Application of umbilical cord mesenchymal stem cells in the treatment of early-onset ovarian insufficiency

Abstract

This invention discloses an application of umbilical cord mesenchymal stem cells (UC-MSCs) in the treatment of early-onset ovarian insufficiency (POI). The core of this method lies in pretreating UC-MSCs with a TAT-IGF-1R-Wnt3a fusion protein to enhance the therapeutic effect. The fusion protein consists of a TAT transmembrane peptide, an IGF-1R active fragment, and a Wnt3a active fragment linked in tandem via a flexible linker (G4S)2. It is obtained through recombinant expression and purification using an *E. coli* expression system. Transplanting UC-MSCs pretreated with this fusion protein into a POI mouse model significantly improves estrous cycle disorders, regulates serum hormone levels (decreasing FSH and increasing AMH, E2, and P), and increases the ovarian index. Its therapeutic effect is superior to that of untreated UC-MSCs and the commonly used estradiol valerate treatment regimen. This invention provides a new clinical treatment strategy for POI and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical treatment technology, and more specifically, to the application of umbilical cord mesenchymal stem cells in the treatment of early-onset ovarian insufficiency, wherein the umbilical cord mesenchymal stem cells are pretreated with TAT-IGF-1R-Wnt3a fusion protein. Background Technology

[0002] Early-onset ovarian insufficiency (POI) is a reproductive health condition with an increasingly high incidence rate in the field of gynecology. It specifically refers to the decline or failure of ovarian reserve in women before the age of 40. Clinical manifestations include oligomenorrhea, amenorrhea, and infertility, accompanied by elevated serum follicle-stimulating hormone (FSH) levels (≥25 mIU / mL) and decreased anti-Müllerian hormone (AMH) levels (<1.1 ng / mL). According to clinical data, the global incidence of POI has reached 1%-3%, and the proportion of young patients continues to increase due to environmental pollutant exposure, mental stress, chemotherapy drug abuse, and genetic factors. It not only seriously affects women's fertility but also induces long-term complications such as osteoporosis and cardiovascular disease due to estrogen deficiency, reducing the quality of life of patients and placing a heavy burden on families and society.

[0003] Current clinical treatment for POI primarily focuses on symptomatic intervention, with hormone replacement therapy (HRT) as the core regimen. Commonly used medications include estradiol valerate and combined estrogen-progestin preparations. While these regimens can temporarily alleviate symptoms of low estrogen such as hot flashes and insomnia, they cannot reverse ovarian tissue damage, and long-term use carries risks of thrombosis, breast hyperplasia, and even cancer, making them particularly unsuitable for patients desiring fertility. Furthermore, while assisted reproductive technologies (such as in vitro fertilization) offer fertility possibilities for some patients, the success rate is less than 10% due to the extremely low number of high-quality follicles in the ovaries of POI patients, making it difficult to meet clinical needs.

[0004] Stem cell therapy, as a core technology of regenerative medicine, offers a new direction for radical treatment of point of injury (POI). Among these, umbilical cord mesenchymal stem cells (UC-MSCs) have become the preferred seed cells for POI cell therapy due to their convenient source (newborn umbilical cord tissue), low immunogenicity (not expressing major histocompatibility complex class II molecules), strong proliferative capacity, and ability to secrete various pro-repair cytokines (such as VEGF and IGF-1). However, current research indicates that simple UC-MSC transplantation suffers from low cell colonization rates (less than 5%), short survival times, and insufficient functional activation, leading to unstable treatment outcomes. In some patients, postoperative ovarian function improvement lasts for less than 6 months. Although some studies have attempted to enhance UC-MSC function through pretreatment with single cytokines (such as IGF-1 and Wnt3a), the limited effectiveness due to single-target action and insufficient signaling pathway activation hinders clinical translation. Therefore, developing a safe and efficient strategy for enhancing UC-MSC function is crucial for solving the challenges of POI treatment. Summary of the Invention

[0005] To address the technical problems of high risks associated with hormone replacement therapy and unstable efficacy of simple umbilical cord mesenchymal stem cell (UC-MSC) transplantation in the treatment of early-onset ovarian insufficiency (POI), this invention first provides an umbilical cord mesenchymal stem cell for the treatment of POI.

