Application of 5-hydroxytryptamine in promotion of cell reprogramming efficiency, cell proliferation and aging resistance, and cell regeneration promotion and reprogramming method

Abstract

Based on metabolic factors having potential correlation with reprogramming in the oocytes, specific metabolites in the oocytes are listed through a metabonomics method, and reductive metabolism characteristics of the oocytes are found from the specific metabolites. The invention provides application of 5-hydroxytryptamine in promotion of cell reprogramming efficiency, cell proliferation and aging resistance and a method for promoting cell regeneration and reprogramming, and relates to the technical field of biology. The invention proves that the cell regeneration can be promoted by various metabolites including nicotinamide mononucleotide, taurine and 5-hydroxytryptamine with proper concentration and the oocyte extract. Particularly, 5-hydroxytryptamine can promote cell reprogramming and cell regeneration, prolong the life of mice and reduce the aging level of tissues and organs. Therefore, the micromolecule metabolite capable of promoting cell reprogramming and cell regeneration is found through the oocyte, and the method is independent of a gene editing method and is safer and more reliable.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of 5-hydroxytryptamine in promoting cell reprogramming efficiency, cell proliferation and anti-aging, and a method for promoting cell regeneration and reprogramming. Background Art

[0002] Aging is an inevitable process that affects all organisms, manifested by multiple organ degeneration and physiological function decline, ultimately leading to chronic diseases and increased risk of death. Therefore, it is very necessary to understand the occurrence and development of aging and how to intervene in aging to extend human life expectancy.

[0003] In recent years, research related to aging has made great progress, including the identification of aging phenotypes, the study of the genetic background behind these phenotypes, the identification of genes related to biological lifespan, and the intervention of lifespan based on the targeting of related genes and pathways. For example, senescent cells are removed (senolysis) through the combination of venetoclax, dasatinib and quercetin (D / Q), fisetin, cardiac glycosides and other drugs, or caloric restriction (CR) is used to inhibit the mTOR signaling pathway and reduce insulin / IGF-1 signals. Recent studies have provided new ideas for anti-aging research based on the fact that reprogramming can restore the epigenetic clock, metabolic homeostasis and transcriptome changes of somatic cells. However, the reprogramming methods used in these studies are all induced reprogramming mediated by Yamanaka reprogramming factors OSKM (Oct3 / 4, Sox2, Klf4 and c-Myc). The risk is that the complete degree of induced reprogramming will cause somatic cells to lose their original identity characteristics and become pluripotent and undergo undirected differentiation, thereby causing the risk of tumorigenesis. Similar to Yamanaka factor-mediated induced reprogramming, somatic cell nuclear transfer (SCNT) is another reprogramming method, which is the process of placing terminally differentiated somatic cell nuclei into enucleated oocytes to ultimately produce totipotent cells. Since the expression level of Yamanaka factors in oocytes is very low, this indicates that there must be other factors in oocytes that promote reprogramming. Combined with the more reduced metabolic characteristics of oocytes compared to other periods of early embryonic development and the precise regulation of metabolism on early embryonic development, the factors that promote totipotency shaping in somatic cell nuclear transfer may be related to epigenetic remodeling, as well as specific metabolites and metabolic pathways. Therefore, the use of oocyte metabolites may be a new idea to promote cell regeneration and reprogramming.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first object of the present invention is to provide a use of serotonin (5-HT) in promoting the efficiency of human cell reprogramming.

[0006] The second object of the present invention is to provide a cell reprogramming method.

[0007] A third object of the present invention is to provide a culture medium for cell reprogramming.

[0008] A fourth object of the present invention is to provide a method for promoting cell regeneration based on oocyte-specific metabolites.

[0009] A fifth object of the present invention is to provide the use of 5-hydroxytryptamine in promoting human cell proliferation.

[0010] The sixth object of the present invention is to provide the use of 5-hydroxytryptamine in the preparation of anti-aging products.

[0011] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides the use of 5-HT in promoting the reprogramming efficiency of human cells; the human cells include human dermal fibroblasts and human skin fibroblasts.

[0012] As a further technical solution, the reprogramming includes OSKM-induced reprogramming.

[0013] In a second aspect, the present invention provides a cell reprogramming method, wherein during OSKM-induced human cell reprogramming, human cells are cultured using a human cell culture medium containing 5-hydroxytryptamine.

[0014] As a further technical solution, the human cells include fibroblasts.

[0015] In a third aspect, the present invention provides a culture medium for cell reprogramming, wherein the culture medium is a human cell culture medium containing 5-hydroxytryptamine.

[0016] In a fourth aspect, the present invention provides a method for promoting cell regeneration based on oocyte-specific metabolites, wherein human cells are cultured using a human cell culture medium containing oocyte-specific metabolites; The oocyte-specific metabolites include 5-hydroxytryptamine.

[0017] As a further technical solution, the human cells include mesenchymal stem cells.

[0018] In a fifth aspect, the present invention provides the use of 5-hydroxytryptamine in promoting the proliferation of human cells; the cells include mesenchymal stem cells.

