A method for preparing a composition for ovarian transplantation and the composition

Abstract

The application discloses a preparation method of a composition for ovary transplantation and the composition. The composition comprises an antioxidant peptide and a colony stimulating factor. The antioxidant peptide in the composition of the application is attached to the colony stimulating factor, so that the antioxidant peptide can reach a ROS site, the antioxidant peptide can remove active oxygen generated by the ROS site and weaken the oxygen generation capacity of the ROS site, the antioxidant effect is improved, follicle loss is reduced, more follicles are protected, and finally the effect of frozen-thawed ovary tissue transplantation is improved.

Description

Technical Field

[0001] This invention relates to the field of peptides, and more specifically to a method for preparing a composition for ovarian transplantation and the composition thereof. Background Technology

[0002] Currently, the number of newly diagnosed malignant tumor patients is rising, with an increasing proportion of female patients under the age of 30. Given that most young patients still wish to have children, and considering that cytotoxic drugs and radiation therapy used in cancer treatment can severely impair their fertility and even lead to early-onset ovarian insufficiency, fertility preservation is particularly important for these patients. Among existing methods for female fertility preservation, ovarian tissue cryopreservation has become the best choice for young malignant tumor patients due to its advantage of not requiring ovulation induction pretreatment and immediately protecting the patient's endocrine function and fertility. To date, there have been over 200 successful live births worldwide achieved through ovarian tissue cryopreservation.

[0003] Although there are still some technical limitations to the freezing method of ovarian tissue, the main problem that determines the survival of ovarian tissue is that during the frozen-thawed ovarian tissue transplantation process, since ovarian cortical fragments without vascular anastomosis are usually used for transplantation, the graft will experience several days of ischemia and hypoxia in the early stage before the new blood vessels are fully established, resulting in the loss of about 50%-90% of primordial follicles during the ischemia-reperfusion process after transplantation.

[0004] Currently, in the process of frozen-thawed ovarian tissue transplantation, the antioxidant vitamin E is injected locally before transplantation. Vitamin E can reduce the generation sites of reactive oxygen species (ROS) in mitochondria and improve follicle survival rate. However, primordial follicles have poor permeability, making it difficult for vitamin E to reach the ROS generation sites in mitochondria. Therefore, its antioxidant effect is poor, leading to follicle loss during ischemia-reperfusion after transplantation. Summary of the Invention

[0005] This invention aims to address the problem of poor antioxidant effect of vitamin E in the frozen-thawed ovarian tissue transplantation process in existing technologies, and provides a novel composition comprising antioxidant peptides and colony-stimulating factors. In this composition, the antioxidant peptides attach to the colony-stimulating factors, ensuring that the antioxidant peptides can reach ROS sites. The antioxidant peptides can remove reactive oxygen species generated by ROS sites and weaken the ability of ROS sites to generate oxygen, thereby improving the antioxidant effect, reducing follicle loss, protecting more follicles, and ultimately improving the transplantation effect of frozen-thawed ovarian tissue.

[0006] The technical method of the present invention is as follows:

[0007] A composition for ovarian transplantation includes an antioxidant peptide and a colony-stimulating factor.

[0008] The colony-stimulating factor is selected from any one of macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor.

[0009] Furthermore, the ratio of colony-stimulating factor to antioxidant peptide is 1 mL: 10–100 mg.

[0010] Furthermore, the ratio of colony-stimulating factor to antioxidant peptide is 1 mL: 50 mg.

[0011] The antioxidant peptide is SS31.

[0012] Furthermore, the composition includes granulocyte-macrophage colony-stimulating factor and SS31.

[0013] The present invention also provides a method for preparing the above composition, wherein the method comprises fusing antioxidant peptides and colony-stimulating factors to prepare the composition.

[0014] The fusion time is 30 to 40 minutes.

[0015] The present invention provides the use of the above composition in transplanted ovaries.

[0016] The present invention provides that the above composition is traced using rhodamine.

