Application of uridine in promoting regeneration or proliferation of spermatogonial stem cell injury

Abstract

The invention discloses an application of uridine in promotion of injury regeneration or proliferation of spermatogonial stem cells, and discloses a new application of uridine in promotion of injury regeneration or proliferation of spermatogonial stem cells for the first time. The same effect is also achieved in the aspects of promoting proliferation of spermatogonial stem cells cultured in vitro, spermatogonial stem cells in testis organs cultured in vitro and spermatogonial stem cells in senescent testis, and the problem that the number of spermatogonial stem cells is reduced due to testis injury caused by chemotherapeutic drugs can be solved; therefore, a new solution is provided for clinical treatment of male infertility and male fertility protection.

Description

Technical Field

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

[0002] About 50% of the factors leading to infertility 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 oligozoospermia, asthenozoospermia and teratozoospermia. In addition, many acquired environmental factors (such as tumor chemotherapy, etc.) can lead to the loss of spermatogonial stem cells (SSC) and thus cause 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 testicles, which can self-renew and produce differentiated germ cells for spermatogenesis. Therefore, the in vitro culture of spermatogonial stem cells and their transplantation into patients is the core method for treating any male infertility problems, including male fertility damage caused by chemotherapy drugs. However, the in vitro culture method of human spermatogonial stem cells has not yet been established, which greatly limits the application of spermatogonial stem cells in clinical intervention and treatment of male infertility.

[0003] Nutrition and metabolite perception 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, the development and application of methods or products that promote the regeneration or proliferation of damaged spermatogonial stem cells will provide a realistic path to solving the problem of male infertility. In addition, one of the important causes of male infertility is poor sperm quality, and the decline in fertility caused by aging or bad living habits are the core factors leading to poor sperm quality. Therefore, discovering key metabolic factors and applying them to male fertility protection is of great significance to 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 art, the purpose of the present invention is to provide the use of uridine in promoting the regeneration or proliferation of spermatogonial stem cells after injury. The present invention discloses for the first time a key metabolic factor (uridine) that can promote the regeneration or proliferation of spermatogonial stem cells after injury. To this end, the present invention proposes that supplementing uridine can promote the long-term proliferation of spermatogonial stem cells in the body and regeneration after injury, can resist and improve the spermatogonial stem cell damage caused by chemotherapeutic drugs and promote spermatogenesis recovery, can promote the continuous proliferation and long-term culture of spermatogonial stem cells in vitro, can promote the continuous proliferation of spermatogonial stem cells in in vitro cultured testicular organoids and the long-term culture of testicular organoids themselves, and 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 objectives:

[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 regeneration and / or proliferation of damaged spermatogonial stem cells.

[0007] Furthermore, the spermatogonial stem cell damage is spermatogonial stem cell damage caused by chemotherapeutic drugs, spermatogonial stem cell damage caused by mechanical force and / or spermatogonial stem cell damage caused by non-chemotherapeutic drugs.

[0008] Furthermore, the chemotherapy drug is busulfan, cyclophosphamide, cisplatin, carboplatin, bleomycin, doxorubicin, methotrexate, fluorouracil, gemcitabine, vincristine, vinblastine, paclitaxel or docetaxel.

[0009] Furthermore, the derivative is nanoparticle-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.

[0010] In the present invention, uridine is a ribonucleoside, which is formed by connecting uracil and ribose through a β-N1-glycosidic bond. Uracil is a nitrogen-containing heterocyclic compound belonging to the pyrimidine class. Its chemical structure is characterized by a pyrimidine ring with nitrogen atoms and carbonyl groups at different positions of the ring. Ribose is a pentose sugar with multiple hydroxyl groups in its structure. These hydroxyl groups play an important role in the chemical properties and physiological functions of uridine. Its corresponding CAS number is 58-96-8 and its molecular formula is C9H 12 N2O6, 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 means those carboxylates, amino acid addition salts, etc. of the compound (uridine) described in the present invention, which are suitable for contact with patients within the scope of sound 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 use, including (if possible) zwitterionic forms of the compounds described in the present invention.

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

[0013] In some embodiments, 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, base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and isolating the free acid.

[0015] In some embodiments, the salt can be a bisulfate, sulfite, bisulfite, pyrophosphate, monohydrogenphosphate, sulfate, nitrate, phosphate, pyrosulfate, bromide, chloride, dihydrogenphosphate, metaphosphate, iodide prepared from an inorganic acid, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like.

