CN116916946A

CN116916946A - Use of nerve growth factor in the preparation of drugs for treating or improving reproductive system diseases

Info

Publication number: CN116916946A
Application number: CN202280008244.7A
Authority: CN
Inventors: 李加斌; 朱庆; 李佳腊
Original assignee: Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Staidson Beijing Biopharmaceutical Co Ltd
Priority date: 2021-11-19
Filing date: 2022-11-17
Publication date: 2023-10-20
Also published as: WO2023088351A1; TW202330578A

Abstract

提供了神经生长因子在制备用于治疗生殖系统疾病或改善生殖系统疾病的药物中的用途。Provided are uses of nerve growth factors in preparing medicaments for treating reproductive system diseases or improving reproductive system diseases.

Description

Use of nerve growth factor in preparing medicine for treating or improving genital system diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of nerve growth factor in preparation of a medicament for treating or improving reproductive system diseases.

Background

Nerve Growth Factor (NGF) was the first member of the family of neurotrophic factors found in murine sarcoma cells in 1953 by Italian scientist Levi-Montelcini. Nerve growth factor is a nerve growth regulator, has double biological functions of providing nutrition for neurons and promoting neurite growth, and plays an important role in regulating the development, differentiation, growth, regeneration and functional expression of central and peripheral neurons. Nerve growth factor comprises three subunits, α, β and γ. The beta subunit is an active region and is formed by combining two single chains through non-covalent bonds.

For the nervous system, the nerve growth factor has the functions of promoting neuron development, axon growth, transmitter synthesis, inhibiting nerve cell apoptosis and the like; for other systems such as cardiovascular, immune and reproductive systems, nerve growth factors are mainly used for regulating the functions of the immune system, inhibiting the mitosis of part of tumor cells, promoting wound healing and the like. Studies have shown that nerve growth factors and their receptors are also widely present in testicular tissue, in addition to being more distributed in the nervous system; nerve growth factor mRNA transcripts and proteins are expressed in both fetal and adult human ovaries. However, there is no report as to whether nerve growth factor can be used to treat diseases of the reproductive system.

Genital diseases generally comprise the following classes: (1) Tumors of the reproductive system, such as testicular cancer, prostate cancer, uterine fibroids, ovarian tumors, endometrial cancer, fallopian tube cancer, ovarian cancer, uterine cervical cancer, and the like; (2) Inflammation of the reproductive system such as epididymitis of testis, seminal vesicle, prostatitis, colpitis, metritis, pelvic inflammatory disease, adnexitis, endometritis, vulvitis, etc.; (3) Tuberculosis of genital tract such as tuberculosis of prostate, tuberculosis of seminal vesicle, tuberculosis of testis, tuberculosis of epididymis, etc.; (4) Genital tract injuries such as testis contusion, penis fracture, urethra fracture, etc.; (5) Deformity of the reproductive system such as hidden penis, webbed penis, cryptorchy, etc.; (6) Sexual dysfunction diseases such as male erectile dysfunction, premature ejaculation, hyposexuality, anejaculation, delayed ejaculation, etc.; (7) Diseases related to reproductive system diseases such as varicocele, oligospermia, congenital vas deferens obstruction, vas deferens deficiency, oviduct obstruction, immune infertility, and premature ovarian failure.

Premature ovarian failure (premature ovarian insufficiency, POI) refers to the natural amenorrhea before age 40 due to ovarian failure. Often accompanied by a decrease in estrogen levels, an increase in folliculogenesis levels, and an increase in gonadotrophin levels, the etiology and mechanism of which are complex. According to statistics, the incidence rate of the people is 1% -3%, the incidence rate of the people accounts for 2% -10% in amenorrhea patients, and the incidence rate in recent years is in an ascending trend. Premature ovarian failure causes fertility loss and a low estrogen state, and becomes a non-negligible factor affecting female reproductive health and social stability.

