Application of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells

Abstract

The present invention discloses the application of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. The present invention discovers for the first time this new use of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. Uridine not only has an obvious promoting effect on the proliferation of spermatogonial stem cells in vivo, but also has the same effect on promoting the proliferation of spermatogonial stem cells in vitro cultured spermatogonial stem cells, spermatogonial stem cells in testicular organoids cultured in vitro, and spermatogonial stem cells in senescent testes, and can repair problems such as the reduction in the number of spermatogonial stem cells caused by testicular injury induced by chemotherapeutic drugs, thereby providing a new solution for the clinical treatment of male infertility and the protection of male fertility.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. Background Art

[0002] Data released by the World Health Organization in 2023 shows that one-sixth of the reproductive-age population globally is troubled by infertility. According to the 2023 China Reproductive Health Industry Research Report, the infertility rate in China reached 18% in 2022, with the number of patients exceeding 50 million. Infertility has become the third major disease threatening people's lives and health after cardiovascular and cerebrovascular diseases and tumors. Among the factors causing infertility, approximately 50% are attributed to male factors. In addition to known causes such as vas deferens obstruction, hypogonadism, and sexual dysfunction, up to 60% of male patients have unknown causes, mainly manifested as idiopathic azoospermia or severe oligo-, astheno-, teratozoospermia, etc. Moreover, many acquired environmental factors (such as tumor chemotherapy, etc.) can lead to the loss of spermatogonial stem cells (SSC), thereby causing Sertoli cell-only syndrome. Since these patients lack functional sperm, they cannot obtain biological offspring even with the help of assisted reproductive technology. Male fertility is maintained by spermatogonial stem cells in the testis, and these cells can self-renew and produce differentiated germ cells for spermatogenesis. Therefore, in vitro culture of spermatogonial stem cells and transplantation into patients is the core method for treating any male infertility problem, including male fertility damage caused by chemotherapy drugs. However, currently, the in vitro culture method of human spermatogonial stem cells has not been established, which greatly limits the application of spermatogonial stem cells in the clinical intervention and treatment of male infertility.

[0003] Nutrient and metabolite sensing is one of the most basic biological processes of cells, and metabolic remodeling plays an important role in stem cell differentiation and the occurrence and development of diseases. Therefore, based on the discovery of key metabolic factors, developing and applying methods or products to promote the injury regeneration or proliferation of spermatogonial stem cells will provide a practical path for solving male infertility problems. In addition, an important current cause of male infertility is low sperm quality, and declining fertility caused by aging or bad living habits are the core factors leading to low sperm quality. Therefore, discovering key metabolic factors and applying them to male fertility protection is of great significance for clinical medicine and even social and economic development. Summary of the Invention

[0004] In view of this, in order to overcome the above-mentioned technical problems existing in the current field, the purpose of the present invention is to provide the use of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. The present invention discloses for the first time a key metabolic factor (uridine) that can promote the injury regeneration or proliferation of spermatogonial stem cells. To this end, the present invention proposes that supplementing uridine can promote the long-term proliferation of spermatogonial stem cells in vivo and the regeneration after injury, can resist and improve the injury effect of chemotherapeutic drugs on spermatogonial stem cells and promote spermatogenesis recovery, can promote the continuous proliferation and long-term culture of spermatogonial stem cells in vitro, and can promote the continuous proliferation of spermatogonial stem cells in testicular organoids cultured in vitro and the long-term culture of the testicular organoids themselves. The present invention has extremely high drug development and clinical application value for solving male infertility.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention purpose:

[0006] The first aspect of the present invention provides the use of uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates, crystalline forms or derivatives thereof in the preparation of a drug for promoting the injury regeneration and / or proliferation of spermatogonial stem cells.

[0007] Further, the injury of the spermatogonial stem cells is the injury of spermatogonial stem cells caused by chemotherapeutic drugs, the injury of spermatogonial stem cells caused by mechanical force and / or the injury of spermatogonial stem cells caused by non-chemotherapeutic drugs.

[0008] Further, the chemotherapeutic drugs are busulfan, cyclophosphamide, cisplatin, carboplatin, bleomycin, doxorubicin, methotrexate, fluorouracil, gemcitabine, vincristine, vinblastine, paclitaxel or docetaxel.