[0006] In some embodiments, the core improvement of the umbilical cord mesenchymal stem cells lies in the pretreatment with the TAT-IGF-1R-Wnt3a fusion protein: the fusion protein is composed of a TAT membrane-penetrating peptide, an IGF-1R active fragment, and a Wnt3a active fragment connected in series via a flexible linker (G4S)2. In some embodiments, the amino acid sequence of the TAT-IGF-1R-Wnt3a fusion protein is as shown in SEQ ID NO:4.

[0007] In some embodiments, the amino acid sequence of the TAT transmembrane peptide, as shown in SEQ ID NO:1, can promote the entry of the fusion protein into the cell to exert its function; the amino acid sequence of the IGF-1R active fragment, as shown in SEQ ID NO:2, can regulate cell proliferation and survival; and the amino acid sequence of the Wnt3a active fragment, as shown in SEQ ID NO:3, can activate ovarian repair-related signaling pathways.

[0008] In some embodiments, the pretreatment process specifically involves: seeding logarithmically growing umbilical cord mesenchymal stem cells into a culture plate, culturing for 24 hours until the cell confluence reaches 60%-70%, adding the TAT-IGF-1R-Wnt3a fusion protein at a final concentration of 10 μg / mL, incubating in a 37°C, 5% CO2 incubator for 24 hours, and then gently washing twice with PBS buffer to remove residual protein, thus obtaining pretreated umbilical cord mesenchymal stem cells. These cells exhibit significantly enhanced proliferation activity compared to untreated cells, and a marked increase in the secretion of repair-related cytokines such as VEGF and IGF-1.

[0009] This invention also provides a method for preparing the above-mentioned TAT-IGF-1R-Wnt3a fusion protein, the method comprising the following steps:

[0010] Recombinant vector construction: Based on the amino acid sequence (SEQ ID NO:4) of the TAT-IGF-1R-Wnt3a fusion protein, the corresponding nucleotide sequence was synthesized. This nucleotide sequence and the pET-28a (+) vector were double-digested with BamHI and XhoI (the digestion system was 50 μL, containing 2 μg of vector, 5 μg of target gene, 2 μL each of the two restriction endonucleases, and 5 μL of 10× digestion buffer), and incubated at 37℃ for 4 h. Subsequently, the digested vector and the target gene were mixed at a molar ratio of 1:3, T4 DNA ligase was added, and ligation was carried out overnight at 16℃ to construct the recombinant expression vector pET-28a-TAT-IGF-1R-Wnt3a. Sequencing verification confirmed that the target gene was free of mutations and the insertion direction was correct.

[0011] Induction of expression by engineered bacteria: The recombinant expression vector was transformed into *E. coli* BL21 (DE3) competent cells using the heat shock method. 5 μL of the recombinant vector was mixed with 100 μL of competent cells, incubated on ice for 30 min, followed by heat shock at 42°C for 90 s, and then immediately incubated on ice for 2 min. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. 200 μL of the recovery solution was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12-16 h to screen for positive clones. Single positive clones were picked and inoculated into LB liquid medium containing kanamycin and shaken at 37°C and 220 rpm until OD (out of 1). 600 =0.6-0.8, add IPTG to a final concentration of 0.5mM, and induce at 25℃ and 180r / min for 8h;