[0019] In a sixth aspect, the present invention provides the use of 5-hydroxytryptamine in the preparation of anti-aging products.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on the metabolic factors in oocytes that are potentially relevant to reprogramming. The specific metabolites in oocytes are listed by metabolomics methods, and the reductive metabolic characteristics of oocytes are found. Using mesenchymal stem cells as an in vitro cell model and G608G premature aging mice as an in vivo model, it is proved that various metabolites including nicotinamide mononucleotide, taurine, 5-hydroxytryptamine at appropriate concentrations and oocyte extracts themselves can promote cell regeneration. Among them, 5-hydroxytryptamine in particular, treating cells with a concentration of 5 μM for 72 hours can promote cell reprogramming and cell regeneration, and feeding G608 mice with 0.375 mg / kg / day can extend the life of mice and reduce the aging level of tissues and organs of G608G premature aging mice and naturally aged mice. Therefore, the present invention finds small molecule metabolites that can promote cell reprogramming and cell regeneration through oocytes, which is independent of the gene editing method and is safer and more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 A in the figure is the principal component analysis plot of targeted metabolomics, depicting the clustering of three biological replicates of MII oocytes and two-cell stage embryos (n = 124 metabolites per sample); Figure 1 B in the figure is the volcano plot analysis of various metabolites in MII oocytes and two-cell embryos. Blue dots indicate metabolites with higher content in the two-cell stage, red dots indicate metabolites specifically enriched in the MII oocyte stage, and gray dots indicate metabolites with little difference in abundance between the two stages. Figure 2 AF in the figure is the comparison of the relative contents of specific metabolites in MII oocytes and two-cell embryos, and statistical analysis was performed using unpaired t-test, with three biological replicates in each group; Figure 3 RT-qPCR was used to verify the effects of oocyte extract, nicotinamide mononucleotide, taurine, and 5-HT on the transcriptional levels of aging-related factors and inflammation-related factors; Figure 4: The effect of 5-hydroxytryptamine on OSKM reprogramming efficiency; A is lentivirus-infected human dermal fibroblasts for reprogramming induction, the bar graph represents the number of clones and alkaline phosphatase levels after induction (14th day of reprogramming), the third picture from the left is a bright field schematic diagram of the clones in the control group, the fourth picture from the left is a bright field schematic diagram of the clones in the 5-hydroxytryptamine treatment group, paired t-test was used for statistical analysis, and there were 3 biological replicates in each group; B is lentivirus-infected human skin fibroblasts for reprogramming induction, the bar graph represents the number of clones and alkaline phosphatase levels after induction (14th day of reprogramming), the third picture from the left is a bright field schematic diagram of the clones in the control group, the fourth picture from the left is a bright field schematic diagram of the clones in the 5-hydroxytryptamine treatment group, paired t-test was used for statistical analysis, and there were 3 biological replicates in each group; Figure 5 The figure is a validation of the effect of 5-HT on the delayed aging of the pathological model of mesenchymal stem cells from patients with progeria; A is the SA-β-Gal staining (upper) and statistics (lower) of mesenchymal stem cells in each group, and one-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; B is the CCK-8 test results of mesenchymal stem cells in each group, and two-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; C is the RT-qPCR detection of the transcription levels of IL1B, IL6, CDKN2A, CDKN1A and TP53 in mesenchymal stem cells in each group, and one-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; Figure 6 This is the verification of 5-HT delaying aging of normal human mesenchymal stem cell replicative model, where A is SA-β-Gal staining (upper) and statistics (lower) of mesenchymal stem cells in each group, statistical analysis was performed using one-way ANOVA test, with 3 biological replicates in each group; B is the CCK-8 test results of mesenchymal stem cells in each group, statistical analysis was performed using two-way ANOVA test, with 3 biological replicates in each group; C is RT-qPCR detection of IL1B, IL6, CDKN2A, CDKN1A and TP53 transcription levels in mesenchymal stem cells in each group, statistical analysis was performed using one-way ANOVA test, with 3 biological replicates in each group; Figure 7To verify the effect of 5-HT on delaying aging at the individual level in the in vivo model of G608G premature aging mice; A is the survival curve of 23 G608G mice fed with 5-HT and 22 G608G mice not fed with 5-HT; B is the open field trajectory of mice in each group; C is the Masson staining of the lung, muscle and aorta of mice in each group; D is the quantitative analysis of the degree of fibrosis in the lung, muscle and aorta in C, and one-way ANOVA test was used for statistical analysis, with 3 biological replicates in each group; Figure 8 A in the figure is the HE staining image of the liver, spleen and aorta of mice in each G608G group; Figure 8 B in the figure is a quantitative analysis of the number of central veins per unit area of ​​the liver, the number of vascular smooth muscle cells per unit area of ​​the aorta, and the percentage of the area of ​​the germinal center of the spleen in A. Statistical analysis was performed using the one-way ANOVA test, with 5 biological replicates per group; Fig. 9 To verify the effect of 5-HT on delaying aging at the individual level in an in vivo model of naturally aged mice; A is the Masson staining of the lungs, muscles and aortas of mice in each group; B is the quantitative analysis of the degree of fibrosis in the lungs, muscles and aortas in A, using one-way ANOVA test for statistical analysis, with 3 biological replicates in each group; Fig.10 A in the figure is the HE staining image of the liver, spleen and aorta of mice in each group of natural aging; Fig.10 B in the figure is a quantitative analysis of the number of central veins per unit area of ​​the liver, the number of vascular smooth muscle cells per unit area of ​​the aorta, and the percentage of the area of ​​the germinal center of the spleen in A. Statistical analysis was performed using the one-way ANOVA test, with three biological replicates in each group; Fig.11 A in the figure is the protein levels of mitochondrial regeneration-related factors in three groups of cells, WT MSCs, G608G MSCs and G608G MSCs+5-HT, detected by Western Blot method; Fig.11 B in the figure shows the results of ATP level detection in three groups of cells: WT MSCs, G608G MSCs and G608G MSCs+5-HT; Fig.11 C in the figure shows the OCR level test results of three groups of cells: WT MSCs, G608G MSCs and G608GMSCs+5-HT; Fig.11 D in the figure is the statistical result of the basic respiration value and the maximum respiration value in C; Fig.12 A in the figure is the qPCR result of all 5-HTR isoforms; Fig.12 B in the figure shows the transcriptome analysis results of all 5-HTR isoforms; Fig.12 C in the figure shows the CCK8 cell proliferation results of four groups of cells: G608G MSCs, G608G MSCs+5-HT, and G608G MSCs+5-HT+SB; Fig.12 D in the figure is the expression results of proteins related to mitochondrial regeneration function in four groups of cells: WT MSCs, G608G MSCs, G608G MSCs+5-HT and G608G MSCs+5-HT+SB; Fig.13 A in the figure is the GO analysis of upregulated genes in G608G MSCs vs WT MSCs; Fig.13 B in the figure is the GO analysis of down-regulated genes in G608GMSCs+5-HT vs G608G MSCs; Fig.14 A in the figure is the flow chart of click chemistry experiment; Fig.14 B in the figure is the qualitative analysis of mass spectrometry of serotonylated peptides enriched by click chemistry (top 20 proteins); Fig.14 C in the figure further detected 5-hydroxytryptamine HSP90AB1 by IP / WB; Fig.15 Mass spectrometry analysis showed that the Gln (Q) residue was serotonylated at amino acid position 207; Fig.16 Mass spectrometry analysis showed that the Gln (Q) residue was serotonylated at amino acid position 493; Fig.17 G608GshHSP90AB1 was used as the control group, and G608GshHSP90AB1+WT, G608GshHSP90AB1+Q207A, and G608GshHSP90AB1+Q493A cells were used as experimental groups to detect the transcription levels of ER stress-related genes including DDIT3, ATF4, and PPP1R15A under 5-HT treatment or without treatment; Fig.18 In the figure, G608GshHSP90AB1 was used as the control group, G608GshHSP90AB1+WT, G608GshHSP90AB1+Q207A, and G608GshHSP90AB1+Q493A were used as the experimental groups, and the ratio of β-galactosidase-positive cells in each group was detected under the condition of 5-HT treatment or no treatment, wherein A is the β-gal bright field result diagram; Fig.18 B in Fig.18 Statistical graph of A in . DETAILED DESCRIPTION