[0009] Further, the derivatives are uridine modified with nanoparticles, uridine modified with liposomes, exosomes encapsulating uridine, micelles encapsulating uridine, viral vectors encapsulating uridine, protein microspheres encapsulating uridine and / or PEG-modified proteins containing uridine.

[0010] In the present invention, the uridine is a ribonucleoside formed by connecting uracil and ribose through a β-N1-glycosidic bond. Uracil is a nitrogen-containing heterocyclic compound belonging to pyrimidines. Its chemical structure features a pyrimidine ring with nitrogen atoms and carbonyl groups and other functional groups at different positions on the ring. Ribose is a pentose sugar with multiple hydroxyl groups in its structure, and these hydroxyl groups play an important role in the chemical properties and physiological functions of uridine. Its corresponding CAS number is 58-96-8, the molecular formula is C9H 12 N2O6, and the molecular weight is 244.2. The present invention has no particular limitation on the specific source of the uridine.

[0011] In some embodiments, the pharmaceutically acceptable salt refers to a pharmaceutically acceptable salt of uridine, which refers to those carboxylates, amino acid addition salts, etc. of the compound (uridine) of the present invention, which are applicable to contact with patients within the scope of reliable medical judgment, do not produce undue toxicity, irritation, allergic reactions, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended applications, including (if possible) the zwitterionic form of the compound of the present invention.

[0012] In some embodiments, examples of the pharmaceutically acceptable salts of uridine include, but are not limited to: salts having (as counterions) alkali metal ions such as Li + , Na + or K + , or salts having alkaline earth metal ions such as Mg 2+ or Ca 2+ , or salts having any other pharmaceutically acceptable metal ions such as Zn 2+ or Al 3+ ; or pharmaceutically acceptable salts formed with organic bases such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine or tromethamine.

[0013] In some embodiments, the pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metals and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are magnesium, potassium, sodium, calcium, etc. Examples of suitable amines are N,N'-dibenzylethylenediamine, diethanolamine, chloroprocaine, choline, ethylenediamine, N-methylglucamine or procaine.

[0014] In some embodiments, the base addition salts of acidic compounds can be prepared by the following method: contacting the free acid form with a sufficient amount of the required base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid and then separating the free acid.

[0015] In some embodiments, the salts can be bisulfates, sulfites, bisulfites, pyrophosphates, monohydrogen phosphates, sulfates, nitrates, phosphates, pyrosulfates, bromides, chlorides, dihydrogen phosphates, metaphosphates, iodides prepared from inorganic acids, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc.

[0016] In some embodiments, representative salts include: hydrobromides, acetates, oxalates, maleates, fumarates, valerates, hydrochlorides, sulfates, stearates, laurates, oleates, palmitates, borates, benzoates, lactates, phosphates, tosylates, bisulfates, nitrates, citrates, succinates, naphthoates, etc.

[0017] In some embodiments, the salt can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, hydroxyalkanoic acids, aromatic acids, alkane diacids, phenyl-substituted alkanoic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, octanoate, isobutyrate, oxalate, propionate, octanedioate, fumarate, malonate, succinate, maleate, sebacate, mandelate, benzoate, etc.

[0018] In some embodiments, pharmaceutically acceptable salts can include cations based on alkali metals and alkaline earth metals, such as sodium, potassium, calcium, lithium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to: ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, etc. Also included are salts of amino acids, such as gluconate, arginine salt, galacturonate, etc.

[0019] In some embodiments, the hydrate refers to a compound obtained by combining the compound (uridine) of the present invention with water. Generally, the ratio of the number of water molecules contained in the hydrate of the compound to the number of compound molecules in the hydrate is determined. Thus, the hydrate of the compound can be represented, for example, by the general formula R·xH2O, where R is the compound (uridine) and x is a number greater than 0.

[0020] In some embodiments, the solvate refers to a solvate addition form of a compound containing a stoichiometric or non-stoichiometric amount of a solvent, including any solvated form of the compound (uridine) of the present invention. Conventional solvents include but are not limited to: water, ethanol, acetic acid, methanol, DMSO, THF, ether, etc. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0021] In some embodiments, the crystalline form refers to the crystalline form of uridine, in which uridine molecules are interconnected by intermolecular forces such as hydrogen bonds. Both the ribose moiety and the uracil moiety in the uridine molecule participate in these interactions. For example, hydrogen bonds may form between the hydroxyl group (-OH) on the ribose and the carbonyl group (C=O) or other suitable groups in adjacent uridine molecules, and this hydrogen bond network helps to stabilize the crystal structure.