[0017] The present invention has the following beneficial effects:

[0018] In the composition of this invention, the antioxidant peptides attach to the colony-stimulating factor, ensuring that the antioxidant peptides can reach the ROS sites. The antioxidant peptides can remove the reactive oxygen species generated by the ROS sites and weaken the ability of the ROS sites to generate oxygen, thereby improving their antioxidant effect, reducing follicle loss, protecting more follicles, and ultimately improving the effect of frozen-thawed ovarian tissue transplantation. Attached Figure Description

[0019] Figure 1 This is a diagram illustrating the preparation method of the composition;

[0020] Figure 2 This is a tracer map of the composition;

[0021] Figure 3 The fluorescence signal of 10 mg of SS31 is shown in the image.

[0022] Figure 4 The fluorescence signal of SS31 at 30 mg is shown in the image.

[0023] Figure 5 The image shows the fluorescence signal of 50 mg of SS31. Detailed Implementation

[0024] Currently, the possible mechanisms of follicle loss after ovarian tissue cryopreservation and transplantation include: (1) Apoptosis mechanism: mitochondrial damage caused by oxidative stress during ovarian tissue cryopreservation and transplantation induces cell apoptosis through the mitochondrial pathway; (2) Overactivation mechanism: primordial follicles in a quiescent state are overactivated and enter the growth stage, causing a "burst growth" of primordial follicles in a short period of time, followed by atresia of most follicles, resulting in premature depletion of the follicle reserve pool.

[0025] To address the issue of primordial follicle depletion during freezing or embryo transfer, the inventors experimented with adding antioxidants to vitrification solutions. They discovered that adding SS31 could improve the vitrification and cryopreservation of ovaries to some extent. SS31 (HD-Arg-Dmt-Lys-Phe-NH) is a novel mitochondrial-targeting antioxidant peptide belonging to a small-molecule, cell-permeable peptide family. It can target and accumulate on the inner mitochondrial membrane, selectively binding to cardiolipin and accumulating 1000 to 5000 times in mitochondria. Mitochondrial uptake of SS31 is independent of mitochondrial transmembrane potential. However, while adding SS31 to the vitrification solution can improve ovarian freezing to some extent, the results are not ideal.

[0026] The inventors then attempted to study the effect of SS31 on the effects of frozen-thawed ovarian tissue by intraperitoneal injection of SS31 (5 mg / kg / day). The results showed that SS31 could improve the transplantation effect to a certain extent, but the effect was still not obvious. It is speculated that the possible reason is that SS31, as an active peptide drug, has a short half-life, is unstable in blood circulation, and is easily enzymatically degraded, resulting in low bioavailability, which affects the maintenance of the antioxidant stress effect of SS31.

[0027] To improve the bioavailability of SS31, the inventors used a combination of SS31 and human granulocyte-macrophage colony-stimulating factor (GM-CSF) for ovarian tissue transplantation and found that the combination significantly reduced the follicle apoptosis rate.

[0028] Therefore, the present invention provides a composition for ovarian transplantation, comprising an antioxidant peptide and a colony-stimulating factor. Here, the antioxidant peptide is located on a colony-stimulating factor carrier, which enables the antioxidant peptide to remain stable in the bloodstream during transplantation and is less prone to enzymatic degradation.

[0029] In some specific embodiments of the present invention, the colony-stimulating factor is selected from any one of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF). Here, M-CSF, M-CSF, and G-CSF are small molecule proteins or peptides produced and secreted extracellularly by cells, capable of regulating the growth, differentiation, survival, or function of other cells. These small molecule proteins or peptides can maintain blood stability and respond to external stimuli (such as infection, trauma, etc.), thus ensuring that the antioxidant peptides can be stably present at the transplantation site, thereby continuously exerting antioxidant effects during ischemia-reperfusion after ovarian transplantation.

[0030] Preferably, the colony-stimulating factor is granulocyte-macrophage colony-stimulating factor. Here, GM-CSF can act on multiple cell types, especially macrophages and eosinophils, and has a broader immunomodulatory effect. Therefore, the harm to the human body via the GM-CSF carrier is minimal.

[0031] In some specific embodiments of the present invention, the antioxidant peptide is SS31. Specifically, S31 is a novel mitochondrial-targeting antioxidant peptide that can target and locate the inner mitochondrial membrane, selectively bind to cardiolipin, and accumulate 1000 to 5000 times in mitochondria. SS31 can directly scavenge reactive oxygen species (ROS) at the site of ROS generation and bind to cardiolipin in the inner mitochondrial membrane through electrostatic and hydrophobic interactions, thereby protecting mitochondrial function, improving the efficiency of mitochondrial electron transport, and reducing the ability of mitochondria to produce ROS. SS31 can also inhibit the mitochondrial permeability transition pore (mPTP), reduce the production of ROS in mitochondria, and prevent mitochondrial swelling.

