Application of FOS / AP-1 inhibitor in preparation of medicine for improving ovarian tissue vitrification freezing injury

Abstract

The invention relates to application of an FOS / AP-1 inhibitor in preparation of a medicine for improving ovarian tissue vitrification injury. The invention provides a new idea and an intervention target for female fertility preservation based on ovarian vitrification, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to the application of a FOS / AP-1 inhibitor in the preparation of a drug for improving ovarian tissue vitrification and freezing injury. Background Art

[0002] Over the past two decades, advances in cancer treatment have significantly improved survival rates. The reproductive needs of young female cancer patients are increasingly valued. However, treatments such as chemotherapy (especially alkylating agents), radiotherapy, and surgery may cause severe damage to ovarian function, causing primary ovarian insufficiency (POI). At the same time, fertility preservation should be considered before any treatment that may damage the gonads, especially for adolescents and women of childbearing age. Embryo and egg cryopreservation are recognized fertility preservation technologies, while ovarian tissue cryopreservation (OTC) is an emerging and promising method.

[0003] Ovarian tissue cryopreservation has been used for fertility preservation for more than 20 years. Since the first report of successful live birth after autologous transplantation of thawed ovarian cortex in 2004, this technology has been recognized and widely used worldwide. At present, OTC and subsequent transplantation are the only clinical strategies that can simultaneously restore fertility and ovarian endocrine function and avoid POI-related diseases. Cohort meta-analysis showed that more than 90% of women recovered ovarian function after OTC-transplantation. However, the transplantation results of frozen ovaries are significantly inferior to those of fresh ovaries, indicating that there are adverse factors in the OTC process. The live birth rate of in vitro fertilization (IVF) after ovarian tissue cryopreservation and transplantation is only 21%, while the live birth rate after embryo cryopreservation is 41%. Therefore, in order to improve the quality of post-transplant follicle development, it is necessary to clarify the cellular analysis mechanism of damage to internal ovarian cells caused by temperature stress and cryoprotectant toxicity. Finding key targets and drugs to improve ovarian tissue freezing injury caused by toxic substances such as dimethyl sulfoxide (DMSO) in cryoprotectants and drastic temperature changes is an important technical breakthrough at this stage. Summary of the invention

[0004] The present invention provides the use of a FOS / AP-1 inhibitor in the preparation of a drug for improving ovarian tissue vitrification and freezing injury, thereby solving the technical problem in the prior art that key genes regulating ovarian tissue injury and their specific action mechanisms are unclear.

[0005] The present invention provides the use of a FOS / AP-1 inhibitor in preparing a medicine for improving ovarian tissue vitrification and freezing injury.

[0006] Preferably, the FOS / AP-1 pathway is used as an intervention target for reversing vitrification and freezing-thawing damage of ovarian tissue.

[0007] Preferably, the FOS / AP-1 inhibitor is T-5224. Specifically, T-5224 inhibits FOS / AP-1, targets and regulates its downstream molecule EGR1, inhibits the excessive accumulation of inflammatory factors ILs and MMPs in frozen-thawed ovaries, and improves the in vitro culture state of frozen-thawed ovaries, which is mainly manifested in the maintenance of blood vessels and the reduction of apoptotic cells.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0010] The present invention also provides the use of the FOS / AP-1 inhibitor in the preparation of ovarian tissue cryoprotective solution or ovarian 3D culture solution.

[0011] Preferably, the final concentration of T-5224 added to the culture medium is 80 uM.

[0012] The present invention also provides a method for observing that FOS / AP-1 inhibitor improves phenotypes related to ovarian tissue vitrification freeze-thaw injury, comprising:

[0013] (1) In vitro 3D culture and phenotypic observation of mouse ovaries after freezing and thawing;

[0014] (2) Total RNA and protein extraction from mouse ovarian tissue for real-time quantitative PCR and western blot analysis;

[0015] (3) Whole-tissue immunofluorescence staining of mouse ovarian tissue was used to mark blood vessels and extracellular matrix components.