[0016] In some embodiments, representative salts include: hydrobromide, acetate, oxalate, maleate, fumarate, valerate, hydrochloride, sulfate, stearate, laurate, oleate, palmitate, borate, benzoate, lactate, phosphate, tosylate, bisulfate, nitrate, citrate, succinate, naphthoate, and the like.

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

[0018] In some embodiments, pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations, such as sodium, potassium, calcium, lithium, magnesium, etc., and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, methylamine, dimethylamine, tetraethylammonium, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as gluconate, arginate, 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 molecules of the compound in the hydrate is determined. Therefore, the hydrate of the compound can be represented by the general formula R·xH2O, for example, wherein R is the compound (uridine) and x is a number greater than 0.

[0020] In some embodiments, the solvate refers to a solvent addition form of a compound containing a stoichiometric or non-stoichiometric solvent, including any solvated form of the compound (uridine) described herein. Conventional solvents include, but are not limited to, water, ethanol, acetic acid, methanol, DMSO, THF, ether, and the like. 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 a crystalline form of uridine, in which the uridine molecules are connected to each other by intermolecular forces such as hydrogen bonds in the crystal structure. Both the ribose moiety and the uracil moiety in the uridine molecule participate in these interactions. For example, a hydrogen bond may be formed between the hydroxyl group (-OH) on the ribose and the carbonyl group (C=O) or other suitable groups in the adjacent uridine molecule, and this hydrogen bond network helps stabilize the crystal structure.

[0022] In some embodiments, the derivative refers to a uridine derivative, and any modified uridine obtained after uridine is modified falls within the protection scope of the present invention. Exemplarily, the uridine derivative includes but is not limited to: nanoparticle-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 salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof in any of the following aspects:

[0024] (1) Application in the preparation of efficient in vitro culture products of mammalian spermatogonial stem cells;

[0025] (2) Application in the preparation of efficient in vitro culture products of mammalian testicular organoids;

[0026] (3) Application in the preparation of in vitro culture products for promoting rejuvenation of aged spermatogonial stem cells;

[0027] (4) Use in the preparation of products for the treatment, prevention, alleviation and / or improvement of male infertility caused by chemotherapy drugs;

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

[0029] (6) Use in the preparation of products for enhancing the fertility of elderly men.

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

[0031] Furthermore, the product is a medicine, a health product or a food.

[0032] Furthermore, the derivative is nanoparticle-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 chemotherapy 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 of spermatogonial stem cells in vivo and regeneration after injury can be specifically embodied as at least one of the following:

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

[0036] B2) Increased expression of spermatogonial stem cell markers (Gfra1, Nefm, Lhx1, Id4, etc.) in the testis;

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

[0038] B4) Increased number of spermatogonial stem cells in the testes.

[0039] In some embodiments, the ability to resist and improve the damage of spermatogonial stem cells caused by chemotherapeutic drugs and promote spermatogenesis recovery can be specifically embodied in at least one of the following:

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

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

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

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

[0044] C5) Compared with the chemotherapy drug group, it can significantly shorten the spermatogenesis recovery time;

[0045] C6) After mating with normal wild-type C57BL6J female mice, they were able to become pregnant and give birth earlier than the chemotherapy drug-affected group.

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

[0047] D1) Increased expression of proliferation marker Mki67 in spermatogonial stem cell population;

[0048] D2) The expression of new spermatogonial stem cell markers (Gfra1, Nefm, Lhx1, Id4, etc.) increases;

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

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

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

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

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

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

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

[0056] E1) Increased expression of proliferation marker Mki67 in testicular organoids;

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

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

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

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

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

[0062] In some embodiments, the present invention has 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 the cell system, subcellular system, tissue system or organ system contains spermatogonial stem cells.

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

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

[0065] In some embodiments, the pharmaceutical composition or pharmaceutical formulation further comprises a pharmaceutically acceptable adjuvant and / or excipient.

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

[0067] In some embodiments, the present invention has no particular restrictions on the pharmaceutically acceptable adjuvants and / or excipients. In addition, the pharmaceutically acceptable adjuvants and / or excipients may also contain liquids such as water, saline, glycerol and ethanol.