The oligospermia is a relatively common disease of men, and is mainly characterized by reduced sperm quantity or reduced sperm motility, which can seriously affect physical and mental health of patients, and also can be accompanied with diseases such as endocrine, nervous system and the like, thereby further causing the reduction of life quality of the patients. The clinical treatment of oligospermia is mainly carried out by two modes of medicine and operation: 1. drug treatment: depending on the specific circumstances of the patient, if the patient is oligospermia due to low gonadotrophin levels, treatment with gonadotrophin may be performed; however, the oligospermia with the cause can not be found more frequently in clinic, and the medicine such as carnitine is mainly used; 2. surgical treatment: if the oligospermia of the patient is caused by varicocele, performing surgical treatment of the varicocele; if the disease is caused by the cryptorchid, the cryptorchid is treated by operation.

There is a great need in the art for a medicament and method for effectively treating or ameliorating reproductive system diseases such as premature ovarian failure and oligospermia.

Disclosure of Invention

In a first aspect, the invention provides the use of a nerve growth factor or nerve growth factor fusion protein in the manufacture of a medicament for the treatment of a disease of the reproductive system or for improving a disease of the reproductive system.

In some embodiments, the reproductive system disease includes, but is not limited to, diminished ovarian reserve, premature Ovarian Failure (POF), ovarian hyperstimulation syndrome, residual ovarian syndrome, ovarian follicular dysplasia, spermatogenic disorder; preferably, the insemination disorder is a disorder including, but not limited to: azoospermia or oligospermia, azoospermia, teratospermia, oligospermia (OAT syndrome).

In some embodiments, the amelioration of a reproductive disease includes, but is not limited to, one or more of the following improvements:

(1) Promote proliferation of ovarian granulosa cells and/or estrogen secretion; (2) Preventing/reversing a decrease in the number and/or function of follicles, or increasing the number and/or function of follicles; (3) Improving testicular seminiferous tubule atrophy, seminiferous tubule seminiferous disorder and/or epididymal duct cell debris; (4) Improving the reduction in sperm count and/or motility, or improving sperm count and/or motility; preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.

In a second aspect, the invention provides the use of a nerve growth factor or nerve growth factor fusion protein in the manufacture of a medicament for ameliorating a disorder or condition associated with or caused by a disorder or condition of the reproductive system.

In some embodiments, the reproductive system diseases include, but are not limited to: premature ovarian failure or oligospermia, reduced ovarian reserve, ovarian hyperstimulation syndrome, ovarian residual syndrome, ovarian follicular dysplasia, dysspermia; preferably, the insemination disorder includes, but is not limited to: oligospermia, azoospermia, teratospermia, oligospermia.

In some embodiments, the disorder or symptom includes, but is not limited to: a reduced proliferation rate of ovarian granulosa cells and/or reduced estrogen secretion, reduced follicular number and/or reduced function, a reduced testicular seminiferous tubule atrophy, a seminiferous tubule seminiferous disorder and/or epididymal duct cell debris, a reduced sperm number and/or reduced motility; preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.

In a third aspect, the invention provides a method of preparing a nerve growth factor or nerve growth factor fusion protein for promoting proliferation of ovarian granulosa cells and/or estrogen secretion in a patient; preventing/reversing a decrease in the number and/or function of follicles, or increasing the number and/or function of follicles; improving testicular seminiferous tubule atrophy, seminiferous tubule seminiferous disorder and/or epididymal duct cell debris; use in medicine for improving the reduction of sperm count and/or the reduction of motility, or for increasing sperm count and/or motility, etc.; preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.

In some embodiments, the patient is a patient suffering from or associated with a disease of the reproductive system, including, but not limited to, a patient suffering from reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, residual ovarian syndrome, ovarian follicular dysplasia, spermatogenic disorder; preferably, the insemination disorder includes, but is not limited to: oligospermia, azoospermia, teratospermia, oligospermia.

Nerve growth factors described herein include, but are not limited to, wild-type nerve growth factor (wild-type NGF), recombinant nerve growth factor (recombinant NGF), nerve growth factor mutant (NGF mutant).

