Application of UBE2N as biomarker and therapeutic target in immune aging

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of UBE2N as a biomarker and a therapeutic target in immune aging. According to the invention, UBE2N is used as a biomarker and a therapeutic target of POI and immune aging for the first time, and is used for early diagnosis of POI and assessment of immune aging by detecting the expression level of UBE2N. Based on UBE2N as a target spot for POI treatment, after intravenous infusion of hUC-MSCs, the estrus cycle and normal ovarian form of a POI model mouse after pZP3 immunization can be obviously recovered, follicular development and granulosa cell proliferation in an ovarian are promoted, fertility is improved and obviously improved, the proportion of Th17 / Treg cells is adjusted, ovarian inflammation (IL-17, IFN-gamma) and DNA damage (UBE2N) is inhibited, and symptoms of the POI mouse are improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of UBE2N as a biomarker and therapeutic target in immune aging. Background Technology

[0002] Premature ovarian failure (POI) is a common gynecological condition characterized by ovarian dysfunction in women before the age of 40. Its pathological basis includes insufficient primordial follicle reserve, altered recruitment of dominant follicles, impaired follicular maturation, and accelerated follicular atresia. Currently, the exact cause of most cases of POI remains unclear. Early diagnosis and treatment assessment of POI primarily rely on hormone level testing (such as FSH and AMH) and imaging studies. However, these methods have limitations, including low sensitivity (unable to detect abnormalities before significant ovarian function decline) and poor specificity (susceptible to interference from other factors, making accurate assessment of ovarian function difficult). They also cannot promptly capture molecular biological changes within ovarian tissue (such as DNA damage and inflammatory responses) and cannot reflect changes at the cellular and molecular level.

[0003] Studies have shown that umbilical cord mesenchymal stem cells (hUC-MSCs) can differentiate into ovarian tissue cells, replacing damaged ovarian cells and restoring ovarian function. Simultaneously, they can regulate the populations of macrophages, regulatory T lymphocytes (Tregs), and related cytokines, secreting various growth factors, cytokines, and other bioactive substances to promote follicle development and ovulation, thereby improving fertility. Furthermore, umbilical cord mesenchymal stem cells can also regulate the patient's immune function, inhibit follicular apoptosis and inflammatory responses, and alleviate symptoms.

[0004] Therefore, the field expects to develop a detection method and evaluation system for POI based on human umbilical cord mesenchymal stem cells (hUC-MSCs) treatment. By detecting target biomarkers, the system can assess ovarian function status and immune aging at an early and sensitive time, while monitoring the efficacy of stem cell therapy, thus providing a scientific basis for the clinical treatment of POI. Summary of the Invention

[0005] The first objective of this invention is to provide an application of the UBE2N gene as a biomarker in the detection of immune aging, for the early diagnosis of POI and assessment of immune aging; The second objective of this invention is to provide an application of the UBE2N gene as a therapeutic target in the preparation of drugs for treating immunosenescence diseases; The third objective of this invention is to provide a diagnostic / detection product for assessing ovarian function and the degree of immune aging, which has the advantages of early detection and high sensitivity.

[0006] To address the aforementioned technical problems, this invention provides an application of the UBE2N gene as a biomarker in the detection of immune aging.

[0007] Specifically, the immunosenescence includes premature ovarian insufficiency.

[0008] Specifically, the UBE2N is derived from ovarian tissue or peripheral blood.

[0009] This invention also provides the application of a reagent for detecting the biomarker UBE2N in the preparation of immunoaging detection products: The testing products include those for the early diagnosis of symptoms of early-onset ovarian insufficiency; and / or, The reagent for detecting the biomarker UBE2N includes substances capable of detecting the expression level of the UBE2N biomarker; and / or, The detection products include detection kits, detection strips, detection chips, or detection systems; and / or, The test samples for the testing products are derived from ovarian tissue or peripheral blood.

[0010] This invention also provides the application of the UBE2N gene as a therapeutic target in the preparation of drugs for treating immunoaging.

[0011] Specifically, the immunosenescence includes premature ovarian insufficiency.

[0012] The present invention also provides the use of umbilical cord mesenchymal stem cells (hUC-MSCs) for the preparation of drugs for the treatment of early-onset ovarian insufficiency.

[0013] Specifically, the functions of the umbilical cord mesenchymal stem cells (hUC-MSCs) include: (1) Restore the estrous cycle and normal ovarian morphology; (2) Promotes follicular development and granulosa cell proliferation in the ovary; (3) Improve fertility; (4) Regulate the Th17 / Treg cell ratio; (5) Inhibits ovarian IL-17 inflammation and IFN-γ inflammation; (6) Inhibits DNA damage to the UBE2N gene.

[0014] The present invention also provides a medicament for treating early-onset ovarian insufficiency, wherein the active ingredient of the medicament includes umbilical cord mesenchymal stem cells (hUC-MSCs).

[0015] Specifically, the drug uses the umbilical cord mesenchymal stem cells (hUC-MSCs) as its sole active ingredient.