[0068] In some embodiments, the present invention has no particular limitation on the dosage form of the pharmaceutical preparation. Exemplarily, the dosage form of the pharmaceutical preparation includes but is not limited to: solutions, emulsions, suspensions, controlled release preparations, tablets, pills, powders, granules, capsules, lozenges, syrups, mists, films, injections, intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, nasal preparations or pulmonary preparations, etc., for patient ingestion.

[0069] Those skilled in the art will understand that, although the pharmaceutical composition or pharmaceutical preparation mentioned above in the present invention may further contain pharmaceutically acceptable adjuvants and / or excipients, 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 adjuvants and / or excipients.

[0070] In some embodiments, suitable administration methods for the pharmaceutical compositions or pharmaceutical formulations described herein include any of the various methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical compositions or pharmaceutical formulations described herein into the subject, including but not limited to oral administration, topical administration, parenteral administration, administration by inhalation spray, rectal administration, nasal administration, buccal administration, or administration by an implanted reservoir.

[0071] In some embodiments, oral administration or injection administration can be selected, wherein injection administration includes the form of rapid injection or continuous infusion. In the case of a pharmaceutical composition or pharmaceutical preparation 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 agents, such as preservatives, stabilizers, suspending agents and / or dispersing agents.

[0072] In some embodiments, when the pharmaceutical composition or pharmaceutical preparation is actually used, its administration regimen and dosage regimen can be selected according to a variety of factors, including the type, species, age, weight, sex and type of disease to be treated of the subject; the severity of the disease to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof used. A dosing and / or dosage regimen can be used, for example, to prevent a disease, inhibit (completely or partially inhibit) a disease, or stop the development of the disease. In a specific embodiment of the present invention, the disease specifically refers to a male infertility-related disease.

[0073] In addition, the present invention also provides a kit comprising uridine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, a pharmaceutical composition or pharmaceutical preparation as described above, that is, by formulating uridine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, a pharmaceutical composition or pharmaceutical preparation as described above in separate unit preparations, and then combining these separate unit preparations to form a kit. In this case, uridine or its pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof, a pharmaceutical composition or pharmaceutical preparation as described above in the separate unit preparations 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 chemotherapy drugs, the method comprising: administering a preventive and / or therapeutically effective amount of uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates, crystalline forms or derivatives thereof, the pharmaceutical composition or pharmaceutical preparation as described above to a subject in need.

[0075] In addition, the present invention also provides a method for enhancing the quantity, quality, fertility and / or testicular function of sperm production in men, the method comprising: administering a preventive and / or therapeutically effective amount of uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates, crystalline forms or derivatives thereof, the pharmaceutical composition or pharmaceutical preparation as described above to a subject in need.

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

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

[0078] In some embodiments, the effective amount refers to an amount having a preventive or therapeutic effect or an amount required to produce a preventive or therapeutic effect in a subject. For example, a pharmaceutically effective amount for prevention or treatment refers to the amount of a drug required to produce the desired preventive or therapeutic effect, which can be reflected by the results of clinical trial results, 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 (such as height, weight, sex, age, and medication history), the type of disease, and the severity of the disease.

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

[0080] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0081] The present invention discloses for the first time the application of uridine in promoting the regeneration or proliferation of damaged spermatogonial stem cells. The present invention creatively discovers for the first time through experimental verification that uridine has a significant 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 cultured in vitro and spermatogonial stem cells in testicular organoids cultured in vitro. It can repair problems such as the reduction in the number of spermatogonial stem cells caused by testicular damage caused by chemotherapy 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 A schematic diagram of constructing an animal experimental model for spermatogonial stem cell proliferation and injury regeneration in vivo in an embodiment of the present invention;

[0083] Figure 2 The figure is a quantitative result of mRNA of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of mouse spermatogonial stem cells after in vivo injection of 600 μg (150 μL) of uridine in the embodiment of the present invention;

[0084] Figure 3The figure is a graph showing the quantitative results of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of spermatogonial stem cell in mice after in vivo injection of 600 μg (150 μL) of uridine in a spermatogonial stem cell injury model established after treatment with the chemotherapeutic drug busulfan in the embodiment of the present invention;

[0085] Figure 4 This is a diagram showing the production and pregnancy results of the chemotherapy drug-affected group and the uridine supplemented group in the embodiment of the present invention;

[0086] Figure 5 This is a schematic diagram of the culture process of primary spermatogonial stem cells supplemented with uridine in an embodiment of the present invention;