[0016] Beneficial Effects

[0017] The present invention firstly performs transcriptome (scRNA) and spatial transcriptome sequencing (Stereo-seq) analysis at the single-cell level, and compares the changes in cell expression profiles of ovarian tissue after freeze-thaw injury in detail and systematically. It is found that in the more complex operation process of ovarian vitrification and thawing, FOS / AP-1 is a sensitive and rapidly activated signal molecule, and expands the accumulation of inflammatory factors through cascade reactions. Furthermore, an in vitro 3D culture model of mouse ovarian tissue after vitrification was constructed to verify the regulatory role of FOS / AP-1 in ovarian cryoinjury. By taking FOS / AP-1 as the target for improving ovarian damage, T-5224, a FOS / AP-1 inhibitor, is added to the ovarian cryoprotectant and 3D culture medium, the over-expression of EGR1 can be inhibited by inhibiting the over-activation of FOS / AP-1, and finally the excessive accumulation of inflammatory factors such as ILs and MMPs can be reversed. It is manifested as the slowing down of the degradation of the vascular network of the frozen-thawed ovaries cultured in vitro and the reduction of apoptotic cells. Overall, this scheme provides new concepts and intervention targets for female fertility preservation based on ovarian vitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 AE showed that scRNA and Stereo-seq sequencing analysis revealed that FOS / AP-1 was rapidly activated after ovarian freezing and thawing, and targeted EGR1, promoting the accumulation of inflammatory factors such as ILs and MMPs, thereby accelerating ovarian damage after freezing and thawing.

[0019] Figure 2 AC shows the construction of an in vitro 3D culture model after mouse ovarian freezing and thawing, and verifies that the FOS / AP-1 pathway is activated during the ovarian freezing and thawing recovery process.

[0020] Figure 3 This is the processing flow chart of the vitrification thawing + T-5224 culture group.

[0021] Figure 4 AB showed that in the in vitro 3D culture model after mouse ovarian freezing and thawing, the addition of T-5224 inhibited AP-1 and thus improved the phenotype of ovarian damage after freezing and thawing. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] The present invention provides FOS / AP-1 as an important target for improving frozen-thaw ovarian tissue damage. FOS / AP-1 is a dimer formed by the combination of transcription factor c-fos and c-jun protein family, which can be combined in the promoter region and participate in the transcriptional regulation of downstream genes.

[0024] The present invention also provides T-5224 as an inhibitor of FOS / AP-1, which specifically inhibits the binding of FOS to DNA and inhibits the overexpression of its downstream gene EGR1, thereby reducing inflammation accumulation, improving the frozen-thawed ovarian cortex microenvironment, and thereby improving the negative effects of vitrification and protective agent toxicity on ovarian tissue.

[0025] The molecular formula of FOS / AP-1 inhibitor T-5224 is as follows:

[0026]

[0027] The present invention also provides the concentration ratio and use strategy of T-5224 added into ovarian tissue cryoprotective solution and ovarian 3D culture solution.

[0028] The present invention also provides a method for observing T-5524 improving phenotypes related to freeze-thaw injury of ovarian tissue, which is as follows:

[0029] (1) In vitro 3D culture and phenotypic observation of mouse ovaries after freezing and thawing;

[0030] (2) Total RNA and protein extraction from mouse ovarian tissue for real-time quantitative PCR and western blot analysis;

[0031] (3) Whole-tissue immunofluorescence staining of mouse ovarian tissue was used to mark blood vessels and extracellular matrix components.

[0032] FOS (also known as c-Fos) belongs to the activator protein 1 (AP-1) transcription factor superfamily. In order to function, FOS needs to form a heterodimeric complex with another basic leucine zipper domain transcription factor Jun family protein to form an active AP-1 complex. AP-1 is a dimeric protein complex composed of Fos (Fos, FosB, Fosl1, Fosl2), Jun (Jun, Junb, Jund), ATF and JDP family members. FOS / AP-1 has been shown to be widely involved in processes such as cell proliferation, differentiation, hypoxia, angiogenesis, steroidogenesis and prostaglandin (PG) production. These processes are particularly critical for follicular development, ovulation and corpus luteum development. The regulation of FOS / AP-1 activity depends on the concentration and activation level of AP-1 protein after extracellular stimulation. Under physiological conditions, the concentration and activity of FOS / AP-1 are extremely low; when cells are stimulated, the level of FOS / AP-1 protein complex will increase briefly and rapidly. FOS / AP-1 transcription factors can be activated by a variety of factors, including chemokines, growth factors, cytokines, hormones and environmental stress. Activated FOS / AP-1 enters the nucleus and induces the expression of inflammatory cytokines such as ILs, IFN-g and TNF-α, participating in the inflammatory response. Therefore, by inhibiting the activation of FOS / AP-1, the inflammatory response can be reduced, which is beneficial to the maintenance of homeostasis during the in vitro culture of frozen-thawed ovaries.