[0016] This invention also provides a kit for the early diagnosis of premature ovarian insufficiency, comprising: (1) qPCR primers or hybridization probes for specific detection of the UBE2N gene; (2) Primary antibody and HRP-labeled secondary antibody targeting UBE2N protein; (3) DAB chromogenic solution, buffer solution, and mounting medium; (4) Positive control, negative control and standard curve samples.

[0017] The present invention also provides a detection system for early diagnosis of premature ovarian insufficiency, the system comprising at least the following modules: The UBE2N expression data module is used to provide UBE2N expression data in the sample to be tested. The data analysis module is used to calculate the disease probability of the test sample based on the expression level of UBE2N in the test sample using a logistic regression model, thereby determining the risk of disease.

[0018] This invention is the first to use UBE2N as a biomarker for POI and immunosenescence. By detecting UBE2N expression levels, it can be used for early diagnosis of POI and assessment of immunosenescence, enabling early detection of ovarian dysfunction and immunosenescence. UBE2N is a key enzyme in K63 ubiquitination and participates in DNA damage repair, inflammatory signaling pathways, and cellular homeostasis regulation. In the ovarian tissue of POI model mice, UBE2N expression was significantly increased and positively correlated with the levels of inflammatory factors (IL-17, IFN-γ), suggesting that it can serve as a biomarker reflecting ovarian damage and immunosenescence. By detecting UBE2N mRNA or protein levels, ovarian function status and the degree of immune inflammation can be indirectly assessed.

[0019] This invention provides a novel, easy-to-use, highly sensitive, and specific diagnostic product and method for early diagnosis of POI and assessment of immune aging by detecting the expression level of UBE2N. It also enables dynamic monitoring of the effects of stem cell therapy, providing a scientific basis for the clinical treatment of POI and allowing for dynamic monitoring of treatment effects (such as stem cell therapy).

[0020] This invention uses UBE2N as a biomarker for point of origin (POI) and immune aging. UBE2N expression changes significantly in POI models, outperforming traditional hormone markers. It offers advantages in high sensitivity and specificity, and can detect molecular-level abnormalities before morphological changes occur. It is applicable to various sample types, including tissues and blood, and can be used to evaluate the efficacy of treatments such as stem cell therapies. The kit provides a standardized procedure for easy clinical application.

[0021] This invention uses UBE2N as a target for POI treatment. After intravenous infusion of hUC-MSCs, it can significantly restore the estrous cycle and normal ovarian morphology in pZP3-immunized POI model mice, promote follicular development and granulosa cell proliferation in the ovary, improve fertility and significantly enhance fertility, regulate the Th17 / Treg cell ratio, inhibit ovarian inflammation (IL-17, IFN-γ) and DNA damage (UBE2N), improve symptoms in POI mice, and the treatment process is safe and non-toxic. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a flowchart of the experimental intervention in Example 1; Figure 2 The changes in body weight of mice before and after modeling and after intervention in Example 2; Figure 3 This is the standard observation under a microscope during a normal estrous cycle in Example 3; Figure 4 The results of the comparison of estrous cycles in the three groups of mice in Example 3; Figure 5 This is a comparison of the wet weight of the ovaries of the three groups of mice in Example 4; Figure 6 This is a comparison of the ovaries of the three groups of mice in Example 4; Figure 7 This is a comparison of the average number of pups born in the three groups of mice in Example 5; Figure 8 The results show the levels of Treg cells and Th17 cells in the peripheral blood of mice in each group in Example 6; where A is the level of Treg cells detected by flow cytometry; B is the level of Th17 cells detected by flow cytometry; C is the result of changes in Treg cell levels; and D is the result of changes in the Th17 / Treg ratio. Figure 9 The figures show the morphological examination results of ovarian tissue from three groups of mice in Example 7; where (a), (b), and (c) are the results of the blank group, model group, and treatment group, respectively; the total length of the scale bar in the figure represents 100 μm. Figure 10 The figures show the IL-17 expression results in the ovaries of each group in Example 9; where (a), (b), and (c) are the results of the blank group, model group, and treatment group, respectively; the total length of the scale bar in the figure represents 250 μm. Figure 11 The figures show the IFN-γ expression results in each ovarian group in Example 9; where (a), (b), and (c) are the results of the blank group, model group, and treatment group, respectively; the total length of the scale bar in the figure represents 250 μm. Figure 12 The expression results of UBE2N in each group of ovaries in Example 9; among them, (a), (b), and (c) are the results of the blank group, the model group, and the treatment group respectively; the total length of the scale bar in the figure represents 250 μm. Specific implementation manners

[0023] In the following examples of the present invention, by constructing an animal model of premature ovarian insufficiency (POI), the changes in the estrous cycle, ovarian tissue morphology, litter size, etc. of mice after infusion of hUC-MSCs were observed, and the tolerance and effectiveness of intravenous injection of human umbilical cord mesenchymal stem cells in the treatment of premature ovarian insufficiency were explored.

