can reduce the G0 / G1 phase ratio of the cell cycle of human umbilical cord mesenchymal stem cells at the P10 generation, and simultaneously increase the G2 / M phase and S phase ratios of the cell cycle.

10. The method according to any one of claims 2 to 4, characterized in that: The method can increase the relative length of telomeres of P10 human umbilical cord mesenchymal stem cells and reduce telomere loss.

Images