[0033] Therefore, the present invention aims to clarify the cascade signaling pathway in which FOS / AP-1 is rapidly activated and initiates the accumulation of ovarian inflammation. By constructing a 3D culture model of mouse ovaries after vitrification and freezing and thawing, T-5224 is used to inhibit FOS / AP-1 activation, inhibit EGR1 overexpression, reduce the accumulation of inflammation after ovarian vitrification and freezing and thawing, and explore methods to improve ovarian damage after vitrification and freezing and thawing.

[0034] The present application is described below in conjunction with specific embodiments:

[0035] Example 1

[0036] This example provides scRNA and Stereo-seq sequencing analysis to reveal that FOS / AP-1 is rapidly activated after ovarian freezing and thawing, and targets EGR1, promoting the accumulation of inflammatory factors such as ILs and MMPs, thereby accelerating ovarian damage after freezing and thawing. Specifically, the following steps are included:

[0037] 1. Single cell preparation and library construction (for scRNA-seq):

[0038] First, the ovarian tissue was peeled off to obtain ovarian cortical tissue of about 1 mm thickness, and then divided into a fresh group (Fresh Group) and a vitrified freezing group (Cryo Group). The Cryo group was processed according to the normal ovarian vitrification operation and then frozen in liquid nitrogen for 2 weeks. The Fresh group was treated with collagenase digestion (2 mg / ml collagenase IV was dissolved in DPBS, a total of 3 mL, and preheated to 37°C in a water bath). After obtaining a single cell suspension, DAPI was stained and flow cytometry was performed to obtain DAPI. - of living cells. Before passing through the 10XGenomics microfluidic instrument, the cell concentration was set to 1000 / uL, and the final cell loading was 10,000. Subsequently, GEMs were made and collected, barcodes were added for reverse transcription, and the first-strand cDNA was purified using magnetic beads. After quality inspection and quantification of cDNA, library construction was performed according to the standard protocol (10X Genomics). The library was sequenced using the Illumina NovaSeq platform.

[0039] 2. Library construction and sequencing of single-cell spatial transcriptome (for Stereo-seq):

[0040] Fresh ovarian cortical tissue was rinsed twice in pre-cooled PBS to eliminate surface impurities, and any residual fluid or blood was gently wiped dry. Next, one part was processed according to the standard human ovarian cortex vitrification and thawing procedures. The other part was embedded in pre-cooled OCT, and the ovary was positioned with a blunt metal needle to ensure correct positioning. The entire OCT block was then quickly frozen in isopentane pre-cooled liquid nitrogen and transferred to a -80°C freezer for storage before cryosectioning. Two weeks later, the vitrified group was processed and embedded in OCT in the same way as the fresh group.

[0041] To minimize RNA degradation, both fresh and frozen groups were operated within 30 minutes, and the whole process was carried out in a low-temperature environment of -20 degrees. The OCT blocks were cut into 10 μm thickness transversely using a Leica CM1950 cryo-microtome (Leica). Total RNA was then extracted from the sections using the RNeasy Mini Kit (Qiagen, USA). Samples with an RNA integrity number (RIN) of 7-10 measured using a 2100Bioanalyzer (Agilent, USA) were subjected to subsequent Stereo-seq sequencing analysis. The target sections were directly adhered to the surface of a sequencing chip (BGI, Qingdao, China) with a capture probe containing a 25 bp coordinate identification barcode (CID), a 10 bp molecular identifier (MID), and a 22 bp polyT for in situ mRNA hybridization. Adjacent sections were stained with H&E for later histological examination. The sections on the chip were incubated at 37°C for 3 minutes, fixed in precooled methanol at -20°C for 30 minutes, and then stained with nucleic acid dye (Thermo Fisher Scientific) for ssDNA visualization. ssDNA and histological imaging were performed using a Ti-7 Nikon Eclipse microscope (Nikon, Japan). Finally, library construction and sequencing were completed with the help of Shanghai Ouyi Biotechnology Co., Ltd.

[0042] Third, we integrated scRNA-seq and Stereo-seq data to systematically analyze the expression profile changes of FOS / AP-1 and its downstream gene EGR1 during ovarian vitrification and thawing. At the same time, we used the algorithm "SCENIC" to clarify that FOS targeted regulation of EGR1.