[0024] In the following examples of the present invention, a mouse model of premature ovarian insufficiency was constructed by mixing and emulsifying pZP3 with complete Freund's adjuvant to make an immunizing reagent.

[0025] In the following examples of the present invention, the information on experimental animals, experimental cells, and experimental drugs is as follows, and the information on experimental reagents is shown in Table 1 below.

[0026] Experimental animals: Strains of Balb / c mice, source: Guangdong Provincial Center for Medical Experimental Animals, 6 - 8 weeks old, body weight 18.46 ± 0.8 g, gender: female, grade: SPF level, feed provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., production certificate number: 20 - SM23111505. Breeding place: Barrier mouse breeding room IVC cage box, grade: SPF level, usage license number: SYXK (Guangdong) 2023 - 0249, address: Building J5, No. 1 Jianta Mountain Road, Huangpu District, Guangzhou City, postal code: 510000.

[0027] Experimental cells: Human umbilical cord mesenchymal stem cells.

[0028] Experimental drugs: Modeling drug pZP3: The 330 - 342 amino acid sequence of mouse ZP3, manufacturer: Qiangyao Biotechnology Co., Ltd., product number: 04010006993, specification 20 mL; physiological saline: 0.9% sodium chloride injection (Sichuan Kelun), stored at room temperature; PBS phosphate buffer, manufacturer: Huayun Biology, product number HY401S, stored at 40 °C.

[0029] Table 1 Experimental reagents Name Manufacturer and Catalogue Number IL-17 Huabio, ER1902-37 IFNy Affinity, DF6045 UBE2N Sigma, 10243-1-AP-50UL Goat anti-rabbit-HRP Abeam, ab205718 Rabbit anti-goat-HRP Abbkine, A21040 DAB developing kit Fuzhou Mayxin, DAB-4033 In the following examples of the present invention, a mouse model of premature ovarian insufficiency was constructed based on mixing and emulsifying pZP3 with complete Freund's adjuvant to make an immunizing reagent, and the role of UBE2N and the therapeutic effect of human umbilical cord mesenchymal stem cells were verified.

[0030] In the following embodiments of the present invention, observation index data are collected, and detailed records are made of the basic information of mice, weight change data, estrous cycle changes and the number of pups born during the experiment. Flow cytometry experimental records and pathological tissue scan data are saved, and the data are organized using an electronic Excel spreadsheet. The data results are visualized using software analysis.

[0031] In the following embodiments of the present invention, statistical analysis of the data was performed using SPSS 25.0 software, and graphPad Prism 8.3.0 was used for plotting. First, a normality test was performed. If the data followed a normal distribution, a homogeneity of variance test was performed. If homogeneity of variance was satisfied (p>0.05), a one-way ANOVA was performed. If the data was not normally distributed or had unequal variances (p<0.05), a Kruskal-Wallis H test was performed. If the Kruskal-Wallis H test result was significant (p<0.05), a pairwise comparison test was further performed using the Mann-Whitney U test. A p<0.05 was considered statistically significant; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0032] Example 1 In this embodiment, an immunoassay reagent was prepared by mixing and emulsifying pZP3 with complete Freund's adjuvant to construct a mouse model of early-onset ovarian insufficiency.

[0033] In this study, mice were randomly divided into three groups: a blank control group, a model group, and a human umbilical cord mesenchymal stem cell therapy group, with 10 mice in each group.

[0034] Preparation of modeling drugs: 10 mg mouse pZP3 was added to 10 ml of triple-distilled water and emulsified with complete Freund's adjuvant at a ratio of 1:1 to prepare an immunomodulatory reagent; 10 mg mouse pZP3 was added to 10 ml of triple-distilled water and emulsified with incomplete Freund's adjuvant at a ratio of 1:1 to prepare an immunomodulatory enhancer; both were sterilized by filtration (0.22 μm filter).

[0035] Experimental Model Establishment: SPF-grade Balb / c female mice (6-8 weeks old) were selected. After one week of acclimatization culture, vaginal exfoliative cytology was performed to select 20 mice with normal estrous cycles for the experiment. Before model establishment, the skin on the back of the mice was shaved and disinfected with alcohol swabs. 0.15 mL of pZP3 immunoassay reagent was injected subcutaneously at multiple sites on the paw pads and abdomen of the mice. At 2 and 4 weeks, 0.15 mL of pZP3 immunoassay reagent was injected at the same sites for enhancement. Vaginal exfoliative cytology was performed daily at 8:00 AM to observe changes in the estrous cycle. The successful establishment of a early-onset ovarian insufficiency (POI) model was defined as two consecutive cycles of estrous cycle disorder. In this example, 20 mice successfully established the model.