[0023] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] Terminology explanation: Oocyte: An oogonia that undergoes meiosis during oogenesis. Oocytes are divided into primary oocytes, secondary oocytes, and mature oocytes. MII: A mature oocyte that is arrested at the metaphase of the second meiotic division. Zygote: A fertilized egg at the cell stage. Two-cell: After embryo fertilization, the initial division produces two cells, forming a two-cell embryo.

[0025] Reprogramming: refers to the process of inducing differentiated cells back to the state of stem cells by changing epigenetic modifications without changing the gene sequence, so that they have pluripotency or even totipotency. Currently, there are two main types of reprogramming: one is induced reprogramming mediated by Yamanaka reprogramming factor OSKM (Oct3 / 4, Sox2, Klf4 and c-Myc); the other is somatic cell nuclear transplantation (SCNT) technology.

[0026] Rejuvenation: refers to the process of restoring vitality of cells or collective tissues and organs through some intervention methods such as reprogramming and small molecules.

[0027] In a first aspect, the present invention provides the use of 5-HT in promoting the reprogramming efficiency of human cells; the human cells include human dermal fibroblasts and human skin fibroblasts.

[0028] The inventors have found that the efficiency of reprogramming can be significantly improved by adding additional 5-hydroxytryptamine to the culture medium during human cell reprogramming.

[0029] In some optional embodiments, the reprogramming comprises OSKM-induced reprogramming.

[0030] In some optional embodiments, the human cells include but are not limited to fibroblasts, or other cells known to those skilled in the art that can be induced to reprogram by OSKM.

[0031] In a second aspect, the present invention provides a cell reprogramming method, wherein during OSKM-induced cell reprogramming, human cells are cultured in a human cell culture medium containing 5-hydroxytryptamine.

[0032] During OSKM-induced cell reprogramming, culturing fibroblasts in human cell culture medium containing 5-hydroxytryptamine can effectively improve the cell reprogramming efficiency.

[0033] In some optional embodiments, the human cells include but are not limited to fibroblasts, or other cells known to those skilled in the art that can be induced to reprogram by OSKM.

[0034] The present invention does not impose any specific limitation on the concentration of serotonin in the culture medium, and a suitable concentration can be selected according to the cells to be reprogrammed. In some optional embodiments, the concentration of serotonin in the culture medium can be, for example, 5 μM.

[0035] In some optional embodiments, the human cell culture medium is mTeSR™1Basal Medium (STEMCELL, 85851) containing 5 μM 5-HT.

[0036] In a third aspect, the present invention provides a culture medium for cell reprogramming, wherein the culture medium is a human cell culture medium containing 5-hydroxytryptamine.

[0037] This culture medium is used for cell reprogramming and can effectively improve the reprogramming efficiency of fibroblasts and the like.