In some embodiments, wild-type nerve growth factors include wild-type nerve growth factors isolated from a variety of sources including, but not limited to, livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., humans and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters).

In some embodiments, the nerve growth factor is a murine wild-type nerve growth factor or a human wild-type nerve growth factor.

In some embodiments, the murine wild-type nerve growth factor or the human wild-type nerve growth factor refers to a natural nerve growth factor derived from mice or humans, including but not limited to a complete nerve growth factor, a nerve growth factor with 1 or 2 amino acids deleted at the C-terminus, a nerve growth factor with 8 amino acids deleted at the N-terminus, a nerve growth factor with 1 amino acid deleted at the C-terminus and 8 amino acids deleted at the N-terminus, and a nerve growth factor glycosylation product and/or a nerve growth factor oxidation product.

In some embodiments, the human wild-type nerve growth factor comprises a sequence as set forth in SEQ ID NO. 1 or 2.

The recombinant nerve growth factor can be prepared by recombinant expression or a synthetic method. In some embodiments, the recombinant nerve growth factor is a murine recombinant nerve growth factor or a human recombinant nerve growth factor.

The nerve growth factor of the present invention may be a nerve growth factor mutant. The mutant is a nerve growth factor mutant with most or all biological activities of wild nerve growth factor. Nerve growth factor mutants include mutations at one or more amino acid positions of nerve growth factor (e.g., mature β -NGF). In some embodiments, the mutant is a variant of a wild-type nerve growth factor obtained by deletion, substitution, and/or insertion of one or more amino acid residues. In some embodiments, the nerve growth factor mutant has reduced side effects (e.g., pain) or is painless compared to the wild-type nerve growth factor.

In some embodiments, the nerve growth factor mutant relative to the position of the wild-type nerve growth factor sequence of human origin (SEQ ID NO:1 or 2) comprises one or more of the following amino acid site mutations: F12E, K G, K L, K Y, R59L, R A, D65A, D G, K74L, K88 3987 88L, K88 3788G, Q96E, R V, R F, R G, R L and F101A.

In some embodiments, the nerve growth factor mutant comprises any one of the amino acid sequences shown in SEQ ID NOs 3-40.

The nerve growth factor fusion protein (NGF fusion protein) disclosed by the invention refers to a fusion protein comprising nerve growth factor and other proteins or polypeptides.

In some embodiments, the other proteins or polypeptides include, but are not limited to, fc portions, human serum albumin, and the like.

In some embodiments, the nerve growth factor fusion protein of the present invention comprises a nerve growth factor, an optional peptide linker, and an Fc portion from the N-terminus to the C-terminus.

In some embodiments, the optional peptide linker may be of any length. In some embodiments, the linker length does not exceed the length necessary to prevent domain interactions and/or optimize biological function and/or stability. In some embodiments, the peptide linker is a hinge region of human IgG1, igG2, igG3, or IgG 4. In some embodiments, the peptide linker is a mutated human IgG1, igG2, igG3, or IgG4 hinge region. In some embodiments, the peptide linker comprises any one of the amino acid sequences of SEQ ID NOs 41-45. In some embodiments, the peptide linker comprises an amino acid sequence (GGGGS) n (SEQ ID NO: 43), where n is any integer of 1, 2, 3, 4, 5, or 6, preferably n is any integer of 2 to 6, more preferably n is an integer of 3 or 4. In some embodiments, the peptide linker comprises the amino acid sequence SEQ ID NO 41 or 42.

The Fc portion may be derived from humans, including but not limited to IgA, igD, igE, igG and IgM and any of their subclasses. The Fc portion may comprise one or more mutations or glycosylation modifications relative to the native Fc structural amino acid site, including insertions, deletions, and/or substitutions, among others. In some embodiments, the Fc portion is from an IgG1Fc, an IgG4Fc, or comprises one or more mutations or glycosylation modifications relative to the native IgG1Fc, igG4Fc amino acid site. In some embodiments, the Fc portion comprises the amino acid sequence shown as SEQ ID NO 46 or 47.