[0022] In some embodiments, the derivative refers to a uridine derivative, and any modified uridine obtained after modification of uridine falls within the protection scope of the present invention. Exemplarily, the uridine derivative includes but is not limited to: nanoparticles-modified uridine, liposome-modified uridine, exosomes encapsulating uridine, micelles encapsulating uridine, viral vectors encapsulating uridine, protein microspheres encapsulating uridine, PEG-modified proteins containing uridine, or uridine modified in other forms.

[0023] The second aspect of the present invention provides the use of uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereoisomers, solvates, crystalline forms or derivatives thereof in any of the following aspects:

[0024] (1) Use in the preparation of an efficient in vitro culture product for mammalian spermatogonial stem cells;

[0025] (2) Use in the preparation of an efficient in vitro culture product for mammalian testicular organoids;

[0026] (3) Use in the preparation of an in vitro culture product for promoting the rejuvenation of senescent spermatogonial stem cells;

[0027] (4) Use in the preparation of a product for treating, preventing, alleviating and / or improving male infertility caused by chemotherapeutic drugs;

[0028] (5) Use in the preparation of a product for enhancing the quantity, quality, fertility and / or testicular function of sperm production in males in vivo;

[0029] (6) Use in the preparation of a product for enhancing the fertility of elderly male populations.

[0030] Furthermore, the in vitro culture product is a culture reagent, a culture solution, a culture substrate or a culture device.

[0031] Furthermore, the product is a drug.

[0032] Furthermore, the derivative is nanoparticles-modified uridine, liposome-modified uridine, exosomes encapsulating uridine, micelles encapsulating uridine, viral vectors encapsulating uridine, protein microspheres encapsulating uridine and / or PEG-modified proteins containing uridine.

[0033] Furthermore, the chemotherapeutic drug is busulfan, cyclophosphamide, cisplatin, carboplatin, bleomycin, doxorubicin, methotrexate, fluorouracil, gemcitabine, vincristine, vinblastine, paclitaxel or docetaxel.

[0034] In some embodiments, according to the above application, the promotion of long-term proliferation and post-injury regeneration of spermatogonial stem cells in vivo can be specifically manifested as at least one of the following:

[0035] B1) The expression level of the proliferation marker Ki67 increases in the testicular spermatogonial stem cell population;

[0036] B2) The expression levels of spermatogonial stem cell markers (such as Gfra1, Nefm, Lhx1, Id4, etc.) increase in the testis;

[0037] B3) The number of Ki67-positive cells increases in testicular spermatogonial stem cells;

[0038] B4) The number of spermatogonial stem cells increases in the testis.

[0039] In some embodiments, the ability to resist and improve the damage effect of chemotherapeutic drugs on spermatogonial stem cells and promote spermatogenesis recovery can be specifically manifested as at least one of the following:

[0040] C1) The expression level of the proliferation marker Mki67 increases in the spermatogonial stem cell population after testicular injury;

[0041] C2) The expression levels of spermatogonial stem cell markers (such as Gfra1, Nefm, Lhx1, Id4, etc.) increase after testicular injury;

[0042] C3) The number of Mki67-positive cells increases in spermatogonial stem cells after testicular injury;

[0043] C4) The number of spermatogonial stem cells increases after testicular injury;

[0044] C5) It can significantly shorten the spermatogenesis recovery time compared with the chemotherapeutic drug-affected group;

[0045] C6) After mating with normal wild-type C57BL6J female mice, it can get pregnant and give birth earlier compared with the chemotherapeutic drug-affected group.

[0046] In some embodiments, the ability to promote the continuous proliferation and long-term culture of spermatogonial stem cells in vitro can be specifically manifested as at least one of the following:

[0047] D1) The expression level of the proliferation marker Mki67 increases in the spermatogonial stem cell population;

[0048] D2) The expression levels of neonatal spermatogonial stem cell markers (such as Gfra1, Nefm, Lhx1, Id4, etc.) increase;

[0049] D3) The number of Mki67-positive cells increases in spermatogonial stem cells;

[0050] D4) The number of neonatal spermatogonial stem cells increases;

[0051] D5) The expression level of the proliferation marker Mki67 increases in the spermatogonial stem cell line C18-4;

[0052] D6) The expression levels of spermatogonial stem cell markers (such as Gfra1, Nefm, Lhx1, Foxc2, etc.) in the spermatogonial stem cell line C18-4 increase;

[0053] D7) The number of Mki67-positive cells in the spermatogonial stem cell line C18-4 increases;

[0054] D8) The number of newborn cells increases.