[0012] Protein purification and desalting: Centrifuge at 6000×g for 10 min to collect induced bacterial cells. Resuspend the cells in pre-chilled PBS buffer (pH 7.4) containing 1 mM PMSF at a ratio of 1:10 (g / mL). Sonicate at 300W power for 30 min with 3 s working time followed by 5 s intervals (ice bath to prevent protein denaturation). Centrifuge at 12000×g for 20 min at 4℃ to collect the supernatant. Purify using a Ni-NTA affinity chromatography column (5 mL). Wash with equilibration buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0) containing 20 mM imidazole until the baseline stabilizes. After loading the sample, elute with equilibration buffer containing 50 mM imidazole, followed by elution with elution buffer containing 250 mM imidazole. Transfer the eluted target protein to a dialysis bag with a molecular weight cutoff of 10 kDa and elute with PBS at 4℃. Dialyze the protein in buffer (pH 7.4) for 24 hours (replace the buffer every 4 hours) to obtain TAT-IGF-1R-Wnt3a fusion protein with a purity ≥ 95%.

[0013] Finally, this invention provides an application in which the above-mentioned umbilical cord mesenchymal stem cells pretreated with TAT-IGF-1R-Wnt3a fusion protein are used in the preparation of a drug for treating early-onset ovarian insufficiency.

[0014] In some embodiments, the preparation of the drug requires first digesting pretreated umbilical cord mesenchymal stem cells with 0.25% trypsin-EDTA digestion solution at 37°C for 5 min, then centrifuging at 1000×g for 5 min to collect the cells, resuspending them in physiological saline, and adjusting the cell concentration to 1×10⁻⁶. 6 Cell viability was ≥90% as determined by trypan blue staining; the drug was administered via tail vein injection, with each subject receiving 0.2 mL (containing 2 × 10⁻⁶ cells / mL). 5 (Pretreated umbilical cord mesenchymal stem cells), administered once weekly for 4 consecutive weeks; the drug can treat early-onset ovarian insufficiency by improving the estrous cycle, reducing serum follicle-stimulating hormone (FSH) levels, increasing anti-Müllerian hormone (AMH), estradiol (E2) and progesterone (P) levels, and increasing the ovarian index. Its efficacy is superior to simple umbilical cord mesenchymal stem cell transplantation and the clinically commonly used estradiol valerate treatment regimen.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] This invention is the first to construct a TAT-IGF-1R-Wnt3a fusion protein, which combines a membrane-penetrating peptide with two functionally active fragments to achieve multi-target synergistic effects. The pretreatment effect is significantly better than that of a single component. The fusion protein is prepared using an E. coli expression system, which is simple, low-cost, and achieves a purity of over 95%, making it suitable for large-scale production. Umbilical cord mesenchymal stem cells pretreated with this fusion protein show significantly enhanced proliferation activity, clear therapeutic effects on POI after transplantation, and high safety with no significant adverse reaction risk. This invention provides a new treatment option for early-onset ovarian insufficiency, overcomes the limitations of existing treatment methods, and has important clinical translational value. Attached Figure Description

[0017] Figure 1 This is an SDS-PAGE electrophoresis image of the TAT-IGF-1R-Wnt3a fusion protein.

[0018] Figure 2 To investigate the effect of fusion protein pretreatment on the proliferation activity of umbilical cord mesenchymal stem cells using the CCK-8 assay.

[0019] Figure 3 To verify the activation effect of the TAT-IGF-1R-Wnt3a fusion protein using Western blot.

[0020] Figure 4 A comparison chart of estrous cycle recovery rates in POI mice from different groups.

[0021] Figure 5 A comparison chart of ovarian indices in mice from different groups. Detailed Implementation

[0022] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1: Preparation of TAT-IGF-1R-Wnt3a fusion protein

[0024] Based on NCBI information, the amino acid sequence information of the active fragments of TAT, IGF-1R, and Wnt3a was retrieved:

[0025] TAT: YGRKKRRQRRR (SEQ ID NO:1);

[0026] Insulin-like growth factor 1 receptor (PDB: 3F5P_F) active fragment (activity verified by Swiss-Model structure prediction):

