Application of mesenchymal stem cells in medicine for treating thin endometrium and uterine cavity adhesion

Abstract

The invention belongs to the field of medicines, and particularly provides application of mesenchymal stem cells in a medicine for treating thin endometrium and uterine cavity adhesion. The invention further provides a PDGF-BB monoclonal antibody, and the antibody can be specifically combined with PDGF-BB so as to inhibit corresponding protein activity. After the monoclonal antibody is used independently or in combination with stem cells, the effects of effectively promoting gland growth and reducing the fibrosis degree can be achieved, which shows that the monoclonal antibody can be used independently or in combination with the stem cells for treating thin endometrium and uterine cavity adhesion, and the application prospect is wide.

Description

Technical Field

[0001] This invention relates to the field of medicine. Background Technology

[0002] Intrauterine adhesions are abnormal adhesions formed after damage to the basal layer of the endometrium. They can cause infertility, recurrent miscarriages, menstrual irregularities, and other problems, seriously threatening women's reproductive health. Their harm is mainly reflected in their impact on fertility, pregnancy outcomes, and quality of life. Intrauterine adhesions can lead to thinning or scarring of the endometrium, affecting embryo implantation. Severe adhesions may completely block the uterine cavity, preventing sperm and egg from meeting. Recurrent miscarriages, even if pregnancy occurs, can lead to abnormal uterine cavity morphology or insufficient blood flow caused by adhesions, potentially resulting in poor embryonic development or fetal arrest. A thin endometrium usually affects pregnancy. Insufficient endometrial thickness may affect embryo implantation, leading to infertility or early miscarriage. A thin endometrium may be related to endocrine disorders, intrauterine surgical damage, inflammation, and other factors. The endometrium is a crucial site for embryo implantation, and its thickness directly affects the success rate of pregnancy. Clinically, an endometrial thickness of 8-14 mm during ovulation is considered ideal; a thickness below 7 mm may affect embryo implantation. A thin endometrium leads to reduced uterine blood flow, making it difficult for the embryo to obtain sufficient nutritional support for development. Some patients may experience accompanying symptoms such as scanty menstruation and irregular cycles. Therefore, thin endometrium and intrauterine adhesions are important factors leading to infertility in women of reproductive age, and effective treatment is currently a hot research topic.

[0003] Medications that improve endometrial blood flow are a direction for clinical treatment. For example, aspirin: it has platelet-lowering and anti-inflammatory effects, and low-dose aspirin is recommended to promote endometrial growth; however, 13 randomized controlled trials have concluded that low-dose aspirin has no definite efficacy in improving blood flow or increasing endometrial thickness. Sildenafil citrate: It can enhance the vasodilatory effect of nitric oxide; vaginal administration is superior to oral administration, has fewer side effects in treating TE, and has some effect on patients with TE and recurrent implantation failure. Vitamin E: It effectively inhibits oxidative stress, thereby protecting embryo implantation, and can significantly enhance the endometrial response, producing some therapeutic effect. Acetylthecobalamin: A methylxanthine derivative, its treatment of TE requires a long cycle and high doses, placing a certain psychological burden on patients; its efficacy is still unclear, and it is not recommended clinically.

[0004] Stem cell therapy is a hot research area, bringing hope to these patients. Many types of stem cells have been proven to be related to endometrial renewal and regeneration, and can be applied to endometrial damage repair, such as bone marrow-derived stem cells, umbilical cord mesenchymal stem cells, menstrual blood stem cells, adipose-derived mesenchymal stem cells, and human embryonic stem cells. One report describes injecting green fluorescent protein-labeled adipose-derived stem cells into rats with endometrial damage. After 30 days, the microvascular density, endometrial thickness, and glandular number in the damaged endometrium increased. Furthermore, in subsequent pregnancy experiments, 60% of the rats successfully conceived, a statistically significant difference compared to the control group. In another study, UC-MSCs were injected via the tail vein into rats with endometrial damage. In SD rats, fibrosis and inflammation at the site of endometrial damage were significantly alleviated, and the number of endometrial cells and new blood vessels significantly increased. However, using stem cell transplantation alone results in a low cell implantation rate and a low efficiency in promoting endometrial repair and regeneration. One study obtained adipose tissue from patients through liposuction, and injected the tissue containing adipose-derived stem cells and vascular components into the uterus via the cervix, followed by estrogen replacement therapy. Of the five patients, two regained menstruation, three experienced increased menstrual flow, and the endometrial thickness increased from (3.0±1.0) mm to (6.9±2.9) mm. After embryo transfer, one patient became pregnant but suffered a spontaneous abortion at the 9th week of gestation. This indicates that adipose tissue treatment can restore menstruation, but it does not significantly improve pregnancy rates. Future research should focus on whether ADSCs treatment can improve pregnancy rates.