[0055] In some embodiments, the ability to promote the continuous proliferation of spermatogonial stem cells and the long-term culture of testicular organoids themselves in vitro can be specifically embodied as at least one of the following:

[0056] E1) The expression level of the proliferation marker Mki67 in testicular organoids increases;

[0057] E2) The expression levels of spermatogonial stem cell markers (such as Gfra1, Nefm, Lhx1, Foxc2, etc.) in testicular organoids increase;

[0058] E3) The number of Mki67-positive cells in spermatogonial stem cells in testicular organoids increases;

[0059] E4) The number of spermatogonial stem cells in testicular organoids increases.

[0060] The third aspect of the present invention provides a method for promoting the injury regeneration and / or proliferation of spermatogonial stem cells in vitro for non-therapeutic purposes.

[0061] Furthermore, the method includes: treating a system in need with uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof.

[0062] In some embodiments, the present invention places no particular limitation on the system, as long as it is a system containing spermatogonial stem cells, it is a system in need. Exemplarily, the system includes a cell system, a subcellular system, a tissue system or an organ system, and spermatogonial stem cells are included in the cell system, subcellular system, tissue system or organ system.

[0063] The fourth aspect of the present invention provides a pharmaceutical composition or a pharmaceutical preparation.

[0064] Furthermore, the pharmaceutical composition or the pharmaceutical preparation contains a prophylactically and / or therapeutically effective amount of uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof.

[0065] In some embodiments, the pharmaceutical composition or the pharmaceutical preparation further contains a pharmaceutically acceptable excipient and / or vehicle.

[0066] In some embodiments, the pharmaceutically acceptable excipients and / or carriers are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used as needed to assist in the stability of the drug or to enhance the activity of the active ingredient (i.e., uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates, crystalline forms or derivatives thereof as described above in the present invention). Such substances include, but are not limited to: diluents, surfactants, humectants, binders, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents. The pharmaceutical composition or formulation thus prepared can be administered by any suitable administration method known to those skilled in the art as needed.

[0067] In some embodiments, the present invention places no particular limitation on the pharmaceutically acceptable excipients and / or carriers. In addition, the pharmaceutically acceptable excipients and / or carriers may further contain liquids such as water, physiological saline, glycerol and ethanol.

[0068] In some embodiments, the present invention places no particular limitation on the dosage form of the pharmaceutical formulation. Exemplarily, the dosage forms of the pharmaceutical formulation include, but are not limited to: solutions, emulsions, suspensions, controlled release formulations, tablets, pills, powders, granules, capsules, lozenges, syrups, aerosols, films, injections, intravenous drip agents, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, nasal formulations or pulmonary formulations, etc., for ingestion by patients.

[0069] Those skilled in the art can understand that although the pharmaceutical composition or formulation mentioned above in the present invention may further contain pharmaceutically acceptable excipients and / or carriers, when uridine is used as a drug for humans or animals, it can also be administered in its own form, that is, the present invention can be achieved without adding any of the above-mentioned pharmaceutically acceptable excipients and / or carriers.

[0070] In some embodiments, the suitable administration methods of the pharmaceutical composition or formulation of the present invention include any one of various methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical composition or formulation of the present invention into a subject. Such administration methods include, but are not limited to: oral administration, topical administration, parenteral administration, administration by inhalation spray, rectal administration, nasal administration, buccal administration or administration through an implanted reservoir device.

[0071] In some embodiments, oral administration or injection administration can be selected, wherein injection administration includes forms of rapid injection or continuous infusion. When the pharmaceutical composition or pharmaceutical preparation is used for injection administration, it can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it can contain formulation reagents such as preservatives, stabilizers, suspending agents and / or dispersing agents.