[0032] Preferably, the composition comprises granulocyte-macrophage colony-stimulating factor and SS31.

[0033] Optionally, the ratio of colony-stimulating factor to antioxidant peptide is 1 mL: 10–100 mg. For example, the ratio of colony-stimulating factor to antioxidant peptide can be 1 mL: 20 mg, 1 mL: 50 mg, or 1 mL: 80 mg.

[0034] In some specific embodiments of the present invention, the composition is a semi-solid. Here, 1 mL of colony-stimulating factor gel contains 10–100 mg of antioxidant peptides.

[0035] In this invention, the composition may also use a carrier or excipient.

[0036] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners, reagents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilizers, antioxidants, tonicity controlling agents, absorption delay agents, etc. The use of these media and reagents in pharmaceutically active substances is well known in the art. These materials are compatible with other components and harmless to their receptors.

[0037] like Figure 1 As shown, GM-CSF and SS31 are fused together, and SS31 is attached to the GM-CSF carrier.

[0038] The present invention also provides a method for preparing a composition by fusing antioxidant peptides and colony-stimulating factors to obtain the composition.

[0039] The fusion temperature is between 30 and 40°C. For example, the temperature is 37°C.

[0040] The present invention also provides a use of the above-described composition for ovarian transplantation. This use may also include ovarian cryopreservation.

[0041] Here, the composition is applied to the ovarian transplantation site. Specifically, SS31 / GM-CSF significantly increased the number of new blood vessels at the ovarian tissue transplantation interface, suggesting that GM-CSF gel promotes angiogenesis. At 3 and 7 days post-transplantation, the MDA content in the grafts of the SS31 / GM-CSF group significantly decreased, indicating a reduction in oxidative stress levels in the early post-transplantation period. Histological examination clearly showed a higher graft survival rate in the SS31 / GM-CSF group, and most importantly, a significantly increased number of primordial follicles remaining in the grafts. Furthermore, the study found that the mechanism by which SS31 improves graft survival is related to SS31's inhibition of p66Shc phosphorylation during cryopreservation, reducing mitochondrial dynamics disruption, decreasing cytochrome C release and ROS generation, and maintaining intracellular energy metabolism.

[0042] The present invention also provides a method for tracing the above-mentioned composition, wherein the composition is traced using rhodamine. The tracing steps include: mixing an antioxidant peptide (e.g., SS31) with rhodamine, then adding a colony-stimulating factor and mixing to obtain the composition, and then detecting the ovarian transplantation status using the composition.

[0043] Here, the ovaries can also be stained.

[0044] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0045] Unless otherwise stated, the culture media, reagents and solutions used in the following examples are commercially available products or can be prepared by methods known in the art.

[0046] Example 1: Rhodamine-SS31 tracer analysis

[0047] 1. HE staining

[0048] Five ovarian tissue samples from each group were fixed in 4% paraformaldehyde and embedded in paraffin.

[0049] (1) Dewaxing: Place the embedded paraffin slices at 60 degrees Celsius and bake for 1 hour, then soak them in xylene twice, each time for 20 minutes.

[0050] (2) Rehydration: The slices were placed in a gradient of 100%, 100%, 95%, 85%, and 75% alcohol for 5 minutes each time, and finally placed in PBS.

[0051] (3) Staining: The rehydrated paraffin sections were serially sectioned at 4µm, and one section was selected every three sections. The sections were stained with HE and mounted with neutral resin.