[0087] Figure 6 The figure is a quantitative result diagram of the mRNA of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of mouse primary spermatogonial stem cells after culture with different concentrations of uridine in the embodiment of the present invention;

[0088] Figure 7 The figure is a quantitative result diagram of the mRNA of the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Id4, Foxc2) of the mouse spermatogonial stem cell line C18-4 after culture with different concentrations of uridine in the embodiment of the present invention;

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

[0090] Fig. 9 This is a diagram showing the mRNA quantification results of spermatogonial stem cell proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Id4, Foxc2) in mouse testicular organoids after culture with different concentrations of uridine in an example of the present invention. DETAILED DESCRIPTION

[0091] The present invention is further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and cannot be construed as limiting the present invention. It can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and 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 available to those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples 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 examples 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 be obtained from commercial channels. Unless otherwise specified, the specific experimental methods involved in the present invention are all conventional methods, which are carried out according to the methods described in the relevant literature in the field or according to the product instructions. Unless otherwise specified, the experiments involved in the present invention were repeated at least 3 times, and statistical analysis was performed using GraphPad Prism v8.0.2 software. The independent sample t-test was used to compare the differences between the two groups, and the results of the experimental quantitative analysis were expressed as mean ± standard error. ns means that there is no statistical difference in the experimental results; P<0.05 means that the experimental results are statistically different; P<0.01 means that the experimental results are statistically significantly different; P<0.001 means that the experimental results are extremely statistically significantly different.

[0093] The mice in the following examples are as follows: The experimental animals used in this study were all C57BL6J mice purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. The male mice were 8 weeks old when purchased, and the purchased mice were raised in an SPF-grade mouse house with a 12-hour day and night cycle, relative humidity maintained at 30%-40%, and a breeding temperature controlled at 22-24°C. No more than 5 mice were raised in each cage, and the formal experiment began 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] C57BL6J 8-week-old male mice were evenly divided into 2 groups, with 5 mice in each group. The 2 groups were control group (CON) and uridine injection group (CON_U). The dose of uridine injected to each mouse in the uridine injection group was 600 μg (150 μL).

[0100] The control group (CON) and the uridine injection group (CON_U) in the above embodiment were injected intraperitoneally in a general manner, and uridine was dissolved and prepared using 1X PBS; in order to facilitate comparison with the experiment in the subsequent Example 2, CON and CON_U needed to be injected with a corresponding dose of 30% DMSO on the first day, and 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 uridine solution, and the subsequent treatment was to inject the corresponding 1X PBS and uridine solution every other day; the injection time was 3 pm every day, and each mouse was injected 5 times in total within one experimental cycle; at 3 pm on the second day after the last injection, the mice were killed by spinal dislocation and testicular samples were collected for statistical and experimental processing.

[0101] 2. Experimental results

[0102] The results are as follows Figure 1 As shown, the results show the animal experimental treatment process of the control group (CON) and the uridine injection group (CON_U); Figure 2 As shown, the QPCR quantitative statistical results showed that after the injection of 600 μg (150 μL) of uridine in vivo (CON_U), the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of spermatogonial stem cells in the mouse testis were significantly increased compared with the control group (CON), indicating that uridine supplementation 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 after damage caused by chemotherapy drugs

[0104] 1. Experimental methods

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

[0106] The busulfan treatment group (BU) and the uridine injection group (BU_U) in the above embodiment were both injected intraperitoneally in a general manner, busulfan was dissolved and prepared using 30% DMSO, and uridine was dissolved and prepared using 1X PBS; each mouse was weighed on the first day for both BU and BU_U, and then each mouse was injected with a corresponding dose of busulfan according to 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, and the subsequent treatment was to inject the corresponding 1X PBS and uridine solution every other day; the injection time was 3 pm every day, and each mouse was injected 5 times in total within one experimental cycle; at 3 pm on the second day after the last injection, the mice were killed by spinal dislocation and testicular samples were collected for statistical and experimental processing.