[0005] Furthermore, research has found that platelet-derived growth factor (PDGF) is an important regulator of fibrosis formation. Belonging to the vascular endothelial growth factor family, it is a crucial mitogenic factor. Under normal conditions, PDGF is mainly stored in platelet α-granules. When the body is stimulated or damaged, aggregated platelets, epithelial cells, macrophages, etc., secrete PDGF cytokines. These cytokines bind to the PDGF receptor (PDGFR), promoting the proliferation and differentiation of fibroblasts. Studies have found that PDGF signaling molecules are significantly upregulated in the kidneys of humans and rodents with interstitial fibrosis or glomerulosclerosis. Numerous previous experimental studies have shown that PDGF signaling molecules are involved in the progression of various fibrotic diseases. Similarly, in a model of intrauterine adhesions, experiments have shown that overexpressed PDGF induces tissue fibrosis and ECM deposition by inducing various pro-fibrotic genes and related pathways. Inhibiting PDGF expression can improve intrauterine adhesions. However, there are not enough drugs to inhibit PDGF, and the types are not diverse enough. In particular, there is still a need for further research on drugs that are convenient to administer and effective in treating thin endometrium and intrauterine adhesions. Summary of the Invention

[0006] Through previous research, the inventors discovered that PDGF, especially PDGF-BB, is a commonly used target for studying fibrosis mechanisms or evaluating the efficacy of anti-fibrotic drugs. Based on this research, the inventors innovatively proposed an inhibitor of PDGF-BB, namely a monoclonal antibody specifically targeting PDGF-BB.

[0007] In one aspect, the present invention provides a monoclonal antibody specifically targeting PDGF-BB, wherein the light chain variable region is shown in SEQ ID NO: 1 and the heavy chain variable region is shown in SEQ ID NO: 2.

[0008] The monoclonal antibody against PDGF-BB of the present invention can effectively bind to PDGF-BB and inhibit the corresponding protein activity. After treatment with 100 nM concentration for 24 h in rat thoracic aortic vascular smooth muscle cells, it can inhibit 86% of the activity of PDGF-BB protein, showing a good inhibitory effect.

[0009] Specifically, the monoclonal antibody of the present invention can be artificially synthesized or prepared by immunizing mice to obtain ascites fluid.

[0010] Furthermore, the antibody is selected from murine antibodies, immunoglobulin molecules, disulfide-linked Fv, monoclonal antibodies, affinity-matured antibodies, scFv, chimeric antibodies, single-domain antibodies, CDR-transplanted antibodies, biantibodies, humanized antibodies, multispecific antibodies, Fab, bispecific antibodies, DVD, TVD, Fab', bispecific antibodies, F(ab')2, and Fv. The antibody or antibody fragment is murine.

[0011] As used herein, “monoclonal antibody” refers to an antibody derived from a group of substantially homologous antibodies, meaning that individual antibodies within that group are identical except for the possibility of naturally occurring mutations (which may be present in small amounts). Monoclonal antibodies are highly specific against a single antigen. Furthermore, unlike polyclonal antibody formulations, they typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targeting a single determinant on the antigen. Specifically, monoclonal antibodies in this article include “chimeric” antibodies, in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portions of one or more chains are identical or homologous to corresponding sequences in fragments of antibodies derived from another species or belonging to another antibody class or subclass, provided they exhibit the desired biological activity.

[0012] Furthermore, the monoclonal antibody of the present invention may be a variant while retaining its activity.