In some embodiments, the nerve growth factor fusion protein comprises a base acid sequence as set forth in SEQ ID NO. 48 or 49.

The medicine of the invention contains nerve growth factor or nerve growth factor fusion protein with effective treatment amount as main active component and pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are non-toxic to the subject at the dosages and concentrations employed, and are of a type well known in the art, including but not limited to buffers, antioxidants; ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and/or nonionic surfactants, and the like.

The medicament of the present invention may be administered by a variety of methods including, but not limited to: oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, and the like.

The dosage forms of the medicine include, but are not limited to, injection, capsule, tablet, powder injection and the like.

Drawings

FIG. 1 shows the nerve growth factor [ ]Proliferation rate of human ovarian granulosa cells (KGN) in murine NGF or mNGF 118) or in nerve growth factor fusion protein (2-118-L3 Fc10-M3-5 or 2-118-L3G 4-BM).

FIG. 2 shows the nerve growth factor [ ]Murine NGF or mNGF 118) or nerve growth factor fusion protein (2-118-L3 Fc10-M3-5 or 2-118-L3G 4-BM) or human ovarian granulosa cells (KGN).

FIG. 3 shows the nerve growth factor [ ]Murine NGF or mNGF 118) or nerve growth factor fusion protein (2-118-L3 Fc10-M3-5 or 2-118-L3G 4-BM).

Detailed Description

In order to make the technical problems to be solved, the technical scheme adopted and the advantages of the invention more clear, the invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The reagents used in the examples below, unless otherwise specified, were formulated in conventional manner or were commercially available; the experimental methods used, unless otherwise specified, are all conventional; the materials, instruments and the like used, unless otherwise specified, are commercially available.

4-vinylcyclohexene dioxide (4-vinylcyclohexene diepoxide, VCD), cat/specification: 94956-100ml of liquid contains 108g VCD, manufacturer: sigma Co Ltd

The nerve growth factors used in the examples of the present invention were derived as follows:

mouse NGF: from Shutaishen (Beijing) biopharmaceutical Co., ltd

The compositions and amino acid sequences of mNGF118, NGF-Fc fusion protein 2-118-L3G4-BM and NGF-Fc fusion protein 2-118-L3Fc10-M3-5 are shown in Table 1.

TABLE 1 composition and sequence of exemplary nerve growth factor and fusion protein thereof

Example 1: therapeutic effects of NGF and NGF-Fc fusion proteins on premature ovarian failure

Premature Ovarian Failure (POF) refers to the natural amenorrhea before age 40 due to ovarian failure. Often accompanied by a decrease in estrogen levels, an increase in folliculogenesis levels, and an increase in gonadotrophin levels, the etiology and mechanism of which are complex. This example investigated the therapeutic effect of NGF and NGF-Fc fusion proteins on POF by in vitro human ovarian granulose tumor cell line (KGN) proliferation assay and KGN estrogen secretion assay and POF model rats.

For KGN proliferation assay, 100ul of KGN suspension (1X 10) was added to each well of a 96-well plate one day before the assay ⁴ cells/mL). Before the test, the serum-free DMEM medium was replaced. After the culture medium is replaced, NGF is added into each experimental group holeMurine NGF or mNGF 118) or NGF-Fc fusion protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) to a final concentration of 10. Mu.g/mL in the medium, without any treatment after medium change in the negative control group. After 48 hours, 10uL of CCK-8 (Japanese same core chemical institute, #CK04) was added to each well to determine the number of living cells. After 1h incubation, absorbance at 450nm was measured and the recorded data was analyzed using Student t-test and a histogram was drawn using GraphPad Prism 8.0.1.