[0036] Experimental intervention: as attached Figure 1 The experimental flowchart shown illustrates the experiment, which included 30 SPF-grade female Balb / c mice, divided into three groups: Control, Model, and hUC-MSCs. The Control group consisted of untreated female Balb / c mice, while the Model and hUC-MSCs groups were point-of-infection (POI) model mice. After model establishment, the hUC-MSCs group mice were injected via tail vein with 1×10⁻⁶ ppm. 5 One group of mice received umbilical cord mesenchymal stem cells once a week for four consecutive weeks. Two other groups received the same volume of saline as controls, with the same frequency of administration as the stem cell therapy group. After four weeks, seven mice from each group were randomly selected for orbital blood collection. The mice were then sacrificed for tissue analysis, and the remaining mice were paired with Balb / c male mice to observe fertility. Mouse weight, general condition, estrous cycle, and fertility were observed. Ovarian wet weight and peripheral blood Th17 / Treg cell ratio were compared among the three groups. Pathological sections and immunohistochemical results of ovarian tissue were analyzed.

[0037] One hour after injection, the mice showed no abnormalities, and no poisoning or death was observed. Subsequent observation for 28 days after administration also revealed no poisoning or death in the mice. After euthanasia, no tumors or drug-related pathological changes were observed at the injection site or in any of the tissues and organs, including the skin, uterus, ovaries, heart, lungs, liver, and kidneys.

[0038] Example 2 This embodiment is based on the drug administration experiment in Example 1 above, and further observes and examines the general condition of the mice. The weight of each group of mice was recorded every two days at 8:00 AM, accurate to two decimal places, and the mice's activity, eating, defecation, fur, and other general conditions were observed. The results are attached. Figure 2 .

[0039] In this embodiment, the groups were randomly assigned. Before modeling, the weight of the blank group was (18.52±0.87)g, the weight of the model group was (18.19±0.79)g, and the weight of the treatment group was (18.65±0.74)g. There was no statistically significant difference in weight among the three groups (p>0.05).

[0040] After three injections of pZP3 immunoassay reagent to model the mice, the weight of the blank group was (22.55±0.76) g, the weight of the model group was (21.16±1.08) g, and the weight of the treatment group was (20.83±0.86) g. There were significant differences in the weight of mice between the blank group, the model group, and the treatment group. The weight of mice decreased after modeling (p<0.05).

[0041] Four weeks after intervention, the weight of mice in the control group was (24.13±0.89) g, the weight of mice in the model group was (21.61±1.29) g, and the weight of mice in the treatment group was (22.97±0.51) g. The weight of mice in the treatment group recovered to a certain extent. There was no significant difference in weight between the mice and the control group (p>0.05).

[0042] Example 3 This embodiment is based on the drug administration experiment in the aforementioned embodiment 1, and further observes the estrous cycle of mice.

[0043] (1) Vaginal exfoliative cytology examination ① Wipe the glass slide with an alcohol swab at 8:00 AM every day, and then dry it with gauze; ②Use your left thumb and forefinger to hold the mouse's neck in place, and your left little finger to hold the base of the mouse's tail in place, thus exposing the vaginal opening; ③ Moisten a thin cotton swab with physiological saline, insert it into the mouse vagina and slowly rotate it 1-2 times before removing it. At the same time, apply erythromycin ointment to the mouse vulva once a week to prevent vaginal inflammation. ④ Rotate the cotton swab to evenly spread the vaginal secretions onto the glass slide, let it air dry naturally, and then add 75% alcohol to fix it and prevent cell deformation; ⑤ Add Wright-Gymsa A solution to the smear and let the stain cover the entire specimen for 1 minute; ⑥ Add Wright-Gymsa B solution to solution A (the amount added is 2-3 times that of solution A), mix the two solutions thoroughly with a bulb syringe, and stain for 5 minutes; ⑦ Rinse the glass slides under running water. Do not pour out the dye solution before rinsing to prevent sediment from settling. Let them air dry naturally. ⑧ Observe the morphology and distribution of exfoliated cells from the mouse vagina under a 10x light microscope to determine the estrous cycle of the mouse and record the findings.

[0044] (2) Criteria for determining the estrous cycle in mouse vaginal exfoliated cells ① Proestrum (P): Mostly nucleated epithelial cells, occasionally a small number of anucleated keratinized epithelial cells, no leukocytes; ②Estrous phase (estrum, E): consists entirely of anucleate keratinized epithelial cells; ③Metestrus (M): nucleated epithelial cells, anucleated keratinized epithelial cells, and leukocytes; ④ Diestrus (D): A large number of white blood cells and a small amount of mucus.

[0045] Normal estrous cycle in mice: 5-6 days; proestrus: 9-18 hours; estrus: 6-12 hours; metestrus: 30-48 hours; demyelinating period: 36-42 hours. Microscopic observations of the normal estrous cycle are shown in the attached figure.Figure 3 As shown.

[0046] As attached Figure 4 The comparison of estrous cycles in the different groups of mice shows that the estrous cycle in mice is generally 5-6 days and develops regularly. After modeling with pZP3 immunotherapy, compared with the control group, the vaginal exfoliated cell smears of mice in the model group and treatment group showed a decrease in anucleated keratinized epithelial cells and an increase in leukocytes and nucleated epithelial cells, suggesting that the POI model mice have prolonged estrus and estrus cycles, and a gradually prolonged and disordered estrous cycle. After 4 weeks of intervention, the estrous cycle in the control group developed regularly, the estrous cycle in the model group did not improve significantly over time, while the estrous cycle in the treatment group developed regularly.