[0013] The term “variant” is used herein to describe a peptide or polypeptide that differs in its amino acid sequence by the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. Representative examples of “biological activity” include the ability to bind to a specific antibody or to promote an immune response. Variants may also be used herein to describe proteins having a substantially identical amino acid sequence to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally considered in the art to involve minor alterations. These minor alterations can be partially identified by considering the hydrophobicity index of the amino acid, as understood in the art. The hydrophobicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices can be substituted and still retain protein function. In one respect, amino acids with a hydrophobicity index of ±2 are substituted. The hydrophobicity of amino acids can also be used to reveal substitutions that will result in the retention of biological function in the protein. In the context of peptides, considerations of the hydrophilicity of amino acids allow for the calculation of the peptide’s maximum local average hydrophilicity, a useful metric that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values ​​can result in peptides retaining biological activity, such as immunogenicity, as understood in the art. Substitution can be performed with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Based on this observation, as revealed by properties such as hydrophobicity, hydrophilicity, charge, size, and others, biologically compatible amino acid substitutions are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids. “Variant” can also be used to refer to an antigenically reactive fragment of an antiPDGF antibody whose amino acid sequence differs from the corresponding fragment of the antiPDGF antibody but still possesses antigenic reactivity and can competitively bind PDGF to the corresponding fragment of the antiPDGF antibody. “Variant” can also be used to describe a polypeptide or fragment thereof that has been differentially treated, for example by proteolysis, phosphorylation, or other post-translational modifications, but retains its antigenic reactivity.

[0014] On the one hand, the present invention also provides a drug for treating thin endometrium and intrauterine adhesions, containing a monoclonal antibody specifically targeting PDGF-BB as the active pharmaceutical ingredient, wherein the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and its heavy chain variable region is shown as SEQ ID NO: 2.

[0015] In another aspect, the present invention also provides a drug for treating thin endometrium and intrauterine adhesions, containing a monoclonal antibody specifically targeting PDGF-BB and umbilical cord mesenchymal stem cells as active pharmaceutical ingredients, wherein the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and its heavy chain variable region is shown as SEQ ID NO: 2.

[0016] Furthermore, the present invention also provides the application of a PDGF-BB monoclonal antibody in the preparation of a drug for treating thin endometrium and intrauterine adhesions, wherein the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and its heavy chain variable region is shown as SEQ ID NO: 2.

[0017] Furthermore, the present invention also provides the application of PDGF-BB monoclonal antibody and umbilical cord mesenchymal stem cells in the preparation of drugs for treating thin endometrium and intrauterine adhesions, wherein the light chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1, and its heavy chain variable region is shown as SEQ ID NO: 2.

[0018] Specifically, the pharmaceutical products of the present invention can be compositions, which may further comprise one or more additional ingredients selected from various excipients known in the field of pharmaceutical formulations. Depending on the desired properties of the tablets or capsules, and based on their known use in preparing the compositions of the present invention, many ingredients may be selected individually or in combination. Such ingredients include, but are not limited to, water, non-aqueous solvents (such as ethanol), coating agents, capsule shells, colorants, waxes, gelatin, fragrances, preservatives (such as methylparaben, sodium benzoate, and potassium benzoate), antioxidants (such as butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E and vitamin E esters such as tocopherol acetate), flavor enhancers, sweeteners (such as aspartame and saccharin), compression aids, surfactants, etc.

[0019] Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphates), glycine, sorbic acid, potassium sorbate, mixtures of metaglycerides of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0020] Pharmaceutical compositions, such as solid dispersions, such as spray-dried pharmaceuticals, can be formulated into tablets. For example, a spray dispersion of a pharmaceutical can be combined with molten particles, one or more diluents, and / or one or more disintegrants, and one or more lubricants, one or more other excipients, media, carriers, and / or fillers, and compressed into tablet form. The resulting tablets can then be further processed, for example, by applying a coating material, such as a film or shellac, to help maintain the stability or integrity of the tablet, facilitate oral administration of the tablet, mask the taste of the tablet, add flavoring, color the tablet, adjust the release of the drug contained in the tablet upon ingestion, and / or mask the taste of the tablet formulation. Coatings suitable for this purpose (e.g., shellac, casings for adjusting release) are known in the art. If desired, certain sweeteners, flavor enhancers, or colorants may also be added to the tablet or coating.

[0021] According to a preferred embodiment, the compositions of the present invention are formulated for administration to mammals, preferably humans. Although the tablet form provided herein is preferably formulated for oral administration, other formulations are also available.

[0022] Pharmaceutical compositions, such as those containing the solid (e.g., spray-dried) dispersions described herein, can also be prescribed to patients in patient packs, containing more than one dose, preferably the entire course of treatment, in a single package (e.g., a blister pack). In conventional prescriptions, pharmacists divide the patient supply from a bulk-supplied medication, while patient packs are superior to conventional prescriptions in that patients always have access to the package insert, which is normally not included in conventional prescriptions. The inclusion of the package insert has been shown to improve patient compliance with physician instructions. Preferably, the medication is in an oral dosage form, such as an injectable form.