[0027] GSFSAADVYVPDEWEVAREKITMSRELGQGSFGMVYEGVAKGVVKDEPETRVAIKTVNEAASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPTLVIMELMTRGDLKSYLRSLRPEMENNPVLAPPSLSKMIQMAGEIADGMAYLNANKFVHRDLAARNCMVAEDFTVKIGDFGMTRDI (SEQ ID NO: 2);

[0028] Wnt3a (GenBank: BAB61052.1) active fragment (via Swiss-Model Structure prediction verification activity): GPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHN NEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK (SEQ ID NO:3);

[0029] Connectors: Flexible sequence (G4S)2 (GGGGSGGGGS) is used to connect TAT to IGF-1R and IGF-1R to Wnt3a respectively (to reduce spatial steric hindrance).

[0030] Construct the amino acid sequence of the TAT-IGF-1R-Wnt3a fusion protein:

[0031] (SEQ ID NO:4);

[0032] The full-length fusion protein sequence (SEQ ID NO:4) was synthesized by Nanjing Genscript Biotech Co., Ltd. The pET-28a(+) vector was used as the E. coli expression system. This vector contains a His tag for easy affinity purification and has multiple restriction enzyme sites (BamHI and XhoI) for targeted cloning of the target gene. During vector construction, the synthesized nucleotide sequence and the pET-28a(+) vector were first double-digested separately. The digestion system consisted of 50 μL (containing 2 μg of vector, 5 μg of target gene, 2 μL each of the two restriction endonucleases, and 5 μL of 10× digestion buffer), and incubated at 37°C for 4 h. Subsequently, ligation was performed overnight at 16°C using T4 DNA ligase to construct the recombinant expression vector pET-28a-TAT-IGF-1R-Wnt3a. The molar ratio of vector to target gene in the ligation system was controlled at 1:3 to improve ligation efficiency. Finally, sequencing verification confirmed that the target gene was mutation-free and the insertion direction was correct.

[0033] The recombinant vector was transformed into *E. coli* BL21(DE3) competent cells using a heat shock method. The specific procedure was as follows: 5 μL of recombinant vector was mixed with 100 μL of competent cells, incubated on ice for 30 min, then heat-shocked at 42°C for 90 s, followed by an immediate ice incubation for 2 min. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. Subsequently, 200 μL of the recovery solution was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 12–16 h. Positive clones were then screened. Single-positive colonies were picked and inoculated into 5 mL of LB liquid medium containing kanamycin, and shaken at 37°C and 220 rpm until the culture temperature reached OD. 600 =0.6-0.8 (approximately 4-6 hours). This stage is the early logarithmic growth phase, where vigorous cell metabolism is conducive to protein expression. IPTG was added to a final concentration of 0.5 mM for induction at 25°C and 180 rpm for 8 hours. This temperature reduces inclusion body formation. OD was measured every 2 hours during induction. 600 To ensure the cells do not enter the senescence phase, after induction, centrifuge at 6000×g for 10 min to collect the cells, discard the supernatant, and store the cell pellet at -20℃ for later use.

[0034] The frozen bacterial cells were resuspended in pre-chilled PBS buffer (pH 7.4, containing 1 mM PMSF protease inhibitor) at a ratio of 1:10 (g / mL), and vortexed until the cells were completely dispersed. The cells were then sonicated at 300W power for 3 seconds followed by 5 seconds of rest, for a total sonication time of 30 min. The sonication process was performed in an ice bath to prevent protein denaturation. After sonication, the cells were centrifuged at 12000×g for 20 min at 4°C, and the supernatant containing the target protein was collected.