[0052] (4) Observation and Counting: Observe under an optical microscope and classify according to Gougeon's follicle grading criteria: Primordial follicle: Oocyte surrounded by a single layer of flattened granulosa cells; Primary follicle: Oocyte surrounded by a single layer of cuboidal granulosa cells; Secondary follicle: Oocyte surrounded by two or more layers of cuboidal granulosa cells, without sinus cavity; Antral follicle: Oocyte surrounded by two or more layers of cuboidal granulosa cells, with sinus cavity formation. Follicle morphological defect classification is determined according to Neto's classification criteria: Type I: No morphological defects, regular follicle and surrounding granulosa cells, uniform distribution of oocyte cytoplasm; Type II: Abnormal oocyte cytoplasm, vacuolar degeneration or eosinophilic cytoplasm; Type III: Abnormal oocyte nucleus, shrunken nucleus, irregular or missing nuclear membrane; Type IV: Degenerate follicle, abnormal follicle morphology or granulosa cells, abnormal oocyte cytoplasm and nucleus. Because the number of primary and secondary follicles in the rabbit ovarian tissue was too small in this experiment, primary and secondary follicles were collectively counted as follicles in the growth phase. Considering the importance of primordial follicles in ovarian tissue freezing and transplantation, only the morphological normality rate of primordial follicles was counted. This section was reviewed jointly by two experienced pathologists and the author. Under a 200x optical microscope, different sections of three samples from each group were observed. Ten fields of view were randomly selected from each section for primordial follicle counting, and the average number of primordial follicles in each field of view was calculated. Morphological defects of primordial follicles were determined under a 400x optical microscope, i.e., the proportion of Type I primordial follicles to the total number of primordial follicles. To avoid double counting, only primordial follicles with visible oocyte nuclei were counted.

[0053] 2. Transmission electron microscopy of ovarian tissue

[0054] Three ovarian tissue samples from each group were used for transmission electron microscopy.

[0055] (1) Fixation: Ovarian tissue was pre-fixed with 3% glutaraldehyde and then fixed with 1% osmium tetroxide.

[0056] (2) Dehydration: Acetone is dehydrated step by step, with the concentration gradient of the dehydrating agent being 30% → 50% → 70% → 80% → 90% → 95% → 100% (the 100% concentration is changed 3 times).

[0057] (3) Infiltration and embedding: Dehydrating agent and Epon812 embedding agent in ratios of 3:1, 1:1 and 1:3 respectively, and finally Epon812 embedding.

[0058] (4) Ultrathin sectioning: Ultrathin sections of about 60-90 nm are prepared using an ultrathin slicer, spread out, and then placed on a copper mesh.

[0059] (5) Staining: First stain with uranium acetate for 10-15 minutes, then stain with lead citrate for 1-2 minutes at room temperature.

[0060] (6) Observation and Counting: Transmission electron microscopy was used to acquire images of copper mesh and observe the ultrastructure of oocytes, granulosa cells, and their intracellular organelles within primordial follicles. The morphology of mitochondria in oocytes and granulosa cells was assessed. The criteria for mitochondrial morphology were: normal mitochondria had intact structures, complete parallel cristae, and uniform matrix density; abnormal mitochondria exhibited greater heterogeneity in shape, with mitochondrial swelling, rupture of the outer mitochondrial membrane, and breakage or disappearance of the mitochondrial cristae structure. The mitochondrial structure in oocytes and granulosa cells of primordial follicles was observed at 13000x magnification, and identification was based on mitochondrial ultrastructural damage. Three samples were observed in each group, and the mitochondrial structure of oocytes and granulosa cells from four primordial follicles within each group was randomly observed. At least ten mitochondria from five randomly selected regions within each follicle were counted to calculate the abnormal mitochondrial ratio.

[0061] 3. Immunohistochemical detection of TUNEL staining in ovarian tissue

[0062] (1) Dewaxing: The sections were placed in xylene I for 10 min, xylene II for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0063] (2) Proteinase K retrieval: After slightly drying the slides, draw circles around the tissue with a histochemical pen (to prevent fluid from flowing away). Add proteinase K working solution to the circle to cover the tissue, and incubate at 37°C for 22 min. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 min each time. (Preparation method of proteinase K working solution: stock solution: PBS = 1:9).

[0064] (3) Membrane breaking: After slightly drying the slide, add the membrane breaking working solution to the circle to cover the tissue, incubate at room temperature for 20 min, place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker, 5 min each time. (The membrane breaking solution is 0.1% triton. Preparation method: triton stock solution: PBS = 1:1000).

[0065] (4) Room temperature equilibration: After slightly drying the slices, add buffer to the circle to cover the tissue, and incubate the buffer at room temperature for 10 min.