[0047] Example 4 This embodiment is based on the drug administration experiment in the aforementioned embodiment 1, and further measures the wet weight of the mouse ovaries.

[0048] After euthanizing the mice and collecting samples, the left and right ovaries of the mice were separated and weighed, accurate to four decimal places.

[0049] As attached Figure 5-6 The results showed that the ovaries in the blank control group were pink in appearance with protrusions of varying sizes on the surface and weighed (0.1145±0.0124) g. The ovaries in the model group were smaller in volume, white or light pink in color, smooth in surface, and spherical in shape, and weighed (0.0968±0.0238) g. The ovaries in the treatment group were pink, plump and spherical in shape, and weighed (0.1177±0.0487) g. There was no significant difference between the treatment group and the blank control group and the model group (p>0.5).

[0050] Example 5 This embodiment is based on the drug administration experiment in the aforementioned embodiment 1, and further observes the fertility of mice in each group.

[0051] Four weeks after administration, three mice from each group were randomly selected and housed with male Balb / c mice at a female-to-male ratio of 3:1 for two months. The number of offspring per litter in each group was observed and recorded.

[0052] After a two-month cohabitation period, the number of pups born in each group was counted, and the results are shown in the attached figure. Figure 7 As shown in the figure. The results showed that all three groups of mice gave birth to healthy pups without deformities. The birth times of the control group and the treatment group were similar, while the birth time of the model group was one week later than the other two groups. The average number of pups in the control group was (7.67±1.53), the average number of pups in the model group was (4.33±1.15), and the average number of pups in the stem cell treatment group was (7.67±1.15). The number of pups in the model group was significantly reduced, but the number of pups recovered after stem cell treatment, with no significant difference compared to the control group.

[0053] Example 6 This embodiment is based on the drug administration experiment in the aforementioned embodiment 1, and further uses flow cytometry to detect the ratio of Th17 / Treg cells in mouse peripheral blood to evaluate immune regulation.

[0054] Peripheral blood was collected from the orbits of mice using heparin sodium blood collection tubes. After treatment with erythrocyte lysis buffer, cells were collected by centrifugation. Lymphocyte membrane / nucleus fluorescent antibodies were added sequentially for staining. Cells were then resuspended in phosphate-buffered saline (PBS) and transferred to flow cytometry tubes in preparation for further analysis. The Th17 / Treg cell ratios in different groups were recorded. The results are shown in the attached figure. Figure 8 As shown.

[0055] As can be seen, compared with the control group, the proportion of CD4+CD25+Foxp3+Treg cells in the model group was significantly reduced, showing a significant difference. The proportion of peripheral blood Treg cells in the treatment group was slightly lower than that in the control group, but the difference was not statistically significant (p>0.05) (see attached). Figure 8 (A and C). After 4 weeks of intervention, the Th17 / Treg ratio in the treatment group decreased, showing a significant difference compared to the model group (see attached). Figure 8 (B and D in the middle).

[0056] Example 7 This embodiment is based on the drug administration experiment in the aforementioned embodiment 1, and further examines the pathological characteristics of ovarian tissue.

[0057] After euthanizing the mice, the ovaries of both sides were separated, excess adipose tissue was removed, and the samples were weighed to four decimal places. Ovarian specimens were collected, fixed with 4% paraformaldehyde fixative, and embedded in dehydrated paraffin. Paraffin sections were treated with hematoxylin and eosin (HE) staining, and the morphology of the ovaries was observed under a light microscope, including histological changes such as the growth of follicles, granulosa cells, corpus luteum, and stroma. Immunohistochemical staining was performed on the paraffin sections, and the positive expression of IL-17, IFN-γ, and UBE2N was observed under a light microscope. The H-SCORE (histochemistry score) was also assessed. H-score = ∑pi (i+1), where pi represents the percentage of positive cells out of all cells in the section; i represents the staining intensity. H-score = [1 × (percentage of cells with a staining score of 1) + 2 × (percentage of cells with a staining score of 2) + 3 × (percentage of cells with a staining score of 3)], where 1+ is weakly positive, 2+ is positive, and 3+ is strongly positive.

[0058] As attached Figure 9 The results shown, after HE staining and microscopic observation, indicate that the morphology and function of follicles at all stages in the control group are normal, with abundant granulosa cells in the follicles and rounded oocytes visible within the follicles (as shown in the attached image). Figure 9 (a) Compared with the control group, microscopic examination of follicular granulosa cells in the model group mice showed apoptosis, nuclear fragmentation, and lysis (see attached figure).Figure 9 (b) In the treatment group, primordial follicles, primary follicles, and secondary follicles were observed under ovarian endoscopy, with good morphological development, abundant granulosa cells in the follicles, and rounded oocytes within the follicles without obvious necrotic tissue (see attached). Figure 9 (c)

[0059] Example 8 In this embodiment, based on the mice in each group of the aforementioned Example 1, gross dissection, organ weighing and histopathological examination were performed. The mouse euthanasia and specimen collection steps are as follows.