[0043] IV. Immunofluorescence staining

[0044] 1) Take out the tissue sections cut by Leica CM1950 from -20 degrees and let them dry, circle the operation range with a hydrophobic pen, and wash with PBS for 20 minutes; 2) Shake off the water and fix with 4% PFA for 30 minutes to 1 hour, and wash with PBS for 10 minutes*3; 3) Block with 4% BSA containing 0.1% TritonX-100 for 1 hour; 4) Incubate with primary antibody at 4 degrees overnight, and then wash with blocking solution for 5 minutes*5; 5) Incubate with secondary antibody at room temperature for 1 hour, and then wash with blocking solution for 5 minutes*5; 6) DAPI staining for 10 minutes, and wash with PBS for 5 minutes*3; 7) Seal with immunofluorescence mounting medium and examine under a microscope.

[0045] Overall results Figure 1 , the expression of FOS gene family was significantly upregulated in ovarian tissue cells after vitrification and thawing ( Figure 1A and B), immunofluorescence staining also basically confirmed that after ovarian vitrification and freezing and thawing, the protein expression level of FOS, especially in the perivascular cells, was significantly higher than that in the fresh control group ( Figure 1 C). Further, with the help of bioinformatics analysis and the use of the "SCENIC" algorithm, it was found that both FOS and JUND directly target and regulate EGR1 ( Figure 1 D). And the immunolabeling results showed that the protein expression of EGR1 was also upregulated after vitrification and freezing and thawing ( Figure 1 E).

[0046] Example 2

[0047] Establishing an in vitro 3D culture model of mouse ovarian cryopreservation and verifying that the FOS / AP-1 pathway is activated during the ovarian cryopreservation and recovery process:

[0048] Specifically, the following steps are included:

[0049] 1. Preparation of in vitro 3D culture model of mouse ovary after freezing and thawing

[0050] 1. In vitro culture medium: DMEM / F12 1:1+1% P / S+5g / ml ITS+5% FBS+100mlU / ml rhFSH.

[0051] 2. Dissection medium: L-15 + 10% FBS + 0.5% P / S.

[0052] 3. The experimental animals were 2-3 week old female BALB / c mice. Male and female mice were caged at a ratio of 2:1. The birth date of the mouse pups was 0.5 dpp. The ovaries of female mice at 16 dpp were cultured in vitro. 0.4 ml of culture medium was added to the 24-well transwell plate so that the membrane of the insert chamber (0.4 um) was covered with a thin layer of culture medium and placed in the incubator for preheating.

[0053] 4. The process of ovarian tissue freezing + in vitro culture is as follows:

[0054] Vitrification freezing process: 1) Under the stereomicroscope, remove the ovarian tissue, dissect and remove the surrounding tissue + cysts, do not squeeze or poke the ovaries, and then use sharp ophthalmic scissors to divide the ovaries into 4 parts; 2) Transfer the ovaries with a pipette, wash them twice in the culture medium, then wash them once with freezing solution ① in a dish, put them in a new freezing solution ① dish, and shake them gently at room temperature for 10 minutes; 3) Aspirate the ovarian tissue in freezing solution ①, rinse them once in freezing solution ②, transfer them to new freezing solution ②, and rotate them at room temperature for 10 minutes. 4) Put them on a 1ml needle and quickly transfer them into liquid nitrogen and let them stand for 30 minutes (prepare the drug preparation culture medium);

[0055] Resuscitation process: Figure 3As shown, 1) preheat the resuscitation solution ① culture medium at 37℃; prepare resuscitation solutions ②, ③, and ④; 2) remove the tissue from liquid nitrogen and immediately place it in resuscitation solution ① for 1 minute, then transfer it to resuscitation solutions ②, ③, and ④ for 10 minutes each; 3) transfer it to a chamber for culture; add other drugs to the culture medium as needed; 4) change the medium once every 2 days, and harvest the ovaries after 4 days.

[0056] 5. Ovarian culture of fresh group:

[0057] 1) Kill the mice, remove the ovaries of 3dpp mice under a dissecting microscope, and place them in preheated dissecting medium; 2) Use a 20ul cut-end pipette to transfer and clean the ovaries, and use forceps to gently place them on the nested chamber of a 12-well plate and culture them in an incubator at 37°C and 5% CO2; 3) Change the medium every 2 days, and harvest the ovaries after 4 days.

[0058] 2. After freezing and thawing of mouse ovaries, Western blot experiments were used to detect the expression of key proteins in the FS / AP-1 signaling pathway, such as FOS / FOSB / JUN / EGR1 / AKT / p-AKT.