[0066] (5) Add reaction solution: Take an appropriate amount of TDT enzyme, dUTP and buffer from the TUNEL kit according to the number of slides and the size of the tissue, mix them in a ratio of 1:5:50, add them to the circle to cover the tissue, place the slides flat in the humidified box, and incubate at 37°C for 2 hours. Add a small amount of water to the humidified box to maintain humidity.

[0067] (6) Add substrate: Add 50-100 μL of DAB substrate to the tissue and react for 10 min (15-25 °C); rinse 3 times with PBS.

[0068] (7) Counterstaining: Hematoxylin counterstaining, rinse with deionized water immediately after a few seconds, and mount with neutral resin.

[0069] (8) Observation and counting: Observe, count and photograph under an optical microscope. Oocyte apoptosis is characterized by a clear brownish-yellow or brownish-brown nucleus against a purplish-blue background. Mark each slide with the oocyte nucleus as a marker under 200x magnification, and randomly select 10 fields of view to count the total number of oocytes and the number of apoptotic oocytes, and calculate the oocyte apoptosis rate.

[0070] 4. Immunofluorescence assay for TUNEL staining of ovarian tissue

[0071] Steps (1) to (5) are the same as steps (1) to (5) in the TUNEL staining of ovarian tissue by immunohistochemistry.

[0072] (6) DAPI counterstaining of cell nuclei: Wash sections three times with PBS (pH 7.4) for 5 min each time. After removing the PBS, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min.

[0073] (7) Mounting: Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time. Shake the sections slightly dry and mount them with anti-fluorescence quenching mounting medium.

[0074] (8) Microscopic examination and photography: The slides were observed and images were acquired under a fluorescence microscope. (DAPI is excited by ultraviolet light at a wavelength of 330-380nm and emits blue light at a wavelength of 420nm; FITC is excited by ultraviolet light at a wavelength of 465-495nm and emits green light at a wavelength of 515-555nm.)

[0075] (9) Result Interpretation: DAPI-stained cell nuclei appear blue under UV excitation, while the kit uses FITC-labeled fluorescein, resulting in green nuclei for positive apoptotic cells. Three different sections from each group were selected for observation. Five fields of view were randomly selected from each section for photographing. ImageJ software was used to count the total number of positively stained cells and mesenchymal cells. The ratio of TUNEL-positive mesenchymal cells to the total number of mesenchymal cells was calculated.

[0076] 5. Establish a GM-CSF gel-loaded SS31 co-transplantation model with ovarian tissue.

[0077] First, fluorescently tagged SS31 (rhodamine-SS31) was synthesized. Rhodamine-SS31 was then used in vivo to confirm its tracking effect after co-transplantation with the ovary. 50 mg of rhodamine-SS31 was mixed thoroughly with a gel and transplanted with a fresh right ovarian cortex fragment, while the left ovary served as a control and was left untreated. In vivo imaging was performed immediately after the procedure. Figure 2 As shown, in vivo imaging revealed a significant fluorescent signal at the right fresh ovary transplantation site in the rabbit, while no fluorescent signal was observed at the left control site. These results clearly demonstrate that in vivo imaging can be used to trace Rhodamine-SS31.

[0078] 6. Rhodamine-SS31 tracer analysis

[0079] The tracer effect of rhodamine-SS31 was confirmed. Therefore, in vivo imaging was performed 3 days after co-transplantation of fresh ovarian cortical fragments with gels loaded with different doses of rhodamine-SS31. This revealed that... Figure 3 , 4 As shown in Figure 5, when SS31 was 10mg, almost no fluorescence signal was detected 3 days after surgery; when SS31 was 30mg, a weak fluorescence signal was visible 3 days after surgery; and when SS31 was 50mg, a stronger fluorescence signal was visible 3 days after surgery, indicating that 50mg SS31 performed better.

[0080] Example 2 Preparation of Composition

[0081] (1) 50 mg of SS31 was fully fused with 1 mL of GM-CSF gel to prepare an SS31 gel composition.