[0038] In some optional embodiments, the human cell culture medium includes mTeSR™1Basal Medium (STEMCELL, 85851) containing 5 μM 5-HT.

[0039] The present invention does not impose any specific limitation on the concentration of serotonin in the culture medium, and a suitable concentration can be selected according to the cells to be reprogrammed. In some optional embodiments, the concentration of serotonin in the cell culture medium can be, for example, 5 μM.

[0040] In a fourth aspect, the present invention provides a method for promoting cell regeneration based on oocyte-specific metabolites, wherein human cells are cultured using a human cell culture medium containing oocyte-specific metabolites; The oocyte-specific metabolites include 5-hydroxytryptamine.

[0041] The inventors have found that nicotinamide mononucleotide, taurine and 5-hydroxytryptamine can reduce the expression of aging and inflammatory factors to a certain extent, and have the effect of promoting cell regeneration.

[0042] In some alternative embodiments, the human cells include mesenchymal stem cells.

[0043] In some optional embodiments, the culture medium is a low-glucose DMEM medium (Gibco, C11885500BT) containing the oocyte-specific metabolite, 10% fetal bovine serum (FBS), and 100x Gluta-MAX (Gibco, 25030081).

[0044] The present invention does not impose any specific limitation on the concentration of serotonin in the culture medium, and a suitable concentration can be selected according to the cells to be reprogrammed. In some optional embodiments, the concentration of serotonin in the culture medium can be, for example, 5 μM.

[0045] In a fifth aspect, the present invention provides the use of 5-hydroxytryptamine in promoting cell proliferation; the cells include mesenchymal stem cells.

[0046] The inventors have found that treating cells with 5-hydroxytryptamine during the culture process can significantly increase the proliferation rate of cells.

[0047] In a sixth aspect, the present invention provides the use of 5-hydroxytryptamine in the preparation of anti-aging products.

[0048] The inventors have found through research that 5-hydroxytryptamine can reduce the expression of aging-related factors, alleviate tissue aging phenotypes, and can be used to prepare anti-aging drugs.

[0049] The present invention is further illustrated by the following specific examples.

[0050] It should be noted that in the following examples, "sh" represents knockdown.

[0051] Example 1. Collect MII oocytes and 2-cell embryos at two different stages for metabolomics analysis Four-week-old (4W) C57BL / 6 female mice were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 5 IU), and 48 hours later, human chorionic gonadotropin (hCG, 5 IU) was injected. MII oocytes and two-cell embryos were collected 14 hours and 40 hours after human chorionic gonadotropin injection, respectively. The collection method was as follows: MII oocytes: The mice were humanely killed by cervical dislocation, and the oviducts on both sides of the mice were removed. The enlarged ampulla of the oviduct was torn open with forceps under a stereomicroscope to release the oocytes with granulosa cells. The oocytes were placed in 0.3 mg / ml hyaluronidase solution for about 1 minute, transferred to fresh M2 culture medium (sigma, product number M7167), and repeatedly blown several times until the granulosa cells fell off. The oocytes with polar bodies were collected with an oviduct transfer tube, and washed in fresh M2 culture medium for 2-3 times to remove excess impurities, followed by washing three times in 0.9% NaCl to remove excess protein, and then transferred to a low-absorption 1.5 ml EP tube, and an appropriate amount of pre-cooled pure methanol was added. The tubes were stored at -80°C. 100 oocytes were collected from each group, and the results were repeated in triplicate.

[0052] Two-cell embryos: 40 hours after human chorionic gonadotropin injection, two cells were collected by flushing the fallopian tube with a needle. Three replicates were collected, with 100 embryos in each group.

[0053] The collected MII oocytes or two-cell embryos were centrifuged at 15,000 rpm for 15 minutes in a 4°C low-temperature centrifuge. The supernatant was gently transferred to a new centrifuge tube, vacuumed at 30°C, and then 80% methanol was added. The methanol was then evaporated to dryness, and the metabolites after evaporation were resuspended and shaken with 30 μl of 0.03% deionized water, then centrifuged at 15,000 rpm for 15 minutes in a 4°C low-temperature centrifuge, and the supernatant was taken for liquid chromatography-mass spectrometry (LC-MS / MS). The liquid chromatography experiment was performed using an ultra-high resolution liquid chromatography system, with an ACQUITY UPLC HSS-T3UPLC column (150×2.1 mm, 1.8 μm, Waters) and a mobile phase gradient of 0–3 min 1% mobile phase B; 3–15 min 1–99% mobile phase B; 15–17 min 99% mobile phase B; 17–17.1 min 99–1% mobile phase B; 17.1–20 min 1% mobile phase B. Mobile phase A was 0.03% formic acid / water solution, and mobile phase B was 0.03% formic acid / acetonitrile solution. The flow rate was 0.25 ml / min, the column was maintained at 35 °C, the sample in the autosampler was maintained at 4 °C, and the injection volume was 20 μL. Mass spectrometry detection was performed using a QTRAP 6500+ mass spectrometer from SCIEX, using mass spectrometry multiple reaction monitoring (MRM) mode. The chromatograms were examined and the peak areas were calculated using MultiQuant software v.3.0 (SCIEX). The peak area of ​​each detected metabolite was normalized to the total number of ions in the sample, so the content was the ratio of the detected metabolite to the total metabolites.