[0035] The target protein was purified using a Ni-NTA affinity chromatography column (GE Healthcare, 5 mL). The column was first washed with equilibration buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) until the baseline stabilized, at a flow rate of 1 mL / min. The crude extract supernatant or renatured protein solution was slowly loaded, and the breakthrough was collected. Subsequently, impurities were eluted with equilibration buffer containing 50 mM imidazole until A... 280The baseline was restored; finally, the target protein was eluted with elution buffer (50mM Tris-HCl, 300mM NaCl, 250mM imidazole, pH 8.0), collecting 2mL from each tube. Protein purity in each tube was determined by SDS-PAGE, and the fractions containing the fusion protein were combined. The combined fusion protein solution was transferred to a dialysis bag (molecular weight cutoff 10kDa), air bubbles were squeezed out, and the bag was sealed with a dialysis clamp. It was then placed in sufficient PBS buffer (pH 7.4) and dialyzed at 4°C with magnetic stirring. The buffer was changed every 4 hours for a total of 24 hours to completely remove imidazole and high-concentration salt ions. After dialysis, the protein solution was removed and centrifuged at 12000×g for 10 min at 4°C to remove the precipitate, yielding the desalted recombinant fusion protein solution. SDS-PAGE analysis was performed. (See figure). Figure 1 .

[0036] Figure 1 The results showed that specific bands were detected by SDS-PAGE, which were close to the theoretical molecular weight of 59.28 kDa, and the purity was over 95% as determined by grayscale analysis.

[0037] Example 2: Pretreatment of umbilical cord mesenchymal stem cells with TAT-IGF-1R-Wnt3a fusion protein

[0038] Logarithmic growth phase UC-MSCs (purchased from Shanghai Xuanke Biotechnology Co., Ltd., catalog number: XK-XB-2567) were cultured at 5 × 10⁻⁶. 3 Seeds were planted at a density of 2 × 10⁶ cells / well in a 96-well plate. 5 Cells were seeded per well in a 6-well plate, and cultured in complete culture medium for 24 hours. When the cell adhesion and confluence reached 60%-70%, pretreatment was performed.

[0039] The experiment was divided into two groups, with three replicates in each group. ① Negative control group: BSA was added to a final concentration of 10 μg / mL; ② Experimental group: TAT-IGF-1R-Wnt3a fusion protein was added to a final concentration of 10 μg / mL. Both groups were pretreated in a 37℃, 5% CO2 incubator for 24 h.

[0040] After pretreatment, each group of cells was gently washed twice with PBS buffer to remove residual proteins, and then cultured in complete culture medium for subsequent bioactivity assays.

[0041] Proliferation activity assay: The CCK-8 assay was used. After pretreatment, 10 μL of CCK-8 reagent was added to each well after 24 h, 48 h, and 72 h of incubation. The absorbance at 450 nm was measured after 2 h of incubation. (See attached image). Figure 2 .

[0042] Figure 2The results showed that the absorbance values ​​of the experimental group at each time point were higher than those of the negative control group, and the proliferation rate at 72h was significantly higher than that of the blank control group (P<0.01).

[0043] Signaling pathway activation detection: Focusing on the two core pathways PI3K / Akt and Wnt / β-catenin, the activation effect of the fusion protein was verified by Western blot.

[0044] Reagents and Sample Preparation: Specific primary antibodies (all purchased from Cell Signaling Technology) were used: phosphorylated Akt (p-Akt, Ser473, catalog number 4060), total Akt (t-Akt, catalog number 4691), phosphorylated β-catenin (p-β-catenin, Ser552, catalog number 9566), total β-catenin (t-β-catenin, catalog number 8480), and internal control protein GAPDH (catalog number 5174). The secondary antibody was HRP-labeled goat anti-rabbit IgG (catalog number 7074). Cells from each group were collected after 24 hours of pretreatment. 150 μL of IRIPA lysis buffer was added to each well, and lysis was performed on ice for 30 min. The cells were centrifuged at 12000×g and 4℃ for 15 min, and the supernatant was collected. Protein concentration was quantified using the BCA method to ensure standardized loading volume (30 μg protein per well).