[0082] (2) Use ophthalmic scissors to gently scrape the surface of the retroperitoneum in the female rabbit's pelvis, and create a wound of about 2cm×2cm at the retroperitoneum of the ovary; apply the SS31 and GM-CSF gel combination evenly to the wound (the length and width of the application are greater than the wound, and the thickness of the application is about 1mm, to create a transplant "base bed" structure).

[0083] (3) Two fresh ovarian cortical fragments (1-1.5cm long, 0.3-0.5cm wide, and 1mm thick) were attached to the surface of the SS31 gel composition and sutured with two stitches of 6-0 silk thread (one stitch at each end) to fix the ovarian tissue and complete the transplantation surgery; the skin of the rabbit abdomen was sutured, the incision was disinfected, and the rabbit was placed on a warming pad until the anesthesia was relieved.

[0084] Comparative Example 1: Conventional Frozen-Thawed Ovarian Tissue Transplantation Group (OT Group)

[0085] (1) Use ophthalmic scissors to gently scrape the surface of the retroperitoneum in the female rabbit's pelvis and create a wound of about 2cm×2cm at the retroperitoneum of the ovary; apply evenly to the wound (the length and width of the application should be greater than the wound, and the thickness of the application should be about 1mm to create a transplant "base bed" structure).

[0086] (2) Two fresh ovarian cortical fragments (1-1.5cm long, 0.3-0.5cm wide, and 1mm thick) were attached to the peritoneal surface and sutured with two stitches of 6-0 silk thread (one stitch at each end) to fix the ovarian tissue and complete the transplantation surgery; the rabbit abdominal skin was sutured, the incision was disinfected, and the rabbit was placed on a warming pad until the anesthesia was relieved.

[0087] Comparative Example 2OT (frozen-thawed ovarian tissue) and GM-CSF

[0088] (1) Use ophthalmic scissors to gently scrape the surface of the retroperitoneum in the pelvic cavity of the female rabbit and create a wound of about 2cm×2cm at the retroperitoneum of the ovary. Apply GM-CSF gel evenly to the wound (the length and width of the application should be greater than the wound, and the thickness of the application should be about 1mm to create a transplant "base bed" structure).

[0089] (2) Two fresh ovarian cortical fragments (1-1.5cm long, 0.3-0.5cm wide, and 1mm thick) were attached to the peritoneal surface and sutured with two stitches of 6-0 silk thread (one stitch at each end) to fix the ovarian tissue and complete the transplantation surgery; the rabbit abdominal skin was sutured, the incision was disinfected, and the rabbit was placed on a warming pad until the anesthesia was relieved.

[0090] Transplantation effect evaluation

[0091] The transplantation effects were evaluated using Example 2, Comparative Example 2, and Comparative Example 2. Seven days post-transplantation, grafts were subjected to HE staining and TUNEL immunofluorescence staining as described in Example 1 to statistically analyze the primordial follicle density and apoptosis rate of primordial follicles in each group.

[0092] The results showed that the primordial follicle density in the SS31 / GM-CSF+OT group was 40.50±4.95 follicles / unit area, significantly higher than that in the OT group (6.00±1.14) and the GM-CSF+OT group (11.00±2.83), with statistically significant differences (P<0.05). There was no significant difference in primordial follicle density between the OT group and the GM-CSF+OT group (P>0.05).

[0093] The apoptosis rate of primordial follicles in the OT group (0.41±0.02) was significantly higher than that in the SS31 / GM-CSF+OT group (0.10±0.012) and the GM-CSF+OT group (11.00±2.83), with statistically significant differences (P<0.05). The experimental results show that in fresh ovarian transplantation, GM-CSF gel loaded with SS31 can improve the efficacy of autologous ovarian cortex transplantation, and a co-transplantation model of gel-loaded SS31 and ovarian tissue was successfully established.

[0094] To determine the effect of SS31 on the early efficacy of frozen-thawed ovarian tissue transplantation, we performed autologous orthotopic transplantation of rabbit frozen-thawed ovaries using a gel-loaded SS31 transplantation model established in previous experiments. Experimental indicators were measured at two time points: 3 days and 7 days post-transplantation for Example 2, Comparative Example 1, and Comparative Example 2, respectively.