[0054] Through metabolomics analysis, data on 124 metabolites were obtained. Principal component analysis showed that the metabolites of embryos at the two stages could be well distinguished ( Figure 1 A in the figure), compared with the two-cell oocyte, the MII oocyte is rich in more reduction-related metabolites, including nicotinamide adenine dinucleotide (NAD+ / NADH), vitamin C (Ascorbic acid), taurine (Taurine), polyamines (spermine, spermidine), 5-hydroxytryptamine (Serotonin, 5-HT), etc. ( Figure 1 B in Figure 2 AF in ). Metabolite enrichment analysis also showed that the niacin and nicotinamide pathways, glutathione pathways, taurine pathways, arginine pathways, and tryptophan pathways were more active in the oocyte stage. In general, compared with the second cell after fertilization, MII oocytes tend to be enriched in reducing metabolites and have antioxidant properties. This antioxidant property is a common feature of many metabolites that promote reprogramming and anti-aging, such as vitamin C and taurine.

[0055] 2. Promoting cell regeneration using oocyte extracts and oocyte-specific metabolites Mesenchymal stem cells differentiated from iPSCs (induced pluripotent stem cells) derived from normal subjects (WT) and progeria patients (G608G, LMNA c. G608G C>T) were used as in vitro cell models for the study, mesenchymal stem cells derived from normal subjects (WT MSCs) were used as control, and mesenchymal stem cells derived from progeria patients (G608G MSCs) were used as the experimental group to explore the role of metabolites in pathological aging, mainly detecting the expression of β-galactosidase, cell proliferation rate, and the expression of factors related to the senescence-associated secretory phenotype (SASP).

[0056] G608G MSCs were inoculated in medium containing oocyte extract, 5 μM 5-HT, 200 μM nicotinamide mononucleotide or 1.25 Mm taurine (low-glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS) and 100x Gluta-MAX (Gibco, 25030081)) and cultured for 72 h. A control group was set up, and then qPCR was used to detect aging-related factors. CDKN2A (P16), CDKN1A (P21), TP53 (P53) and inflammatory related factors IL6 , IL1B The transcription level of Figure 3As shown in the results, it was found that these metabolites specifically enriched in oocytes can reduce the expression of aging and inflammatory factors to a certain extent. Similarly, oocyte extracts also have a certain effect, but no single metabolite has a significant effect, which may be due to the presence of multiple other unrelated metabolites in oocyte extracts.

[0057] Note: Oocyte extract was obtained by lysing 100 oocytes with 80% pre-cooled methanol, centrifuging, taking the supernatant, aspirating and then reconstituting with culture medium.

[0058] 3. Oocyte-specific metabolite 5-HT promotes reprogramming efficiency The oocyte-specific metabolite 5-HT has the function of promoting reprogramming and cell regeneration.

[0059] After transfecting 293T cells with the Yamanaka factor OSKM lentiviral plasmid to obtain the corresponding virus, human skin fibroblasts (NHDF) from young people and human dermal fibroblasts (AHDF) from the elderly were induced to reprogram. On the first day of reprogramming, additional 5-hydroxytryptamine (5μM, continuously added throughout the reprogramming process) was added to the mTeSR™1 Basal Medium (STEMCELL, 85851) to explore the effect of this metabolite on the reprogramming efficiency. During the experiment, the culture without 5-hydroxytryptamine was used as the control (Ctr). Around the 14th day of reprogramming (clones can be seen under bright field), the number of reprogrammed clones was counted ( Figure 4 A and Figure 4 In B), the level of alkaline phosphatase (AP) ( Figure 4 A and Figure 4 B) etc.

[0060] Reprogramming experiments showed that 5-HT could improve the efficiency of OSKM reprogramming, including an increase in the number of clones and an increase in alkaline phosphatase levels.

[0061] IV. The regeneration-promoting effect of the oocyte-specific metabolite 5-hydroxytryptamine Mesenchymal stem cells differentiated from iPSCs derived from normal humans (WT) and progeria patients (G608G, LMNA c. G608G C>T) were used as in vitro cell models for this study, and G608G MSCs were used as the experimental group to explore the role of serotonin in pathological aging.

[0062] The experimental steps include: G608G MSCs were inoculated in low-glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS) and 100x Gluta-MAX (Gibco, 25030081), and then treated with 5-hydroxytryptamine (final concentration of 5μM) for 72h. WTMSCs and G608G MSCs without 5-hydroxytryptamine treatment were used as controls. Then, a series of cellular senescence-related markers such as β-galactosidase expression, cell proliferation rate, and expression levels of factors related to the senescence-associated secretory phenotype (SASP) were detected.

[0063] The experiments involved mainly include: β-cell Gal-galactosidase staining: SA-β-Gal staining was performed using the SA-β-Gal staining kit (Beyotime, RG0039, China) according to the manufacturer's protocol. Briefly, 50,000 cells / well were cultured in a 12-well plate. After washing once with phosphate-buffered saline (PBS), fixative solution was added, incubated at room temperature for 15 min, and then stained with SA-β-Gal staining solution at pH 6.0, 37°C overnight. Senescent cells were observed under a light microscope.

[0064] CCK8 cell proliferation: 1000 mesenchymal stem cells were seeded in each well of a 96-well transparent culture dish. After the cells adhered, CCK8 solution was added to the culture medium (1:100) and incubated at 37°C for 4 h. The absorbance was measured at 450 nm and the second measurement was performed after an interval of 24 h. The measurement was performed four times in total, and the cell number was calculated based on the standard curve.