[0059] 1) After grinding thoroughly, add lysis buffer and fully lyse on ice for 2 hours (RIPA: Cocktail proteinase inhibitor = 100: 1). Then centrifuge at 12000rpm at 4℃ for 10 minutes. Transfer the supernatant to a new EP tube, add 5 times concentrated SDS loading buffer, mix well and put it in a boiling water bath for 5 minutes to denature the protein. Finally, divide the processed samples and store them in a -80℃ refrigerator for later use. 2) Use BCA for protein quantification for subsequent unified loading. 3) Prepare gel and assemble the electrophoresis device, and add electrophoresis buffer. Inject 30μg protein marker and protein sample into the gel hole in sequence, set the voltage to run the gel, and stop electrophoresis and transfer the membrane when bromophenol blue approaches the bottom of the gel. Cut the filter paper and PVDF membrane into the same size as the gel, and soak the PVDF membrane in methanol for 15 seconds; stack the sponge pad, filter paper, gel, PVDF membrane, filter paper and sponge pad in order, remove the bubbles and put them into the transfer tank, add transfer buffer; block with 5% skim milk powder at room temperature for 2 hours. Dilute the primary antibody (FOS, FOSB, JUN, EGR1, AKT and p-AKT) with the primary antibody diluent according to the proportion of the antibody instructions, and incubate at 4°C overnight. After rewarming, recover the primary antibody and wash the membrane with TBST for 5 minutes 3 times. Incubate with HRP-labeled secondary antibody (1:3000) at room temperature for 1 hour, and wash the membrane with TBST for 5 minutes 3 times. 4) Finally, add an appropriate amount of ECL luminescent liquid to the membrane and take photos using an integrated chemiluminescence instrument.

[0060] See also Figure 2In the vitrified frozen mouse ovaries, the number and quality of mature follicles were significantly lower than those in the fresh group ( Figure 2 A), at the same time, during the entire ovarian cryopreservation process, FOS / AP-1 related proteins (FOS, FOSB, JUN) and their target gene (EGR1) were activated and highly expressed at the beginning and continued throughout the process ( Figure 2 B and C).

[0061] Example 3

[0062] In the in vitro 3D culture model of mouse ovarian cryopreservation, the addition of T-5224 inhibited AP-1 and thus improved the phenotype of ovarian cryopreservation injury:

[0063] 1. Whole-mount fixation and immunofluorescence staining analysis of mouse ovarian tissue

[0064] The mice were randomly divided into 3 groups, 12 in each group. (A) Fresh Group: Fresh ovaries, after dissecting the surrounding tissues, rinsed with culture medium, and directly placed in a chamber for culture; (B) Cryo Group: After the cryo-recovery process, the ovaries were rinsed with culture medium and placed in a chamber for culture; (C) Cryo+T-5224 Group: The cryo-recovery process was completed in the freezing solution and resuscitation solution containing 80uM T-5224, the culture medium was rinsed, and the ovaries were placed in the culture medium containing 80uM T-5224 for culture. After 48h, the culture medium was replaced with the culture medium without T-5224. The specific process is as follows. Figure 3 .

[0065] 2. Whole-body fixation and immunofluorescence staining analysis of mouse ovarian tissue

[0066] 1) Fix the in vitro cultured ovaries in cold 4% paraformaldehyde (PFA) at 4°C overnight, while placing the culture dish on a shaker to ensure that the embryos are completely immersed in PFA; 2) Wash the ovaries in PBS for 5 minutes, 3 times, and then transfer to cold 100% methanol and place at -20°C overnight; 3) Subsequently, rehydrate the ovarian tissue in a series of gradient methanol (MeOH) / PBST (PBS+0.2% Triton X-100). Use 75%, 50%, 25% MeOH / PBST; 5 minutes each time. Finally, wash the ovarian tissue in PBST twice, 5 minutes each time; 4) Block the ovarian tissue in blocking buffer (5% BSA in PBST) at room temperature on a shaker for 2 hours; 5) Incubate the ovarian tissue in an appropriate primary antibody overnight at 4°C as needed; 6) After the tissue has been incubated in the antibody overnight, wash the tissue four times with PBST on a shaker at room temperature (10 minutes each time); 7) Incubate the tissue with a secondary antibody and / or a fluorescent-conjugated secondary antibody at room temperature for 2 hours; 8) Wash the tissue four times in PBST (10 minutes each time), protect from light during the washing process, and finally take pictures and collect data using a laser confocal microscope.