[0060] Place the mouse in a cage. Hold the mouse's neck by the thumb and forefinger of your left hand, and stabilize the tail with your little finger, causing the mouse's eyes to protrude and the retro-orbital venous plexus to become congested. With your right hand, hold a capillary glass tube and insert it into the corner of the mouse's eye at a 45-degree angle, stopping when resistance is felt after 2-3 mm. Collect peripheral blood. After blood collection, hold the mouse's tail base firmly with your right hand and pull it backward forcefully to euthanize the mouse using a cervical dislocation method. Fix the mouse on the operating table, trim the fur in the anterior abdominal region, disinfect with 75% medical alcohol, and cut the skin along the middle of the abdomen to fully expose the abdominal cavity. Locate the ovaries along the characteristic Y-shaped uterus of the mouse, symmetrically shaped like soybeans, surrounded by white adipose tissue and spiral fallopian tubes. Quickly remove the ovarian tissue with scissors, remove excess adipose tissue, and weigh.

[0061] The ovarian tissue was rinsed in pre-cooled saline buffer, and one ovarian tissue was stored at -80°C for later use. The other ovarian tissue was fixed in 4% paraformaldehyde solution at room temperature for 48 hours. The fixed tissue was removed from the 4% paraformaldehyde solution and, in a fume hood, the tissue at the target site was trimmed and smoothed with a scalpel according to the tissue sectioning requirements (note the maximum surface, cross section, and longitudinal section). The trimmed tissue was then placed in a pre-labeled embedding frame.

[0062] HE staining method After washing, the embedding frame containing the tissue was placed in the basket of a dehydrator and dehydrated and then impregnated with paraffin using a gradient of alcohols. The paraffin-impregnated tissue was then embedded in an embedding machine. First, molten paraffin was placed into the embedding mold. Once the paraffin at the bottom of the mold had slightly solidified, the tissue was carefully removed from the embedding frame with forceps and placed back into the embedding mold. The tissue was gently pressed down with the bottom of the forceps to ensure it was completely flat within the mold. The embedding frame was then placed on top of the mold containing the tissue, and the mold was gently moved to a -20°C freezing stage for cooling. After the paraffin solidified, the paraffin block was removed from the mold and trimmed. The trimmed paraffin block was then sectioned using a paraffin microtome to a thickness of 4 μm. The sections were floated on 42°C warm water in a slide spreader to flatten the tissue. The tissue was then vertically lifted using a glass slide, slightly drained, and baked in a 60°C slide oven for 30 min–1 h. The slides were then stored at room temperature.

[0063] Example 9 This embodiment is based on the mice treated in the aforementioned embodiment 8, and the expression of IFN-γ, IL-17, and UBE2N is detected by immunohistochemistry.

[0064] (a) Slices and slides First, use a blade to trim away excess paraffin around the ovarian tissue block, leaving about 2 mm of paraffin. Shape the tissue block into a trapezoidal shape to facilitate separation of the paraffin bands during slide preparation. The tissue section thickness is 5 μm. The slide preparation temperature is generally 42 ℃ to 45 ℃. Place the slides in a slide warmer for warming at 60 ℃ for 2 hours.

[0065] (II) Immunohistochemical experiments (1) Dewaxing and hydration: Dewaxing paraffin sections to water, using environmentally friendly dewaxing agent (three tanks, 10 min, 10 min, 10 min), gradient alcohol (anhydrous, 95%, 75%), 5 min per tank, and rinsing with tap water.

[0066] (2) Antigen retrieval: Add antigen retrieval solution (pH 9.0, EDTA) to the retrieval box. Place the dewaxed and hydrated tissue sections into the retrieval box, ensuring the solution completely covers the sections. Loosely cover the box and place it in a microwave oven. Set the microwave oven to medium power and the time to 12 minutes. After retrieval, remove the retrieval box from the microwave oven and allow the retrieval solution to cool naturally to room temperature (25-30°C, approximately 1 hour). Then, remove the sections from the retrieval solution and rinse them three times with distilled water for 5 minutes each time.

[0067] (3) Blocking: Block endogenous peroxidase with 3% hydrogen peroxide at room temperature (18-30 degrees) for 20 min. Rinse with TBST 3 times, 3 minutes each time.

[0068] (4) Draw circles: Use a pen to draw circles, and pay attention to the appropriate size.

[0069] (5) Blocking: Rabbit-derived antibodies were incubated with 10% goat serum at room temperature for 20 min.

[0070] (6) Incubation of primary antibody: Remove serum, add 100 μL of primary antibody working solution (rabbit anti-UBE2N, Thermo Fisher Scientific, 10243-1-AP) to each tissue, and incubate overnight at 4 °C.