[0045] Electrophoresis and transfer: Prepare a 10% separating gel and a 5% stacking gel. Mix the protein sample with 5× loading buffer at a 4:1 ratio, denature at 95℃ for 5 min, and then load the sample. Electrophore at a constant voltage of 80V until the protein enters the separating gel, then adjust to 120V and continue electrophoresis for 90 min. Transfer the protein to a PVDF membrane (Millipore, catalog number IPVH00010) using the wet transfer method. Transfer at a constant current of 300mA for 90 min. After transfer, block with 5% skim milk at room temperature for 2 h.

[0046] Incubation and development: Dilute the primary antibody at a ratio of 1:1000 and incubate the membrane overnight at 4°C; wash the membrane three times (10 min each time) with TBST buffer, add the secondary antibody diluted 1:5000, and incubate at room temperature for 1 h; after washing the membrane again, add ECL chemiluminescence solution (Thermo, catalog number 32106), acquire images using a chemiluminescence imaging system (Bio-Rad, ChemiDoc XRS+), and quantify the gray values ​​of the bands using ImageJ software.

[0047] Results analysis: The gray values ​​of the target protein were corrected using GAPDH gray values, and the ratios of p-Akt / Akt and p-β-catenin / β-catenin were calculated. (See attached...) Figure 3 .

[0048] Figure 3The results showed that the p-Akt / Akt ratio in the experimental group was 1.82±0.15, which was significantly higher than that in the negative control group (1.05±0.09) (P<0.01); the p-β-catenin / β-catenin ratio was 1.76±0.13, which was also significantly higher than that in the negative control group (1.08±0.10) (P<0.01).

[0049] This indicates that in the TAT-IGF-1R-Wnt3a fusion protein, the IGF-1R active domain specifically binds to the IGF-1 receptor on the surface of UC-MSCs, activating the downstream PI3K / Akt pathway; the Wnt3a active domain, upon binding to the Frizzled receptor, inhibits β-catenin phosphorylation and degradation, increasing its active form. These two pathways synergistically promote UC-MSC proliferation, and the experimental results confirm that the fusion protein enhances the biological activity of umbilical cord mesenchymal stem cells by targeting and activating core pathways.

[0050] Example 3: Experiment on the improvement of premature ovarian insufficiency (POI) mouse model by pretreated umbilical cord mesenchymal stem cells.

[0051] Sixty 6-8 week old SPF-grade female ICR mice, weighing 18-22g, were purchased from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd. All mice were housed in a barrier environment with a temperature controlled at 20-24℃, humidity at 50%-60%, 12-hour alternating light and dark lighting, and ventilation at 15-20 times / hour. They had free access to food and water and underwent one week of acclimatization before the experiments were conducted.

[0052] After one week of acclimatization, mice with normal estrous cycles were selected for modeling by observing vaginal smears for three consecutive days. Modeling method: Each mouse was injected intraperitoneally with cyclophosphamide 150 mg / kg once. Starting on the second day after injection, vaginal smears were taken daily to observe the estrous cycle for seven consecutive days. Successful modeling was defined as estrous cycle disorder (cycle duration >5 days or irregular) or arrest in a certain phase. Mice with successful modeling were included in subsequent experiments.

[0053] Forty-eight POI mice that successfully developed the model were randomly divided into four groups of 12 mice each. Twelve normal mice that did not develop the model were used as a blank control group. The specific groupings are as follows:

[0054] Blank control group: Normal mice, without modeling, were injected with an equal volume of physiological saline via the tail vein once a week for 4 consecutive weeks.

[0055] Model control group: POI model mice were injected with an equal volume of physiological saline via the tail vein once a week for 4 consecutive weeks.

[0056] UC-MSC group: POI model mice were injected with untreated UC-MSC suspension via the tail vein once a week for 4 consecutive weeks.

[0057] Pretreated UC-MSC group (experimental group): POI model mice were injected via tail vein with UC-MSC suspension pretreated with TAT-IGF-1R-Wnt3a fusion protein once a week for 4 consecutive weeks.