[0067] 3. TUNEL staining analysis and statistics of mouse ovarian tissue sections

[0068] Ovarian paraffin tissue sections were operated using the Yeasen Biotechnology (Shanghai) Co., Ltd. TUNEL apoptosis detection kit (FITC), which was simple: 1) Soak the paraffin tissue sections in xylene at room temperature for 5 minutes, repeat once, to completely remove the paraffin; soak the sections in 100% ethanol at room temperature for 5 minutes, repeat once; soak in gradient ethanol (90, 80, 70%) once, 3 minutes each time. 2) Add 100μL Proteinase K working solution to each sample to cover it completely, and incubate at room temperature for 20 minutes. 3) Add 100μL 1×EquilibrationBuffer to each sample for equilibrium. 4) Wash most of the 100μL 1×Equilibration Buffer around the equilibrium area with absorbent paper, and then add 50μL TdT incubation buffer to the cells in an area of ​​5cm X 2. After the operation, the slides should be protected from light and incubated at 37℃ for 60 minutes. 5) After washing with PBS, stain with DAPI for 20 min, then wash the samples and immerse the slides in deionized water for 5 min at room temperature, repeat twice, and wash three times in total. 6) After that, immediately analyze the samples under a fluorescence microscope and observe green fluorescence at 520 ± 20 nm using a standard fluorescence filter set.

[0069] See also Figure 4 In the vitrified frozen mouse ovaries, the overall vascular density of the mouse ovaries was improved after T-5224 treatment ( Figure 4 A), and at the same time, analyzing the cell apoptosis, it can be found that in the vitrified frozen resuscitation group with T-5224 added, the overall cell apoptosis ratio has improved, but is slightly lower than that of the fresh group ( Figure 4 B).

[0070] The human ovarian cortical tissue vitrified in the present invention is discarded ovarian horn cortical tissue donated by female patients with ovarian cysts and tumors after surgery at the International Peace Maternity and Child Health Hospital affiliated to Shanghai Jiao Tong University, and is implemented with the approval of the Ethics Committee of the International Peace Maternity and Child Health Hospital affiliated to Shanghai Jiao Tong University (review number B2022269P).

[0071] The experimental animals used in the mouse ovary vitrification and thawing model were from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and all animal experiments were approved by the Animal Committee of the International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (approval number 202201403).

[0072] The present invention is based on the molecular and cellular responses of human ovarian cortical tissue to freeze-thaw stress and protective agent toxicity. Through 10× single-cell transcriptome sequencing, Stereo-seq spatial transcriptome sequencing technology and immunofluorescence staining localization, it was found that FOS / AP-1 was overactivated in ovarian cells after vitrification and freezing and thawing.

[0073] Furthermore, in the present invention, an in vitro 3D culture model of mouse ovarian cryopreservation was constructed to study the effects of FOS / AP-1 inhibitor (T-5224) on physiological metabolism after ovarian cryopreservation and possible signaling pathways. The ovarian vascular density and the expression of AP-1 core components c-Jun and c-Fos were detected. It was found that pharmacological inhibition of FOS / AP-1 can significantly reverse ovarian cryopreservation damage, which has important theoretical significance and clinical value.

Claims

1. Application of FOS / AP-1 inhibitors in the preparation of drugs to improve ovarian tissue vitrification injury.

2. The use according to claim 1, characterized in that: The FOS / AP-1 pathway serves as an intervention target for reversing ovarian tissue vitrification and freezing-thawing injury.

3. The use according to claim 1, characterized in that: The FOS / AP-1 inhibitor is T-5224.

4. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

6. Application of FOS / AP-1 inhibitors in the preparation of ovarian tissue cryoprotectant or ovarian 3D culture medium.

7. The use according to claim 6, characterized in that: The final concentration of T-5224 added to the culture medium was 80uM.

8. Observe the methods of improving phenotypes related to ovarian tissue vitrification freeze-thaw injury by FOS / AP-1 inhibitors, including: (1) In vitro 3D culture and phenotypic observation of mouse ovaries after freezing and thawing; (2) Total RNA and protein extraction from mouse ovarian tissue for real-time quantitative PCR and western blot analysis; (3) Whole-tissue immunofluorescence staining of mouse ovarian tissue was used to mark blood vessels and extracellular matrix components.

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