[0071] (7) Warm-up: On the second day, take the slices out of the refrigerator and place them at room temperature for 15 minutes to warm up. Rinse them three times with TBST and then soak them three times for three minutes each time.

[0072] (8) Incubation of secondary antibody: Add 100 μL of secondary antibody working solution (goat anti-rabbit-HRP) to each slide, incubate at 37 ℃ for 45 min, rinse 3 times with TBST for 3 minutes each time.

[0073] (9) Color development: Add 100 μL of freshly prepared DAB to each slice, observe under a microscope, and stop the color development with tap water.

[0074] (10) Counterstaining: hematoxylin counterstaining, hydrochloric acid alcohol differentiation, and blueing solution for blueing.

[0075] (11) Drying and mounting: Dehydrate and dry with graded alcohol, mount with environmentally friendly mounting medium, air dry, examine under a microscope, and store in a cool, dry place.

[0076] (12) Take a photo or scan.

[0077] In this embodiment, the aforementioned pathological process was outsourced to Hubei Biosbio Technology Co., Ltd.

[0078] (III) Results (1) Immunohistochemical detection of IL-17 expression in ovarian tissue IL-17 (interleukin-17) is a class of pro-inflammatory cytokines mainly secreted by Th17 cells, playing a central role in autoimmune responses and chronic inflammation. In premature ovarian failure, IL-17A, as the core subtype of this family, accelerates the depletion of follicular reserves by promoting local ovarian inflammation and fibrosis.

[0079] As attached Figure 10 As shown in (a)-(c), IL-17 was located in the cytoplasm of immune cells infiltrating ovarian tissue (such as Th17 cells and macrophages), with positive signals appearing as brownish-yellow granules. Its expression intensity was positively correlated with the degree of ovarian damage. The results indicate that, compared to the control group, the number of IL-17-positive cells in the model group was significantly increased in ovarian tissue. After 4 weeks of treatment with human umbilical cord mesenchymal stem cells, the expression of IL-17-positive cells in the ovaries significantly decreased, suggesting that human umbilical cord mesenchymal stem cells can alleviate the degree of ovarian inflammation.

[0080] (2) Immunohistochemical detection of IFN-γ expression in tissues IFN-γ is a pro-inflammatory cytokine mainly secreted by Th1 cells, NK cells, and macrophages, playing a central role in autoimmune ovarian injury. In premature ovarian failure, IFN-γ accelerates follicular atresia and ovarian fibrosis by activating the inflammatory cascade and disrupting the follicular microenvironment.

[0081] As attached Figure 11As shown in (a)-(c), the IFN-γ positive signal was mainly located in the cytoplasm of ovarian granulosa cells, appearing as brownish-yellow granules, with the deepest staining and highest density in the granulosa cells surrounding the oocytes; simultaneously, a positive fibrous network distribution was observed in the corpus luteum. The results indicate that, compared with the control group, the number of IFN-γ positive cells in the model group was significantly increased in ovarian tissue. After 4 weeks of treatment with human umbilical cord mesenchymal stem cells, the expression of IFN-γ cells in the ovaries significantly decreased, suggesting that human umbilical cord mesenchymal stem cells can alleviate the degree of ovarian inflammation.

[0082] (3) Immunohistochemical detection of UBE2N expression in tissues UBE2N (ubiquitin-binding enzyme E2N) is a key enzyme that performs K63 ubiquitination modification. It regulates DNA damage repair, inflammatory signaling pathways, and protein homeostasis by catalyzing the formation of non-degradable ubiquitin chains.

[0083] As attached Figure 12 As shown in (a)-(c), UBE2N was mainly located in granulosa cells and oocyte cytoplasm, with positive signals appearing as brownish-red granules. The results indicate that, compared to the control group, the number of UBE2N-positive cells in the model group was significantly increased in ovarian tissue. After 4 weeks of treatment with human umbilical cord mesenchymal stem cells, the expression of UBE2N cells in the ovaries significantly decreased, suggesting that human umbilical cord mesenchymal stem cells can reduce DNA damage and maintain protein homeostasis. Therefore, UBE2N reflects pathological changes earlier at the molecular level, superior to traditional indicators such as estrous cycle and ovarian wet weight.

[0084] In summary, after pZP3 immunomodeling, the model group mice exhibited estrous cycle disorder, characterized by a decreased proportion of anucleate keratinized epithelial cells and an increased proportion of white blood cells in vaginal exfoliated cells. However, after hUC-MSCs intervention, the estrous cycle returned to normal (5-6 days / cycle). Fertility assessment showed that the number of litters in the stem cell treatment group was significantly higher than that in the model group, and the delivery cycle was shortened by one week. Ovarian morphological analysis indicated that the ovarian weight and appearance (pink, plump, spherical) in the treatment group were similar to those in the control group, while the ovaries in the model group were smaller. Histopathological examination showed that the follicles in the treatment group had complete follicular development, abundant granulosa cells, and rounded oocytes without nuclear fragmentation or apoptotic bodies. Flow cytometry revealed that hUC-MSCs could regulate the Th17 / Treg cell ratio, and immunohistochemistry confirmed that the expression of IL-17, IFN-γ, and UBE2N in the ovaries of the treatment group was significantly lower than that in the model group. No toxic reactions or mouse deaths were observed throughout the treatment process, and all mice showed no abnormal changes in diet, activity, or coat color, demonstrating safety.