[0023] Beneficial effects This invention provides the application of mesenchymal stem cells in the treatment of thin endometrium and intrauterine adhesions. This invention also provides a PDGF-BB monoclonal antibody that specifically binds to PDGF-BB, thereby inhibiting the activity of the corresponding protein. When used alone or in combination with stem cells, the monoclonal antibody effectively promotes glandular growth and reduces the degree of fibrosis. This indicates that the PDGF-BB-3F7 monoclonal antibody of this invention can be used alone or in combination with stem cells to treat thin endometrium and intrauterine adhesions, showing broad application prospects. Attached Figure Description

[0024] Figure 1 Figure showing the effect of each experimental group on the number of endometrial glands; Figure 2 Figure showing the effect of each experimental group on the ratio of endometrial stromal fibrosis area. Detailed Implementation

[0025] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Unless otherwise specified, the methods, equipment, and materials in the following embodiments are all conventional methods, equipment, and materials in the art and are commercially available.

[0026] Example 1: Preparation of PDGF-BB monoclonal antibody Recombinant rat PDGF-BB (Bio-Levi, product code: GS4863) was used as the antigen and mixed with an equal volume of Freund's complete adjuvant. The mixture was thoroughly emulsified and injected intraperitoneally into three Balb / c mice, 90 μg per mouse. Three weeks later, the same dose of antigen plus an equal volume of incomplete Freund's adjuvant was injected intraperitoneally. Three days before fusion, a booster immunization was administered intraperitoneally with twice the dose of antigen-saline solution. One day before cell fusion, a normal Balb / c mouse was euthanized by cervical dislocation, immersed in 70% ethanol for 5 min, and its abdominal skin was cut open in a laminar flow hood to fully expose the peritoneum. 5 ml of GKN solution was injected into the mouse's peritoneal cavity using a 5 μm syringe. After repeated compression, the fluid was aspirated and transferred to a centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and HAT medium was added to suspend the cells. The cells were then transferred to 96-well culture plates.

[0027] After immunization, mice were bleed thoroughly from the orbital veins, euthanized by cervical dislocation, and immersed in 70% ethanol for 5 minutes. The spleen was then removed in a clean bench, and a cell suspension was prepared. The suspension was centrifuged at 1000 rpm for 10 minutes at room temperature, the supernatant was discarded, and the suspension was washed twice with GKN solution and resuspended in GKN solution. Cells were counted using a fluorescence microscope for later use.

[0028] Mouse spleen cells and SP2 / 0 cells were mixed at a ratio of 10:1 in 50 ml centrifuge tubes. The centrifuge tubes were placed in a 37°C water bath and gently shaken. 1 ml of 50% polyethylene glycol 2000 was slowly added over 1 min, and the mixture was allowed to stand for 1 min. 10 ml of GKN solution was added to terminate cell fusion, and the mixture was allowed to stand for 1 min. Centrifuged at 1000 rpm for 12 min, the supernatant was discarded, and HAT culture medium was added. The mixture was gently stirred with a pipette to form a homogeneous suspension. The suspension was then seeded into 96-well culture plates pre-added with feeder cells and placed in a 37°C, 5% CO2 incubator. The medium was changed every 3 days, and after 7 days, it was changed to HT culture medium. Positive clones were subcloned using the limiting dilution method. Five wells with high antibody titers were selected for further subcloning until the positive rate reached 100%. The PDGF-BB-3F7 hybridoma cells with the highest titers were selected for cryopreservation.

[0029] The PDGF-BB-3F7 monoclonal hybridoma cell suspension obtained from subcloning was centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were washed once with serum-free culture medium. Serum-free culture medium was then added again, and the supernatant was collected after 3 days. Mice were intraperitoneally injected with 0.5 ml of liquid paraffin per mouse, and 6 × 10⁶ hybridoma cell suspensions were injected intraperitoneally 8 days later. 6 Approximately 10 days after injection, significant abdominal swelling was observed in mice. Ascites fluid was extracted, purified using an affinity column, and the concentration was adjusted to 1 mg / mL. The fluid was then stored at -20°C for later use.

[0030] Example 2: Identification of PDGF-BB-3F7 monoclonal antibody subtypes 150 μl of serum-free culture supernatant diluted with 1×PBS was added to the reaction tube and incubated at room temperature for 30 s. The tube was then briefly shaken to completely resuspend the gel at the bottom. After placing the subtype identification test strip into the reaction tube for 12 min, the subtype of the monoclonal antibody was directly determined from the bands displayed on the test strip. The results showed that the heavy chain was IgG1 and the light chain was κ chain.