[0095] On days 3 and 7 post-transplantation, five rabbits from each group underwent laparotomy to observe the grafts and collected them for HE staining. In each group, the ovarian grafts remained in place after the freeze-thawed ovarian tissue transplantation, with no tissue detachment. Macroscopically, on day 3 post-transplantation, the ovarian tissue showed a relatively clear boundary with the surrounding tissue, and the tissue had largely fused with the transplant site. However, some grafts showed partial or complete bulging, loose connections and poor fusion with the underlying transplant site, and the tissue color was dark brown, indicating localized or complete graft necrosis. On day 7 post-transplantation, the boundary between the ovarian tissue and the transplant site was unclear, and the tissue was tightly fused with the underlying tissue, making separation difficult. The tissue color was similar to the surrounding tissue, indicating ovarian graft survival. However, some grafts showed localized dark brown bulging, indicating localized graft necrosis.

[0096] HE staining was performed on the grafts, and graft survival was determined based on tissue morphology. In surviving grafts, cell nuclei stained normally, and primordial follicles were visible. In non-surviving grafts, the entire tissue showed deeply stained cell nuclei, indicating cell apoptosis, and no follicles remained. Therefore, the survival rate of ovarian grafts in each group was statistically analyzed. The results are shown in Table 1. The results show that Example 2 showed the highest ovarian tissue transplantation survival rate.

[0097]

[0098] Table 1

[0099] Calculate the density of primordial follicles 3 days and 7 days after embryo transfer.

[0100] Based on the morphological changes of follicles without growth phase surviving in early tissue sections after frozen-thawed ovarian tissue transplantation, the number and state of primordial follicles remaining in the graft are the most important parameters for evaluating the transplantation effect. At 3 and 7 days after frozen-thawed ovarian tissue transplantation, grafts from 5 rabbits in each group were harvested and stained with hematoxylin and eosin (HE). The number of primordial follicles in each group was counted under a microscope, and the density of primordial follicles in each graft was calculated.

[0101] Three days after embryo transfer, the primordial follicle densities of Comparative Example 1 and Comparative Example 2 were 5.55±0.52 and 7.53±0.52, respectively, with no statistically significant difference between the two groups (P>0.05). The primordial follicle density of Example 2 was 19.21±2.01, which was significantly higher than that of Comparative Example 1 and Comparative Example 2, and the difference was statistically significant (P<0.05).

[0102] Seven days after embryo transfer, the primordial follicle density in Comparative Example 1 was 5.89±2.20, which was significantly lower than that in Comparative Example 2 (21.45±9.06) and Comparative Example 5 (36.11±2.01), and the difference was statistically significant (P<0.05).

[0103] Calculate the apoptosis rate of primordial follicles 3 days and 7 days after embryo transfer.

[0104] To determine the apoptosis status of surviving primordial follicles, TUNEL immunofluorescence staining was performed on surviving grafts from each group, and the apoptosis rate of primordial follicles was calculated at 3 and 7 days after ovarian tissue transplantation in each group.

[0105] Three days after embryo transfer, the apoptosis rates of primordial follicles in Comparative Example 1, Comparative Example 2, and Example 2 were 0.09±0.014, 0.08±0.021, and 0.06±0.014, respectively, with no significant difference among the three groups (P>0.05).

[0106] Seven days after embryo transfer, the apoptosis rates of primordial follicles in Comparative Example 1, Comparative Example 2, and Example 2 were 0.12±0.013, 0.10±0.008, and 0.09±0.012, respectively, with no significant difference among the three groups (P>0.05).

[0107] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the technical solutions and core ideas of this application. Those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. The use of a composition in the preparation of a drug for ovarian transplantation, characterized in that, It includes an antioxidant peptide and a colony-stimulating factor, wherein the antioxidant peptide is SS31 and the colony-stimulating factor is a granulocyte-macrophage colony-stimulating factor, and SS31 is attached to a granulocyte-macrophage colony-stimulating factor gel. The method for preparing the composition includes: The composition was prepared by fusing antioxidant peptides and colony-stimulating factor gels.

2. The use according to claim 1, characterized in that, The ratio of colony-stimulating factor to antioxidant peptide is 1 mL: 10–100 mg.

3. The use according to claim 2, characterized in that, The ratio of colony-stimulating factor to antioxidant peptide was 1 mL: 50 mg.

4. The use according to claim 1, characterized in that, The fusion temperature is 30–40℃.

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