[0072] In some embodiments, when actually using the pharmaceutical composition or pharmaceutical preparation, its dosing regimen and dosage regimen can be selected according to various factors, including the type, species, age, weight, sex of the subject and the type of disease being treated; the severity of the disease being treated; the route of administration; the renal and liver functions of the patient; and the specific compound or its salt used. One dosing and / or dosage regimen can be used, for example, to prevent a disease, inhibit (fully or partially inhibit) a disease or arrest the development of the disease. In a specific embodiment of the present invention, the disease specifically refers to male infertility-related diseases.

[0073] In addition, the present invention also provides a kit comprising uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, and the pharmaceutical composition or pharmaceutical preparation as described above, that is, by formulating uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, and the pharmaceutical composition or pharmaceutical preparation as described above in separate unit dosage forms, and then combining these separate unit dosage forms to form the form of a kit. In this case, the uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, and the pharmaceutical composition or pharmaceutical preparation as described above in the separate unit dosage forms can be easily administered to a subject simultaneously, sequentially or separately in time.

[0074] In addition, the present invention also provides a method for treating, preventing, alleviating and / or improving male infertility caused by chemotherapeutic drugs, the method comprising: administering to a subject in need a prophylactically and / or therapeutically effective amount of uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, and the pharmaceutical composition or pharmaceutical preparation as described above.

[0075] In addition, the present invention also provides a method for enhancing the quantity, quality, fertility and / or testicular function of sperm in males, the method comprising: administering to a subject in need a prophylactically and / or therapeutically effective amount of uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, and the pharmaceutical composition or pharmaceutical preparation as described above.

[0076] In addition, the present invention also provides a method for enhancing the fertility of elderly men, which method comprises: administering to a subject in need a prophylactically and / or therapeutically effective amount of uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, the pharmaceutical composition or pharmaceutical preparation as described above.

[0077] In some embodiments, the prophylaxis and / or treatment further includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or disorder; the term also includes prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving a related disease, pathological condition or disorder. In a specific embodiment of the present invention, the disease particularly refers to male infertility-related diseases.

[0078] In some embodiments, the effective amount refers to an amount having a prophylactic or therapeutic effect or an amount required to produce a prophylactic or therapeutic effect in a subject. For example, a pharmaceutically prophylactically or therapeutically effective amount refers to the amount of a drug required to produce a desired prophylactic effect or therapeutic effect, and the prophylactic effect or therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject to be treated (such as height, weight, gender, age and medication history), the type of disease suffered, and the severity of the disease suffered.

[0079] In some embodiments, the subject (or test subject, or patient) includes one or more animals, including, for example, bovine, equine, ovine, primate, avian and rodent species. The subject can be a mammal, bird, fish, reptile or amphibian. The mammal includes human or non-human mammals. In other embodiments, the subject can be a mouse, rat, hamster, ferret, gerbil, rabbit, monkey, chimpanzee, horse, pony, donkey, sheep, pig, chicken, goat, cat or dog. In a preferred embodiment, the subject is a human.

[0080] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0081] The present invention discloses for the first time the application of uridine in promoting the regeneration or proliferation of damaged spermatogonial stem cells. Through experimental verification, the present invention creatively discovers for the first time that uridine has an obvious promoting effect on the proliferation of spermatogonial stem cells in vivo, and also has the same effect on promoting the proliferation of spermatogonial stem cells in vitro-cultured spermatogonial stem cells and in vitro-cultured testicular organoids, and can repair problems such as the reduction in the number of spermatogonial stem cells caused by testicular damage induced by chemotherapeutic drugs, thereby providing a new solution for the clinical treatment of male infertility and the protection of male fertility. Brief Description of the Drawings

[0082] Figure 1 Schematic diagram for constructing an animal experimental model for in vivo proliferation and injury regeneration of spermatogonial stem cells in the embodiments of the present invention;

[0083] Figure 2 Graph showing the mRNA quantification results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of mouse spermatogonial stem cells after injecting 600 μg (150 μL) of uridine in vivo in the embodiments of the present invention;

[0084] Figure 3 Graph showing the mRNA quantification results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of mouse spermatogonial stem cells after constructing a spermatogonial stem cell injury model by treatment with the chemotherapeutic drug busulfan and then injecting 600 μg (150 μL) of uridine in vivo in the embodiments of the present invention;

[0085] Figure 4 Graph showing the production and pregnancy results of the chemotherapeutic drug influence group and the uridine supplementation group in the embodiments of the present invention;