As shown in FIG. 1, NGF was measured as compared with PBS negative control groupMurine NGF or mNGF 118) and NGF-Fc fusion proteins (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) both significantly promoted KGN proliferation (p)<0.05)。

For KGN estrogen secretion assay, KGN cells, 1X 10, were seeded in 24-well plates ⁵ Cells/well (cell confluence about 80%) and serum-free medium was changed. After the culture medium is replaced, NGF is added into each experimental group holeMurine NGF or mNGF 118) or NGF-Fc fusion protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) to a final concentration of 10. Mu.g/ml in the medium. The negative control group was not subjected to any treatment after medium replacement. After incubation for 18h, the wells were washed twice with 2.2X10 s of each group of 4 duplicate wells ^-8 M testosterone (Beijing Soy Bao technology Co., # IT 0110) and 0.01IU/ml sheep follicle stimulating hormone (Ovine FSH, NHPP Co., USA) for 24hDiluting the supernatant 1.6 times, using R&The estrogen assay kit (KGE 014) produced by D Systems, measured absorbance at 450nm and calculated secreted estrogen concentrations, and the recorded data were analyzed using Student t-test and plotted using GraphPad Prism 8.0.1.

As shown in FIG. 2, NGF was measured as compared with the negative control groupMurine NGF or mNGF 118) and NGF-Fc fusion proteins (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) all significantly promoted KGN estrogen secretion (p)<0.05)。

Deoxyvinylcyclohexene (VCD) selectively destroys primordial and primary follicles in the ovaries of female mice without affecting secondary follicles and antral follicles, resulting in POF in female mice. To further investigate the in vivo therapeutic Effects of NGF and NGF-Fc fusion proteins on POF, SD rats were continuously intraperitoneally injected with VCD for two weeks to construct a POF rat model (see, e.g., muhammad FS et al, effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rates: ovarian, clinical, and pathologic outcomes [ J ]]Comp Med,2009,59 (1): 46-59.). NGF administration was performed immediately after the model was started, and was designated as day 1. Adopts the subcutaneous injection administration mode, NGF # Murine NGF or mNGF 118) or NGF-Fc fusion protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) experimental groups were injected at a dose of 10 μg/kg bw with the same volume of sterile physiological saline as a negative control. NGFMurine NGF, mNGF 118) or sterile saline once every other day, NGF-Fc fusionThe protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) was administered once a week. After 42 days, all rats were euthanized, ovarian tissue was fixed, and normal paraffin embedding, sectioning and H-plating were performed&E (hematoxylin and eosin) staining and counting of each stage of follicles. Recorded data were analyzed using Student t-test and histograms were drawn using GraphPad Prism 8.0.1.

As shown in FIG. 3, NGF was measured as compared with the negative control groupBoth murine NGF or mNGF 118) and NGF-Fc fusion proteins (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) significantly increased the number of primary follicles (p<0.05 Indicating that they have excellent effects in improving the reduction in the number of primary follicles caused by POF. NGF compared with the negative control groupMurine NGF or mNGF 118) or NGF-Fc fusion protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5) treated POF rat models also exhibited higher numbers of primary and secondary follicles.

Example 2: therapeutic effects of NGF and NGF-Fc fusion proteins on oligospermia

Oligospermia is mainly manifested by a reduced number of sperm and/or a reduced sperm motility. Sperm is formed by the proliferation of germ cells in the seminiferous tubules of the testes through a series of divisions, and heat stress can affect the division, differentiation and formation of sperm. This example investigated the therapeutic effect of NGF and NGF-Fc fusion proteins on oligospermia (oligospermia and oligospermia) in a mouse seminiferous model.