[0058] Estradiol group: POI model mice were administered estradiol valerate suspension by gavage daily (the dosage was 0.3 mg·kg⁻¹·d⁻¹ based on the clinical dose), and an equal volume of physiological saline was injected via the tail vein at the same time, once a week for 4 consecutive weeks.

[0059] Preparation of UC-MSC suspension: UC-MSCs in logarithmic growth phase were washed twice with PBS buffer (pH 7.4), digested with 0.25% trypsin-EDTA digestion solution at 37°C for 5 min, and centrifuged at 1000×g for 5 min to collect the cells. The cells were then resuspended in physiological saline and the cell concentration was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL, with trypan blue staining indicating cell viability ≥90%.

[0060] Preparation of UC-MSC suspension for fusion protein pretreatment: Following the method in Example 2, UC-MSCs were prepared at 5 × 10⁻⁶ ppm. 3 Cells were seeded per well in 6-well plates and cultured for 24 hours until confluence reached 60%-70%. Then, TAT-IGF-1R-Wnt3a fusion protein was added to a final concentration of 10 μg / mL, and the cells were pretreated at 37°C in a 5% CO2 incubator for 24 hours. Subsequent cell division included digestion, centrifugation, washing, resuspending, and adjustment of cell concentration to 1 × 10⁻⁶ cells / well. 6 Cells / mL, ensuring cell viability ≥90%.

[0061] Intervention Implementation: Intervention began on day 3 after successful model establishment in each group of mice. In the cell transplantation group, each mouse received a tail vein injection of 0.2 mL of cell suspension (containing 2 × 10⁻⁶ cells). 5 (1 cell), the estradiol group was administered the corresponding dose of estradiol valerate suspension by gavage according to body weight, while the blank control group and the model control group were injected with an equal volume of physiological saline. Gavage was performed once daily, and tail vein injection was performed once a week for 4 consecutive weeks.

[0062] Estrogenic cycle observation: During and after the intervention, vaginal smears were taken at 9:00 AM daily. Cell morphology was observed under a microscope after Wright staining, and estrous cycle changes were recorded. This was observed continuously for 14 days. The number of mice whose estrous cycles returned to normal and the recovery rate (number of mice returning to normal / total number of mice in the group × 100%) were calculated. See [link to relevant documentation]. Figure 4 .

[0063] Figure 4The results showed that the estrous cycle of mice in the blank control group remained normal throughout (recovery rate 100%); the estrous cycle disorder rate in the model control group was 100%, and no mice recovered to normal after intervention (recovery rate 0%); in the UC-MSC group alone, 5 mice recovered to normal estrous cycles, a recovery rate of 41.7%; in the experimental group, 10 mice recovered to normal estrous cycles, a recovery rate of 83.3%; and in the estradiol group, 7 mice recovered to normal estrous cycles, a recovery rate of 58.3%. The recovery rate of estrous cycles in the experimental group was significantly higher than that in the UC-MSC group alone and the estradiol group (P<0.05), and the recovery rate in the estradiol group was higher than that in the UC-MSC group alone (P<0.05).

[0064] Serum hormone level detection: After the intervention, mice were fasted for 12 hours, and blood was collected from the retro-orbital venous plexus. The serum was separated by centrifugation at 2000×g for 10 min at 4℃, aliquoted, and stored at -80℃ for later testing. Serum FSH, AMH, E2, and P levels were detected by enzyme-linked immunosorbent assay (ELISA), strictly following the kit instructions. Each sample was tested in triplicate. The concentrations of each hormone were calculated based on the standard curve, as shown in Table 1.

[0065] Table 1. Serum FSH, AMH, E2, and P levels detected by ELISA in each group.

[0066]

[0067] Note: Compared with the blank control group, P a <0.01, P b <0.05; compared with the model control group, P c <0.01, P d <0.05.