[0086] Figure 5 Schematic diagram of the culture process of primary spermatogonial stem cells supplemented with uridine in the embodiments of the present invention;

[0087] Figure 6 Graph showing the mRNA quantification results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of mouse primary spermatogonial stem cells after culturing with different concentrations of uridine in the embodiments of the present invention;

[0088] Figure 7 Graph showing the mRNA quantification results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Id4, Foxc2) of mouse spermatogonial stem cell line C18-4 after culturing with different concentrations of uridine in the embodiments of the present invention;

[0089] Figure 8 Schematic diagram of the construction and culture process of testis organoids supplemented with uridine in the embodiments of the present invention;

[0090] Figure 9 Graph showing the mRNA quantification results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Id4, Foxc2) of spermatogonial stem cells in mouse testis organoids after culturing with different concentrations of uridine in the embodiments of the present invention. Detailed Description of the Invention

[0091] The present invention will be further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents. The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained from commercial sources. The experimental methods without specific conditions described in the present invention are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers. In particular, the following embodiments are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0092] Unless otherwise specified, the experimental consumables and reagents involved in the present invention can all be obtained from commercial sources. Unless otherwise specified, the specific experimental methods involved in the present invention are all conventional methods and are carried out according to the methods described in relevant literature in the art or according to the product instructions. Unless otherwise specified, the experiments involved in the present invention are repeated at least 3 times, and statistical analysis is performed using GraphPad Prism v8.0.2 software. Independent sample t-tests are used for comparing differences between two groups, and the results of experimental quantitative analysis are expressed as mean ± standard error. n.s. represents that there is no statistical difference in the experimental results; P < 0.05 represents that there is a statistical difference in the experimental results; P < 0.01 represents that there is a significant statistical difference in the experimental results; P < 0.001 represents that there is an extremely significant statistical difference in the experimental results.

[0093] The mice in the following examples are as follows: All experimental animals used in this study are C57BL6J mice, purchased from Jiangsu Jicui Yakang Co., Ltd. Male mice are 8 weeks old when purchased and are housed in an SPF-class mouse house with a 12-hour day-night cycle, a relative humidity maintained at 30%-40%, and a breeding temperature controlled at 22-24°C. No more than 5 mice are housed in each cage, and formal experiments begin after one week of adaptive breeding.

[0094] Uridine was purchased from Sigma, with the product number U3003-5G.

[0095] Busulfan was purchased from Sigma, with the product number B2635-10G.

[0096] Dimethyl sulfoxide (DMSO) was purchased from Sigma, with the product number D2650.

[0097] Example 1: Uridine Promotes the Proliferation of Spermatogonial Stem Cells in Vivo under Physiological Conditions

[0098] 1. Experimental methods

[0099] Eight - week - old male C57BL6J mice were evenly divided into 2 groups, with 5 mice in each group. The two groups were: the control group (CON) and the uridine - injected group (CON_U). The dose of uridine injected into each mouse in the CON_U group was 600 μg (150 μL).

[0100] For the control group (CON) and the uridine - injected group (CON_U) in the above - mentioned examples, the general intraperitoneal injection method was used. Uridine was dissolved and prepared with 1X PBS. For the convenience of comparison with the experiments in Example 2 later, CON and CON_U needed to be injected with 30% DMSO at the corresponding dose on the first day. On the second day, each mouse in the CON group was injected with 150 μL of 1X PBS, and each mouse in the CON_U group was injected with 150 μL of the uridine solution. Subsequently, the corresponding 1X PBS and uridine solution were injected every other day. The injection time was 3:00 p.m. every day, and each mouse was injected 5 times in one experimental cycle. At 3:00 p.m. on the second day after the last injection, the mice were sacrificed by cervical dislocation, and the testis samples were collected for statistics and experimental treatment.

[0101] 2. Experimental results

[0102] The results are as Figure 1 shown. The results show the animal experiment treatment process of the control group (CON) and the uridine - injected group (CON_U). As Figure 2 shown, the QPCR quantitative statistical results show that after injecting 600 μg (150 μL) of uridine in vivo (CON_U), compared with the control group (CON), the proliferation marker Mki67 of spermatogonial stem cells and the stem cell markers (Gfra1, Nefm, Lhx1, Id4) in the testes of mice are significantly increased, indicating that supplementing uridine can effectively promote the proliferation of spermatogonial stem cells in vivo under physiological conditions.