The experiment was performed using C57BL/6 JSHHjh mice (purchased from Shanghai Ji Hui laboratory animal feeding Co., ltd.). For the experimental group, 20 mug/kg bw/dose NGF is injected by subcutaneous injection in groinMurine NGF or mNGF 118) or 60 μg/kg bw/minor NGF-Fc fusion protein (2-118-L3G 4-BM or 2-118-L3Fc 10-M3-5). The normal control group or the insemination disorder model control group is injected with the equal volume of 0.9% sodium chloride injection. The day of first administration (NGF, NGF-Fc fusion protein or sodium chloride) was day 1. To construct an animal model of insemination disorder caused by heat stress of the testes of the mice, the mice are anesthetized after the first administration for 4 hours, and after the testes of the mice descend to the scrotum, the control group of the animal model of insemination disorder, the experimental group of NGF and the experimental group of NGF-Fc fusion protein are immersed in a constant temperature water bath at 42 ℃ for 30 minutes; the lower abdomen (hind limb, tail and scrotum) of the normal control mice was immersed in a constant temperature water bath at 25℃for 30 minutes.Murine NGF or mNGF118 was administered once every day, 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 2 times per week. The normal control group or the model control group is injected with the equal volume of 0.9 percent sodium chloride injection, and the injection is administrated 1 time every other day. The total administration was 5 weeks. Mice were euthanized on day 37 post-dosing, left epididymal tails were weighed and placed in M199 broth preheated at 37 ℃, sheared and placed in an incubator for 5 minutes at 37 ℃, sperm suspensions were aspirated, diluted 1:6 with M199 broth, after mixing, dilutions were taken and sperm count and sperm motility were detected using a TOX IVOS sperm analyzer. Recorded data were analyzed using Student t-test.

As shown in table 2, the number of sperm and sperm motility of the control group of the insemination disorder model were significantly lower than those of the normal control group, indicating that the animal model was successfully constructed. NGF compared with control group of dysspermia modelMurine NGF or mNGF 118) or NGF-Fc fusion protein (2-118-L3 Fc10-M3-5 and 2-118-L3G 4-BM) the sperm count and sperm motility were both significantly increased in the experimental group of mice. As can be seen, the subcutaneous injection of NGF or NGF-Fc fusion protein is effective in improving spermatogenic disorders (such as oligospermia, and oligospermia)Oligospermia) and decreased sperm motility.

TABLE 2 Effect of NGF and NGF-Fc fusion proteins on sperm count and sperm motility in dysspermia mice

To further verify the therapeutic effects of NGF and NGF-Fc, the right testis and epididymis of the euthanized mice described above were weighed, fixed with 10% neutral formalin, embedded, sectioned and H-stained&E staining to evaluate its histopathological lesions. As shown in Table 3, NGF [ ]Murine NGF or mNGF 118) and NGF-Fc fusion proteins (2-118-L3 Fc10-M3-5 and 2-118-L3G 4-BM) showed significant therapeutic effects on symptoms of testicular seminiferous tubule atrophy, seminiferous tubule seminiferous disorders and epididymal duct cell debris caused by heat stress.

TABLE 3 histopathological statistics of NGF or NGF-Fc fusion proteins injected subcutaneously in dysspermia mice (alone)

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

Use of a nerve growth factor or nerve growth factor fusion protein in the manufacture of a medicament for treating or ameliorating a disorder of the reproductive system.
The use according to claim 1, wherein the reproductive system diseases include, but are not limited to: reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian residual syndrome, ovarian follicular dysplasia, and spermatogenic disorder;

preferably, the insemination disorder includes, but is not limited to: oligospermia, azoospermia, teratospermia, oligospermia.
The use according to claim 1, wherein the amelioration of a reproductive disease includes, but is not limited to, one or more of the following improvements: (1) Promote proliferation of ovarian granulosa cells and/or estrogen secretion; (2) Preventing/reversing a decrease in the number and/or function of follicles, or increasing the number and/or function of follicles; (3) Improving testicular seminiferous tubule atrophy, seminiferous tubule seminiferous disorder and/or epididymal duct cell debris; (4) Improving the reduction in sperm count and/or motility, or improving sperm count and/or motility;

preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.
Use of a nerve growth factor or nerve growth factor fusion protein in the manufacture of a medicament for ameliorating a disorder or condition associated with or caused by a disorder or condition of the reproductive system.
The use according to claim 4, wherein the reproductive system diseases include, but are not limited to: reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian residual syndrome, ovarian follicular dysplasia, and spermatogenic disorder;