[0065] RT-qPCR: Total RNA was extracted from cells using the phenol-chloroform method or a pre-made kit and reverse transcribed to obtain cDNA. TB Green premix (RR820A, TAKARA) was used to detect target gene expression on a LightCycler 480 Instrument II System (Roche) or QuantStudio 6 Flex machine (ABI). All genes were normalized and calculated using ACTIN as the internal reference gene.

[0066] Figure 5 β-galactosidase staining assay ( Figure 5 A in the figure), CCK8 cell proliferation assay ( Figure 5 B) and qPCR ( Figure 5C) Experiments in Figure 5 show that 5-HT can reduce the number of β-gal-positive cells in pathological aging, promote cell proliferation and reduce the expression of aging-related factors.

[0067] Mesenchymal stem cells differentiated from iPSCs from normal human (WT) were subcultured to obtain young WT MSCs (denoted as WT Y) at passages P3-P5 and old WT MSCs (denoted as WTO) at passages P12-P15. It should be noted that P here refers to the number of passages. All MSCs are called P0 after iPSC differentiation induction, and one is added for each subsequent passage.

[0068] Then, WT MSCs at the P12-P15 passage were inoculated in low-glucose DMEM medium (Gibco, C11885500BT) containing 10% fetal bovine serum (FBS) and 100x Gluta-MAX (Gibco, 25030081), and then treated with 5-hydroxytryptamine (final concentration of 5 μM) for 72 h. WT Y and WT O without 5-hydroxytryptamine treatment were used as controls, and then a series of cellular senescence-related markers such as β-galactosidase expression, cell proliferation rate, and expression levels of factors related to the senescence-associated secretory phenotype (SASP) were detected.

[0069] Figure 6 β-galactosidase ( Figure 6 A), CCK8 cell proliferation assay ( Figure 6 B) and qPCR detection of aging-related factors ( Figure 6 C) demonstrates that serotonin also plays a role in replicative senescence.

[0070] The LMNA G608G progeria mouse model was used to explore the role of 5-HT in promoting cell regeneration and anti-aging in vivo. Transgenic homozygous G608G progeria mice were used as an in vivo research model. One-month-old homozygous G608G mice were given 5-HT orally (at a dose of 0.375 mg / kg / day until the mice died) to explore the effect of 5-HT on the survival rate of G608G progeria mice ( Figure 7 A in the figure). One-month-old normal mice (denoted as WT) were used as the control group, and one-month-old G608G progeria mice were used as the experimental group. The mice that took 5-HT orally were denoted as G608G+5-HT, and the mice that did not take 5-HT orally were denoted as G608G. The effect of 5-HT on the motor ability of G608G progeria mice was investigated in all groups at 5 months of age ( Figure 7B) The degree of aging at the organ level includes the influence of histology and the expression level of aging molecules.

[0071] The experiments involved mainly include: Tissue staining: Tissue samples were fixed in 4% PFA overnight and dehydrated with 70% ethanol for at least 24 h. The samples were then embedded in paraffin and sectioned for HE or Masson staining.

[0072] Open field test: The locomotor activity of mice was assessed using an open field test in a white plexiglass box with a length, width and height of 40 cm. The mice were placed in the center of the box floor and had 10 minutes to explore freely. Their every move was recorded by a camera. The total distance and movement trajectory of the mice were analyzed using AlsVision software.

[0073] The results of in vivo experiments showed that long-term drinking of 5-HT-containing water in premature aging mice (LMNA G608G) can increase their maximum lifespan ( Figure 7 A in Figure 1 was analyzed using the Log-rank (Mantel-Cox) test. The open field test also showed that 5-HT enhanced the motor ability of premature aging mice ( Figure 7 Masson staining results showed that 5-HT can reduce the degree of fibrosis in the lungs, muscles, and aorta at the tissue level ( Figure 7 CD in the figure), HE staining results further showed that long-term oral administration of 5-HT can increase the blood flow of the liver, the number of vascular smooth muscle cells per unit area of ​​the aorta, and the germinal center of the spleen in premature mice ( Figure 8 AB in).

[0074] In addition, we fed 12-month-old wild-type C57BL / 6 mice with water containing 5-HT (0.375 mg / kg / day) until the mice were 20 months old. Mice that were not fed with 5-HT were used as controls and anatomical experiments were performed. We found that compared with 4-week-old C57BL / 6 mice (denoted as 4W), 20-month-old mice that were not fed with 5-HT (denoted as 20M) showed obvious histological aging characteristics, while 20-month-old mice that were fed with 5-HT (denoted as 20M+5-HT) were able to alleviate the tissue aging phenotype. Masson staining results showed that 5-HT reduced the degree of fibrosis in the lungs, muscles, and aorta at the tissue level ( Fig. 9 AB in the figure), HE staining results further showed that long-term oral administration of 5-HT can increase the blood flow of the mouse liver, the number of vascular smooth muscle cells per unit area of ​​the aorta, and the germinal center of the spleen ( Fig.10 AB in).