[0103] Example 2: Uridine promotes the regeneration of testicular spermatogonial stem cells damaged by chemotherapy drugs in vivo

[0104] 1. Experimental methods

[0105] Eight - week - old male C57BL6J mice were evenly divided into 2 groups, with 5 mice in each group. The two groups were: the busulfan - treated group (BU) and the uridine - injected group (BU_U). Busulfan was injected at a low dose of 15 mg / kg to establish a model of testicular spermatogonial stem cell damage caused by chemotherapy drugs in vivo. The dose of uridine injected into each mouse in the BU_U group was 600 μg (150 μL).

[0106] In the above-mentioned embodiments, both the busulfan treatment group (BU) and the uridine injection group (BU_U) adopted the general intraperitoneal injection method. Busulfan was dissolved and formulated with 30% DMSO, and uridine was dissolved and formulated with 1X PBS; on the first day, each mouse in both BU and BU_U groups needed to be weighed, and then each mouse was injected with the corresponding dose of busulfan according to its body weight, and the maximum volume of the injection solvent did not exceed 200 μL; on the second day, each mouse in the BU group was injected with 150 μL of 1X PBS, and each mouse in the BU_U group was injected with 150 μL of uridine solution. The subsequent treatment was to inject the corresponding 1X PBS and uridine solution every other day; the injection time was 3:00 pm every day, and each mouse was injected 5 times in one experimental cycle; at 3:00 pm on the second day after the last injection, the mice were sacrificed by cervical dislocation and the testis samples were collected for statistical and experimental processing.

[0107] 2. Experimental results

[0108] The results are as Figure 1 shown. The results show the animal experiment processing procedures of the busulfan treatment group (BU) and the uridine injection group (BU_U); as Figure 3 shown, the QPCR quantitative statistical results show that after injecting 600 μg (150 μL) of uridine in vivo (BU_U), compared with the busulfan treatment group (BU), the proliferation marker Mki67 of spermatogonial stem cells and the stem cell markers (Gfra1, Nefm, Lhx1, Id4) in the testes of mice were significantly increased, indicating that supplementing uridine can effectively promote the regeneration and recovery of testicular spermatogonial stem cells damaged by chemotherapy drugs in vivo; as Figure 4 shown, the results show the fertility identification experiment procedures of male mice in the BU group and male mice in the BU_U group. Compared with male mice in the BU group, after male mice in the BU_U group mated with female mice, the pregnancy and production time of female mice were earlier. This result also shows that uridine can resist and improve the damage effect of chemotherapy drugs on spermatogonial stem cells and promote spermatogenesis recovery.

[0109] Example 3: Uridine promotes the proliferation of in vitro cultured spermatogonial stem cells

[0110] 1. Experimental method

[0111] Three 7-day-old mice were taken, and their testes were collected. After removing the tunica albuginea, the tissues were digested into single-cell suspensions. After passing through a 40-μm filter sieve, the cells were seeded into culture dishes, and spermatogonial stem cells were screened using the differential attachment method. The time for the first passage of the cell line depended on the growth of primary spermatogonial stem cell clones, and the subsequent passage time was about 7 days. When the cell line was passaged more than 5 times, the state gradually became stable and was used for experiments. In this example, the 7th-generation spermatogonial stem cells were used. After culturing for three days, uridine was added to the medium at six concentration gradients of 0 μg / mL, 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 200 μg / mL. When the medium was changed on the fifth day, the spermatogonial stem cell medium was replaced again at the six concentrations of 0 μg / mL, 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 200 μg / mL. Samples were collected on the sixth day and used for experiments. The group without uridine addition (0 μg / mL) served as the control, and the groups with uridine addition at 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 200 μg / mL were used to explore the effect of uridine supplementation on primary spermatogonial stem cells cultured in vitro. In this example, the spermatogonial stem cell line C18-4 was also used to explore the effect of uridine supplementation on the proliferation of spermatogonial stem cells. The cryopreserved C18-4 cells were thawed and passaged twice until stable. Then, uridine was added to the medium at six concentrations of 0 μM, 1 μM, 10 μM, 100 μM, 500 μM, and 1000 μM. This cell line was passaged daily, and samples from the third generation were collected for further experiments. The group without uridine addition (0 μM) served as the control, and the groups with uridine addition at 1 μM, 10 μM, 100 μM, 500 μM, and 1000 μM were used to explore the effect of uridine supplementation on the spermatogonial stem cell line C18-4 cultured in vitro.