[0068] Table 1 shows that, compared with the blank control group, the serum FSH level in the model control group was significantly increased (P<0.01), while the AMH, E2, and P levels were significantly decreased (P<0.01). Compared with the model control group, the serum FSH level in the UC-MSC group, the experimental group, and the estradiol group was significantly decreased (P<0.01), while the AMH, E2, and P levels were significantly increased (P<0.05 or P<0.01). Among them, the serum FSH level in the experimental group was lower than that in the estradiol group, while the AMH, E2, and P levels were higher than those in the estradiol group, and the differences were statistically significant (P<0.05).

[0069] Ovarian index calculation: After blood collection, mice were euthanized using the carbon dioxide method. The bilateral ovaries were quickly dissected and dissected, surrounding adipose tissue was removed, and the mice were rinsed thoroughly with PBS buffer. Surface moisture was blotted dry with filter paper, and the wet weight of the ovaries was measured using an electronic balance (accurate to 0.1 mg). The ovarian index was calculated (ovarian index = wet weight of ovary (mg) / mouse body weight (g)). See [link to relevant documentation]. Figure 5 .

[0070] Figure 5 The results showed that the ovarian index of the model control group was significantly lower than that of the blank control group (P<0.01), and the UC-MSC group, experimental group, and estradiol group were all significantly higher than those of the model control group (P<0.05). The ovarian index of the experimental group was not significantly different from that of the blank control group, but was higher than that of the UC-MSC group and the estradiol group (P<0.05).

[0071] This experiment successfully constructed a POI mouse model using cyclophosphamide. After transplantation of umbilical cord mesenchymal stem cells pretreated with TAT-IGF-1R-Wnt3a fusion protein, the activation of the two core pathways PI3K / Akt and Wnt / β-catenin significantly improved estrous cycle disorders, regulated serum hormone balance (decreased FSH, increased AMH, E2, and P), and increased ovarian index in POI mice. The effect was superior to that of untreated umbilical cord mesenchymal stem cells and the clinically commonly used estradiol treatment regimen.

[0072] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An umbilical cord mesenchymal stem cell therapy for treating early-onset ovarian insufficiency, characterized in that, The umbilical cord mesenchymal stem cells were pretreated with the TAT-IGF-1R-Wnt3a fusion protein; the fusion protein consisted of a TAT membrane-penetrating peptide, an IGF-1R active fragment, and a Wnt3a active fragment connected by a flexible linker (G4S) 2. The TAT-IGF-1R-Wnt3a fusion protein is tandemly linked; the amino acid sequence of the TAT-IGF-1R-Wnt3a fusion protein is shown in SEQ ID NO:4; the pretreatment includes: seeding logarithmically growing umbilical cord mesenchymal stem cells into a culture plate, adding the TAT-IGF-1R-Wnt3a fusion protein for co-incubation, and washing to remove residual protein.

2. The TAT-IGF-1R-Wnt3a fusion protein as described in claim 1, characterized in that, The amino acid sequence of the TAT transmembrane peptide is shown in SEQ ID NO:1, the amino acid sequence of the IGF-1R active fragment is shown in SEQ ID NO:2, and the amino acid sequence of the Wnt3a active fragment is shown in SEQ ID NO:

3.

3. The method for preparing the TAT-IGF-1R-Wnt3a fusion protein according to claim 2, characterized in that, include: The corresponding nucleotide sequence was ligated with the pET-28a (+) vector by double enzyme digestion to construct a recombinant vector, which was transformed into E. coli BL21 (DE3) for induction expression. The fusion protein with a purity of ≥95% was obtained by Ni-NTA affinity chromatography and PBS dialysis for desalting.

4. The use of the umbilical cord mesenchymal stem cells according to claim 1 in the preparation of a drug for treating early-onset ovarian insufficiency.

5. The application according to claim 4, characterized in that, The drug is used to improve the estrous cycle in patients with early-onset ovarian insufficiency, regulate serum FSH / AMH / E2 / P levels, or increase the ovarian index.

Images