[0075] 5-HT delays aging through classical and non-classical pathways Classical pathway: 5-HT depends on 5HTR1B to regulate mitochondrial function After verifying the phenotypic effect of 5-HT on regeneration in in vivo and in vitro models, in order to further explore the mechanism of action of 5-HT, transcriptomic analysis was performed on three groups of cells: WT MSCs and G608G MSCs treated or not with 5-HT. The results showed that 5-HT can increase the expression level of factors related to mitochondrial function. In order to further prove the relationship between 5-HT and mitochondria, the Western Blot method was used to detect transcription factors related to mitochondrial regeneration, including PGC1α (peroxisome proliferator-activated receptor γ coactivator 1α), Tomm20 (mitochondrial outer membrane translocase 20), Timm23 (mitochondrial inner membrane translocase 23) ( Fig.11 A in the figure shows that 5-HT can increase the protein expression levels of these genes. At the same time, ATP level detection also shows that 5-HT can increase the ATP content of aging cells ( Fig.11 To further verify the effect of 5-HT on mitochondrial function, the seahorse experiment detected the OCR (oxygen consumption rate) levels of three groups of cells: WT MSCs, G608G MSCs, and G608G MSCs+5-HT. Fig.11 C in the figure). The results showed that compared with WT MSCs, the basal and maximum respiration levels of G608G MSCs were lower, and 5-HT could indeed enhance the respiratory function of mitochondria to a certain extent, including increasing the basal and maximum respiration values ​​( Fig.11 D in.

[0076] The common mode of action of 5-HT is that extracellular serotonin activates intracellular signaling pathways by interacting with multiple subtypes of receptors and then participates in biological processes. In order to further understand which receptor 5-HT uses to alleviate cell aging, the transcriptome data above were used to analyze the transcription levels of all 5-HTR (5-hydroxytryptamine receptor) subtypes and secondary verification was performed using qPCR. The results showed that the receptor subtype with the highest expression in MSCs was 5HTR1B, and the expression levels of other subtypes were very low or even basically non-existent ( Fig.12 A in the figure is the qPCR result. Fig.12B in the figure is the result of transcriptome analysis). Therefore, the competitive inhibitor of 5HTR1B, SB-224289 hydrochloride (MCE, HY-101105A, hereinafter referred to as SB), was selected to explore whether 5HTR1B mediates 5-HT-promoted cell regeneration. Referring to the experimental method in the fourth part above, 2 μM SB was added while G608GMSCs were treated with 5-HT. It was found that the promoting effect of 5-HT on cell proliferation was partially inhibited by SB ( Fig.12 C in the figure is the result of CCK8 cell proliferation), including the upregulation of protein expression related to mitochondrial regeneration function was also inhibited ( Fig.12 D in.

[0077] The main experiments involved include: ATP assay: MSCs were cultured in 12-well plates for 72 h. Whole cells were lysed using lysis buffer according to the instructions of the ATP assay kit (S0026, Bio-Tech). After centrifugation, the supernatant was added to the ATP assay solution, and the luminescence value was read using a Spark multi-function microplate reader. Protein content was determined using the Pierce BCA protein quantification kit.

[0078] Cellular bioenergetics analysis: Real-time mitochondrial respiration analysis was performed using a Seahorse XF96 extracellular flux analyzer (Agilent, SantaClara, USA) following the manufacturer’s protocol. Briefly, the oxygen consumption rate (OCR) per minute in cell cultures was measured by sequentially adding the following respiratory regulators to determine bioenergetics parameters: 1.5 µM oligomycin (ATP synthase inhibitor; Cat. No. 103015-100, Agilent), 1 µM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, ionophore and mitochondrial uncoupler), and 0.5 µM rotenone (mitochondrial complex I inhibitor). MSCs were seeded at a density of 1.2 × 10^4 cells / well in 150 µL of the corresponding culture medium. Before the experiment, the culture medium was changed to DMEM without sodium bicarbonate and supplemented with 1 mM pyruvate and 5 mM glucose. Mitochondrial respiratory parameters (basal respiratory capacity, maximal respiratory capacity) were calculated using OCR values.

[0079] Non-classical pathway: 5-HT inhibits endoplasmic reticulum stress by serotoninating HSP90AB1, thereby delaying aging.

[0080] Further analysis of the RNA-seq above revealed that compared with young cells, G608G MSCs were significantly enriched in genes related to "unfolded protein response" and "endoplasmic reticulum stress", and 5-HT rescued this phenomenon ( Fig.13A in the figure is the GO analysis of the upregulated genes in G608G MSCs vs WT MSCs. Fig.13 B in Figure 2 is the GO analysis of down-regulated genes in G608G MSCs+5-HT vs G608G MSCs). It is known that 5-HT can modify proteins through covalent transamination mediated by transglutaminase (TGM2). Based on the establishment of click chemistry method, G608G MSCs or WT MSCs were treated with 5-PT (5-HT with alkynyl group, MCE, HY-156562A) to label the target protein, and then the target protein was obtained by immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify the 5-hydroxytryptamine protein ( Fig.14 A in Figure 14 is a flowchart of click chemistry experiments). The results showed that the most enriched proteins among the targeted proteins included not only ACTIN, which has been previously shown to be serotonylated, but also HSP90AB1 (heat shock protein 90α family class B member 1), which is highly associated with endoplasmic reticulum stress and unfolded protein response (B in Figure 14). IP and WB further confirmed that 5-HT in WT MSCs and G608G MSCs could serotonylate HSP90AB1 (C in Figure 14, where the upper side shows the binding of 5-HT to HSP90AB1 in G608G MSCs, and the lower side shows the binding of 5-HT to HSP90AB1 in WT MSCs).