[0112] 2. Experimental Results

[0113] The results are as Figure 5 shown. The results show the process of obtaining primary spermatogonial stem cells and culturing and treating them with uridine addition; as Figure 6 shown, the QPCR quantitative statistical results show that compared with the group without uridine addition (0 μg / mL), the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of spermatogonial stem cells cultured in vitro were significantly increased in the uridine addition groups at different concentrations (0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 200 μg / mL). Among them, the uridine at a concentration of 10 μg / mL had the greatest effect on improving the proliferation efficiency of spermatogonial stem cells. The above results indicate that uridine supplementation can effectively promote the proliferation of spermatogonial stem cells cultured in vitro; as Figure 7As shown in the figure, the QPCR quantitative statistical results of the spermatogonial stem cell line C18-4 showed that in the uridine addition groups with different concentrations (1 μM, 10 μM, 100 μM, 500 μM, 1000 μM), compared with the group without uridine addition (0 μM), the Mki67 of the spermatogonial stem cell line C18-4 cultured in vitro and the stem cell markers (Gfra1, Nefm, Id4, Foxc2) were significantly increased. Among them, the uridine at a concentration of 10 μM had the greatest effect on improving the proliferation efficiency of the spermatogonial stem cell line C18-4. The above results indicate that supplementing uridine can also effectively promote the proliferation of the spermatogonial stem cell line C18-4.

[0114] Example 4: Uridine Promotes the Proliferation of Spermatogonial Stem Cells in Testicular Organoids

[0115] 1. Experimental Method

[0116] Ten 3-day-old mice were taken, and their testes were collected. After removing the tunica albuginea, the testes were digested into single-cell state; after centrifuging at 1000 rpm for 3 min, the supernatant was discarded, and then the cells were resuspended with the polymerization solution and inoculated into a U96 well plate. After 48 hours, the spermatogonial stem cell medium was changed and the cells were transferred to an agar block for culture. After 4 days, the differentiation solution was changed and uridine was added to the differentiation solution at four concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, and the samples were collected after continuing to culture for 4 days for further experiments.

[0117] 2. Experimental Results

[0118] The results are as Figure 8 shown. The results show the process of constructing testicular organoids and culturing and treating them with uridine addition; as Figure 9 shown, the QPCR quantitative statistical results showed that in the uridine addition groups with different concentrations (5 μg / mL, 10 μg / mL, 20 μg / mL), compared with the group without uridine addition (0 μg / mL), the proliferation marker Mki67 of spermatogonial stem cells in testicular organoids and the stem cell markers (Gfra1, Nefm, Id4, Foxc2) were significantly increased. Among them, the uridine at a concentration of 10 μg / mL had the greatest effect on improving the proliferation efficiency of spermatogonial stem cells in testicular organoids. The above results indicate that supplementing uridine can effectively promote the proliferation of spermatogonial stem cells in testicular organoids and promote the development and growth of testicular organoids.

Claims

1. Use of uridine or its pharmaceutically acceptable salt in the preparation of a drug for promoting the regeneration and / or proliferation of damaged spermatogonial stem cells; The spermatogonial stem cell damage is spermatogonial stem cell damage caused by chemotherapeutic drugs.

2. The use according to claim 1, characterized in that: The chemotherapy drug is busulfan, cyclophosphamide, cisplatin, carboplatin, bleomycin, doxorubicin, methotrexate, fluorouracil, gemcitabine, vincristine, vinblastine, paclitaxel or docetaxel.

3. Use of uridine or its pharmaceutically acceptable salts in the preparation of a highly efficient in vitro culture product of mammalian spermatogonial stem cells.

4. The use according to claim 3, characterized in that: The in vitro culture product is a culture reagent.

5. A method for promoting the regeneration and / or proliferation of spermatogonial stem cells in vitro for non-therapeutic purposes, characterized in that: The method comprises: treating a system in need thereof with uridine or a pharmaceutically acceptable salt thereof; The spermatogonial stem cell damage is spermatogonial stem cell damage caused by chemotherapeutic drugs.

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