preferably, the insemination disorder includes, but is not limited to: oligospermia, azoospermia, teratospermia, oligospermia.
The use according to claim 5, wherein the disorder or symptom includes, but is not limited to: a reduced proliferation rate of ovarian granulosa cells and/or reduced estrogen secretion, reduced follicular number and/or reduced function, a reduced testicular seminiferous tubule atrophy, a seminiferous tubule seminiferous disorder and/or epididymal duct cell debris, a reduced sperm number and/or reduced motility;

preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.
Nerve growth factor or nerve growth factor fusion protein in the preparation of a medicament for promoting proliferation of ovarian granulosa cells and/or estrogen secretion in a patient; preventing/reversing a decrease in the number and/or function of follicles, or increasing the number and/or function of follicles; improving testicular seminiferous tubule atrophy, seminiferous tubule seminiferous disorder and/or epididymal duct cell debris; use in medicine for improving sperm count reduction and/or motility reduction, or improving sperm count and/or motility, etc.; preferably, the follicle is a primary follicle, a primary follicle and/or a secondary follicle.
The use according to claim 7, wherein the patient is a patient suffering from or associated with a disease of the reproductive system, including but not limited to a patient suffering from reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, residual ovarian syndrome, and/or ovarian follicular dysplasia, or a patient suffering from a spermatogenic disorder; preferably, the insemination disorder includes, but is not limited to: oligospermia, azoospermia, teratospermia, oligospermia.
The use according to any one of claims 1-8, wherein the nerve growth factor comprises a wild-type nerve growth factor, a recombinant nerve growth factor, a nerve growth factor mutant.
The use of claim 9, wherein the wild-type nerve growth factor comprises wild-type nerve growth factor isolated from various sources including livestock, primate, rabbit, and rodent; preferably, the wild-type nerve growth factor is a murine wild-type nerve growth factor or a human wild-type nerve growth factor.
The use according to claim 9, wherein the recombinant nerve growth factor comprises a recombinant nerve growth factor obtained by recombinant expression or synthetic method; preferably, the recombinant nerve growth factor is a murine recombinant nerve growth factor or a human recombinant nerve growth factor.
The use according to claim 9, wherein the nerve growth factor mutant is a nerve growth factor mutant having most or all of the biological activity of a wild-type nerve growth factor, comprising a mutation at one or more amino acid positions of the wild-type nerve growth factor; preferably, the nerve growth factor mutant relative to the wild-type nerve growth factor sequence of human source SEQ ID NO. 1 or 2, including one or more of the following amino acid mutations: F12E, K G, K L, K Y, R59L, R A, D65A, D G, K74L, K88 3987 88L, K88 3788G, Q96E, R V, R F, R G, R L and F101A.
The use according to claim 12, wherein the nerve growth factor mutant comprises any one of the amino acid sequences of SEQ ID NOs 3 to 40.
The use according to any one of claims 1 to 8, wherein the nerve growth factor fusion protein comprises, from N-terminus to C-terminus, a nerve growth factor, optionally a peptide linker and an Fc portion.
The use according to claim 14, wherein the nerve growth factor comprises any one of the amino acid sequences of SEQ ID NOs 1 to 40; preferably, the peptide linker comprises any one of the amino acid sequences of SEQ ID NOS.41-45, wherein n in SEQ ID NO. 43 is an integer of 1, 2, 3, 4, 5 or 6; preferably, the Fc portion comprises the amino acid sequence shown in SEQ ID NO 46 or 47.
The use according to claim 14, wherein the nerve growth factor fusion protein comprises the amino acid sequence shown in SEQ ID No. 48 or 49.
The use according to any one of claims 1 to 16, wherein the medicament comprises a therapeutically effective amount of a nerve growth factor or nerve growth factor fusion protein as a major active ingredient together with a pharmaceutically acceptable carrier.
The use according to claim 17, wherein the pharmaceutical dosage form comprises an injection, a capsule, a tablet or a powder injection.