[0031] Example 3 Western blot detection of PDGF-BB-3F7 monoclonal antibody Recombinant PDGF-BB and BSA were subjected to SDS-PAGE at a concentration of 0.9 mA / cm². 2 Proteins were electroporated onto nitrocellulose membranes and blocked overnight at 4°C. The membranes were then immersed in PDGF-BB-3F7 monoclonal antibody and incubated at room temperature for 1 hour, followed by washing three times for 10 minutes each time. Enzyme-labeled secondary antibody was then added, and the membranes were incubated at room temperature for 45 minutes, followed by washing three times for 10 minutes each time. The membranes were then detected using an ECL Westem blot assay kit. The results showed that only recombinant PDGF-BB showed a specific band, while BSA did not show a band, indicating that the PDGF-BB-3F7 monoclonal antibody of this invention has good specificity.

[0032] Example 4: Affinity Identification of PDGF-BB-3F7 Monoclonal Antibody Anti-mouse IgG secondary antibody was immobilized on a CM5 chip, and PDGF-BB-3F7 antibody was captured using a Biacore T200 microarray. PDGF-BB antigen was used as the analyte and diluted with buffer to concentration gradients of 0, 7.5, 15, and 30 nM. Binding of different antibodies to PDGF-BB antigen was detected using single-cycle kinetics. The final data were analyzed using Biacore Evaluation Software 3.0 with a 1:1 model. The results are shown in Table 1.

[0033] Table 1. Affinity data of PDGF-BB-3F7 monoclonal antibody As can be seen from Table 1, the PDGF-BB-3F7 monoclonal antibody prepared in this invention has good affinity properties and can be used for subsequent experiments.

[0034] Example 5: Activity assay of PDGF-BB-3F7 monoclonal antibody Selected SD pregnant mice, gestational age 15-20 days, weight 420-470g (Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.). Before surgery, the animals were housed individually in a controlled environment with temperature [(25±2)℃] and light (12h light and 12h darkness), with free access to food and water.

[0035] A pregnant rat model of intrauterine adhesions (IUA) was constructed using a method that is well-established in the field. Specifically, pregnant rats were anesthetized by intraperitoneal injection of phenobarbital sodium at a dose of 40 mg / kg. First, the surgical field of the rats was prepared by clipping the hair and exfoliating the skin to an area of ​​approximately 3 cm × 3 cm. After disinfection with povidone-iodine, a midline incision of approximately 4 cm was made in the lower abdomen, allowing for layered access to the abdominal cavity and exposure of both uteri. A 1 cm longitudinal incision was made proximal to both uteri, and the embryos were removed. The uterine cavity was scraped back and forth along different directions using a curette until the walls of the uterine cavity felt rough. The uterine and abdominal incisions were then sutured. Rats that successfully modeled the IUA were randomly divided into the following groups, with 10 rats in each group: Monoclonal antibody treatment group: PDGF-BB-3F7 monoclonal antibody was injected subcutaneously on day 7 after curettage. 50μg / 100g, 0.3mL, injected once every 3 days, for a total of 3 injections; Blank control group: 0.4 mL / 100 g of normal saline was injected subcutaneously on the 7th day after curettage, once every 3 days, for a total of 3 injections; Stem cell therapy group: 1×10⁻⁶ mouse umbilical cord mesenchymal stem cells (catalog number CP-M238, Wuhan Yipu Biotechnology Co., Ltd.) were subcutaneously injected on day 7 after curettage. 6 0.3 mL per 100g, injected once every 3 days, for a total of 3 injections; Stem cell combined with monoclonal antibody therapy group: 1×10⁻⁶ mouse umbilical cord mesenchymal stem cells (catalog number CP-M238, Wuhan Yipu Biotechnology Co., Ltd.) were subcutaneously injected on day 7 after curettage. 6 0.3 mL per 100 g, injected once every 3 days for 3 consecutive injections; 0.3 mL of PDGF-BB-3F7 monoclonal antibody subcutaneously injected half an hour after each injection, once every 3 days for 3 consecutive injections; Positive control group: On the 7th day after curettage, estrogen 50μg / 100g, 0.3mL was injected subcutaneously, once every 3 days, for a total of 3 injections; Rats in all groups were sacrificed on day 14. Bilateral uterine sections were prepared, and the number of endometrial glands in the HE-coated endometrial sections of the female rats in each group was compared. The results are as follows: Figure 1 As shown.