[0085] Example 11 This embodiment also provides a kit for early diagnosis of early-onset ovarian insufficiency, used to detect the expression level of UBE2N, suitable for detecting the expression level of UBE2N in tissue samples (e.g., ovarian tissue) or peripheral blood samples. The kit mainly includes the following components: (1) qPCR primers or hybridization probes for specific detection of the UBE2N gene; (2) Primary antibody against UBE2N protein and HRP-labeled secondary antibody (e.g., HRP-labeled); (3) DAB chromogenic solution, buffer solution, and mounting medium; (4) Positive control, negative control and standard curve samples; (5) User manual: detailed operating steps and result interpretation standards.

[0086] In this embodiment, the kit extracts RNA or protein from tissues or blood for sample processing, and uses qRT-PCR, immunohistochemistry (IHC) or Western blotting for detection. The expression level of UBE2N is quantified by H-score or Ct value, and the results are analyzed.

[0087] Example 12 This embodiment discloses a pharmaceutical preparation using human umbilical cord mesenchymal stem cells (hUC-MSCs) as an active ingredient or as the sole active ingredient. The pharmaceutical preparation has a therapeutic effect on immunosenescence, particularly premature ovarian insufficiency. It is understood that the human umbilical cord mesenchymal stem cells (hUC-MSCs) as the active ingredient of the drug can exert a superior therapeutic effect.

[0088] In this embodiment, the pharmaceutical preparation may select human umbilical cord mesenchymal stem cells (hUC-MSCs) as the sole active ingredient; it may also include other active ingredients known in the art for treating early-onset ovarian insufficiency. For example, it may include drugs known in the art for treating early-onset ovarian insufficiency.

[0089] In this embodiment, the pharmaceutical preparation comprises a therapeutically effective amount of the human umbilical cord mesenchymal stem cells (hUC-MSCs). It should be noted that the term "therapeutically effective amount" refers to the dosage of the drug required to produce an effective effect. This "therapeutically effective amount" can be adjusted and varied according to actual circumstances and is ultimately determined by medical personnel, taking into account factors such as the route of administration and the nature of the preparation, the recipient's weight, age, and other general characteristics, as well as the nature and severity of the disease being treated.

[0090] In this embodiment, the pharmaceutical preparation is a pharmaceutically acceptable intravenous injection formulation. The pharmaceutical preparation also includes other pharmaceutically acceptable carriers and / or excipients compatible with the human umbilical cord mesenchymal stem cells (hUC-MSCs). It should be noted that "other pharmaceuticals" here refers to drugs that are compatible with the human umbilical cord mesenchymal stem cells (hUC-MSCs) and will not cause inactivation, reduced activity, or hydrolysis of the hUC-MSCs, and that, after synergistic action with the hUC-MSCs, can enhance the therapeutic effect of the hUC-MSCs.

[0091] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. Application of UBE2N gene as a biomarker in detection of immunosenescence.

2. Use according to claim 1, characterized in that, The immunosenescence comprises premature ovarian failure.

3. Use according to claim 1 or 2, characterized in that, The UBE2N is derived from ovarian tissue or peripheral blood.

4. Application of a reagent for detecting biomarker UBE2N in preparation of a product for detecting immunosenescence, characterized in that: The detection product comprises a product for early diagnosis of symptoms of premature ovarian failure; and / or, The reagent for detecting biomarker UBE2N comprises a substance capable of detecting the expression level of UBE2N biomarker; and / or, The detection product comprises a detection kit, a detection membrane strip, a detection chip or a detection system; and / or, The detection sample of the detection product is derived from ovarian tissue or peripheral blood.

5. Application of UBE2N gene as a therapeutic target in preparation of a drug for treating immunosenescence.

6. Use according to claim 5, characterized in that, The immunosenescence comprises premature ovarian failure.

7. Use of umbilical cord mesenchymal stem cells in preparation of a drug for treating premature ovarian failure.

8. Use according to claim 7, characterized in that, The umbilical cord mesenchymal stem cells have the following effects: (1) restoring the estrous cycle and normal morphology of the ovary; (2) promoting follicle development and granulosa cell proliferation in the ovary; (3) improving fertility; (4) regulating the ratio of Th17 / Treg cells; (5) inhibiting IL-17 inflammation and IFN-γ inflammation in the ovary; (6) inhibiting DNA damage of UBE2N gene.

9. A medicament for the treatment of premature ovarian failure, characterized in that, The active ingredient of the drug comprises umbilical cord mesenchymal stem cells.

10. The medicament according to claim 9, characterized in that, The drug uses the umbilical cord mesenchymal stem cells as the only active ingredient.

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