[0081] To further identify the site of 5-HT modification on HSP90AB1, an in vitro 5-HT modification experiment was performed using LC-MS / MS. Specifically, the in vitro purified HSP90AB1 protein was incubated with 5-HT and recombinant TGM2 enzyme at 37°C for modification, followed by mass spectrometry analysis. The results showed that the Gln (Q) residue was at the 207th ( Fig.15 ) and the 493rd amino acid ( Fig.16In order to prove which site of 5-HT serotonylation contributes to the cell renewal effect of 5-HT, we further constructed wild-type HSP90β with Flag tag and HSP90β with the 207th or 493rd glutamine mutated to alanine, which were denoted as WT, Q207A and Q493A mutants, respectively. In G608G MSCs with endogenous HSP90AB1 knocked down (G608GshHSP90AB1), the wild type (WT) and Q207A and Q493A mutant HSP90AB1 with Flag were overexpressed, respectively, and recorded as G608GshHSP90AB1+WT, G608GshHSP90AB1+Q207A and G608GshHSP90AB1+Q493A. These three groups of cells were then treated with 5-HT, and G608G MSCs with HSP90AB1 knocked down were used as the control group. It was found that the effect of 5-HT on HSP90AB1 serotoninization was completely inhibited only after the 207th glutamine was mutated to alanine. Moreover, the replacement of endogenous wild-type HSP90AB1 by the mutant Q207A indeed eliminated the effect of 5-HT on promoting regeneration, which was reflected in the loss of regulation of endoplasmic reticulum stress and unfolded protein response genes, including DDIT3 (DNA damage-induced transcription factor 3), ATF4 (transcription activator 4) and PPP1R15A (protein phosphatase 1 regulatory subunit 15A), which were no longer downregulated. The Q493A mutant did not have this effect ( Fig.17 ). According to the same experimental design, the effect of glutamine 207 mutation to alanine on the action of 5-HT was further tested. The results showed that Q207A inhibited the effect of 5-HT on reducing the proportion of β-Gal positive cells ( Fig.18 A in the figure is the bright field result of β-gal, and B is the statistical graph).

[0082] The experiments involved mainly include: 5-PT co-immunoprecipitation assay: Copper-catalyzed click chemistry was used to label serotonylated proteins. 5-PT, an alkyne-functionalized 5-HT derivative, was conjugated to a biotin-azide molecule prepared by MedChemExpress (MCE®). Cells were treated with 10 µM 5-PT for 72 hours. Cell pellets were sonicated in 100 µL click reaction buffer (Thermo, C10643) and then centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatant was transferred to a new 1.5 mL EP tube. 0.25 mM biotin-azide, 0.1 mM CuSO4, and 50 μL 1× click additive were added to the lysate in sequence, and the final volume was adjusted to approximately 500 μL with click reaction buffer. Samples were incubated at 4°C for 2-3 hours. Magnetic streptavidin beads (ThermoFisher, 11206D) were washed twice in PBS. The reaction volume was adjusted to 1 mL with PBS, and 5% was taken as input control before adding the washed beads. 20 μL of the bead suspension was used for each immunoprecipitation, and the samples were incubated with rotation at room temperature for 3 hours. After incubation, the samples were placed on a magnetic stand to separate the beads from the supernatant, and the supernatant was carefully removed. The beads were washed three times with cold RIPA buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM PMSF) containing protease inhibitors, followed by three washes with cold DPBS to remove excess detergent. After the last wash, 2× SDS was added to the beads and boiled at 98°C for 8 minutes, followed by gel electrophoresis and incubation with appropriate primary and secondary antibodies.

[0083] In vitro enzymatic serotonylation assay: 5-HT (5 mmol / L) was co-incubated with 0.25 mg functional TGM2 protein (MCE, HY-P76114) and HSP90AB1 protein (abcam, ab80033) at 37°C to transamidate 5-HT to HSP90AB1. The total reaction volume was 25 µL enzyme reaction buffer (25 mM Tris-HCl, pH 8 and 5 mM CaCl2, containing protease inhibitors). The reaction buffer was incubated at 37°C in the dark for 3 hours, and the samples were boiled with 2×SDS at 95°C for 10 minutes. The serotonylated proteins were visualized by Coomassie Brilliant Blue staining, followed by LC-MS / MS analysis to detect serotonylation modification.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it.

Claims

1. Application of 5-hydroxytryptamine in promoting the reprogramming efficiency of human cells; the human cells include human dermal fibroblasts and human skin fibroblasts.

2. The use according to claim 1, characterized in that: The reprogramming includes OSKM-induced reprogramming.

3. A cell reprogramming method, characterized in that: During OSKM-induced human cell reprogramming, human cells were cultured in human cell culture medium containing 5-HT.

4. The reprogramming method according to claim 3, characterized in that: The human cells include fibroblasts.

5. A cell reprogramming medium, characterized in that The culture medium is a human cell culture medium containing 5-hydroxytryptamine.

6. A method for promoting cell regeneration based on oocyte-specific metabolites, characterized in that: Human cells were cultured in human cell culture medium containing oocyte-specific metabolites; The oocyte-specific metabolites include 5-hydroxytryptamine.

7. The method according to claim 6, characterized in that The human cells include mesenchymal stem cells.

8. Use of 5-hydroxytryptamine in promoting human cell proliferation; the cells include mesenchymal stem cells.

9. Application of 5-hydroxytryptamine in the preparation of anti-aging products.

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