[0036] from Figure 1 The results showed that the number of glands on day 14 post-surgery was (2.38±0.12) in the blank control group, (4.96±0.24) in the monoclonal antibody treatment group, (3.77±0.30) in the stem cell treatment group, and (6.05±0.15) in the stem cell combined with monoclonal antibody treatment group, while the number of glands on day 14 in the positive control group was (4.63±0.18). These results demonstrate that the monoclonal antibody of this invention can effectively increase the number of glands, and the combination of monoclonal antibody and stem cells synergistically increases the number of glands, with significant effects. Both methods are better than the positive control group.

[0037] In addition, paraffin-embedded uterine specimens from each group of rats were serially sectioned at 4 μm and then stained with Masson's stain. Four high-power (×100) fields of view were randomly selected from each Masson's slide. The area of ​​endometrial stromal fibrosis and the sum of the areas of endometrial stroma and glands in each field of view were calculated using the Image-ProPlus 6.0 image analysis system. The fibrosis ratio was obtained by dividing the area of ​​endometrial stromal fibrosis by the total area of ​​endometrial stroma and glands, and the average value was taken for each field of view. The results are as follows: Figure 2 As shown.

[0038] from Figure 2The results showed that on postoperative day 14, the proportion of endometrial stromal fibrosis area in the monoclonal antibody treatment group, stem cell treatment group, positive control group, and stem cell combined monoclonal antibody treatment group was lower than that in the control group, and the differences were statistically significant (P<0.01). In particular, the proportion of endometrial stromal fibrosis area in the stem cell combined monoclonal antibody treatment group on postoperative day 14 was 0.032±0.005, which was significantly lower than that in the blank control group (0.135±0.007) on postoperative day 14 (P<0.01).

[0039] In summary, the results of this experiment indicate that PDGF-BB-3F7 can promote the growth of endometrial glands and reduce the degree of fibrosis in a pregnant mouse model of intrauterine adhesions (IUA) after curettage, thus playing a therapeutic role in IUA. In particular, the combination of this monoclonal antibody and stem cells has a synergistic effect in promoting gland growth and reducing the degree of fibrosis, with significantly improved results compared to the positive control group. This demonstrates that the PDGF-BB-3F7 monoclonal antibody of this invention can be used alone or in combination with stem cells to treat thin endometrium and intrauterine adhesions.

[0040] When implementing or testing embodiments of the present invention, optional methods and materials similar to or equivalent to those described in this specification may be used, although preferred methods, apparatus, and materials are described in this specification. However, before describing the materials and methods of the present invention, it should be understood that the specific sizes, shapes, dimensions, materials, methods, means, etc., described in this specification can be modified according to conventional experimental methods and for optimization purposes; therefore, the present invention is not limited to these. Furthermore, it should be understood that the technical terms used in this specification are only used to describe specific types or embodiments and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.

Claims

1. A monoclonal antibody specifically targeting PDGF-BB, characterized in that, The variable region of the light chain is shown in SEQ ID NO: 1, and the variable region of the heavy chain is shown in SEQ ID NO:

2.

2. A drug for treating thin endometrium and intrauterine adhesions, characterized in that, The active pharmaceutical ingredient contains a monoclonal antibody that specifically targets PDGF-BB, wherein the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and its heavy chain variable region is shown in SEQ ID NO:

2.

3. A drug for treating thin endometrium and intrauterine adhesions, characterized in that, The drug contains a monoclonal antibody specifically targeting PDGF-BB and umbilical cord mesenchymal stem cells as active pharmaceutical ingredients. The light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and its heavy chain variable region is shown in SEQ ID NO:

2.

4. The application of PDGF-BB monoclonal antibodies in the preparation of drugs for treating thin endometrium and intrauterine adhesions, among which, The light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and its heavy chain variable region is shown in SEQ ID NO:

2.

5. The application of PDGF-BB monoclonal antibodies and umbilical cord mesenchymal stem cells in the preparation of drugs for treating thin endometrium and intrauterine adhesions, among which, The light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and its heavy chain variable region is shown in SEQ ID NO:

2.

6. The application as described in any one of claims 4-5, characterized in that... The drug contains a pharmaceutically acceptable carrier.

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