[0107] 2. Experimental results

[0108] The results are as follows Figure 1 As shown, the results show the animal experimental treatment process of the busulfan-treated group (BU) and the uridine-injected group (BU_U); Figure 3 As shown in the figure, the quantitative statistical results of QPCR showed that after the injection of 600 μg (150 μL) of uridine in vivo (BU_U), the proliferation marker Mki67 and stem cell markers (Gfra1, Nefm, Lhx1, Id4) of spermatogonial stem cells in the mouse testis were significantly increased compared with the busulfan treatment group (BU), indicating that uridine supplementation can effectively promote the regeneration and recovery of testicular spermatogonial stem cells in vivo after damage caused by chemotherapeutic drugs; Figure 4 As shown in the figure, the results show the fertility identification experimental process of BU group male mice and BU_U group male mice. Compared with BU group male mice, the female mice became pregnant and gave birth earlier after mating with BU_U group male mice. This result also shows that uridine can resist and improve the damage of spermatogonial stem cells caused by chemotherapy drugs and promote spermatogenesis recovery.

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

[0110] 1. Experimental methods

[0111] Take three mice born 7 days old, collect the testicles, remove the white membrane and digest to a single cell state; inoculate into a culture dish after filtering through a 40 μM filter, and use the differential adhesion method to select spermatogonial stem cells; the time of the first passage of the cell line depends on the clonal growth of the primary spermatogonial stem cells, and the subsequent passage time is about 7 days; when the cell line is passed to more than 5 generations, the state gradually becomes stable and is used for experiments; in this example, we use spermatogonial stem cells of the 7th generation, and after three days of culture, set 0 μg / mL, 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 200 μg / mL 6 concentration gradients of uridine are added to the culture medium. When the medium is changed on the fifth day, the spermatogonial stem cell culture medium is replaced again according to 0 μg / mL, 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 200 μg / mL, 6 concentrations. On the sixth day, samples are collected and used for experiments, and no uridine is added to the group (0 μg / mL) as the control, 0.01 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 200 μg / mL as the uridine-added group, to explore the effect of uridine supplementation on the in vitro culture of primary spermatogonial stem cells. In this example, we also used the spermatogonial stem cell line C18-4 to explore the effect of uridine supplementation on the proliferation of spermatogonial stem cells. The frozen C18-4 cells were revived and passed for two generations until stable. Uridine was added to the culture medium at 0μM, 1 μM, 10 μM, 100 μM, 500 μM, and 1000 μM, 6 concentrations. The cell line was passed for one generation every day, and the samples of the third generation were collected for further experiments. The group without uridine addition (0 μM) was used as the control, and 1 μM, 10 μM, 100 μM, 500 μM, and 1000 μM were uridine-added groups to explore the effect of uridine supplementation on the in vitro culture of spermatogonial stem cell line C18-4.

[0112] 2. Experimental results

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

[0114] Example 4 Uridine promotes the proliferation of spermatogonial stem cells in testicular organoids

[0115] 1. Experimental methods

[0116] Ten mice born on day 3 were selected, and the testes were collected. After removing the tunica albuginea, the testes were digested to a single cell state. After centrifugation at 1000rpm for 3 min, the supernatant was discarded, and the cells were resuspended in polymerase solution and inoculated into U96 well plates. After 48 hours, the spermatogonial stem cell culture medium was replaced and the cells were transferred to agar blocks for culture. After 4 days, the differentiation medium was replaced and uridine was added to the differentiation medium at four concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. After 4 days of continuous culture, samples were collected for further experiments.

[0117] 2. Experimental results

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

Claims

1. 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 regeneration and / or proliferation of damaged spermatogonial stem cells.

2. The use according to claim 1, characterized in that: The spermatogonial stem cell damage is spermatogonial stem cell damage caused by chemotherapeutic drugs or non-chemotherapeutic drugs, or spermatogonial stem cell damage caused by mechanical force.

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

4. The use according to claim 1, characterized in that: The derivative is nanoparticle-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.

5. Use of uridine or its pharmaceutically acceptable salts, hydrates, enantiomers, diastereomers, solvates, crystalline forms or derivatives thereof in any of the following aspects: (1) Application in the preparation of efficient in vitro culture products of mammalian spermatogonial stem cells; (2) Application in the preparation of efficient in vitro culture products of mammalian testicular organoids; (3) Application in the preparation of in vitro culture products for promoting rejuvenation of aged spermatogonial stem cells; (4) Use in the preparation of products for the treatment, prevention, alleviation and / or improvement of male infertility caused by chemotherapy drugs; (5) Use in the preparation of products for enhancing the quantity, quality, fertility and / or testicular function of sperm production in men; (6) Use in the preparation of products for enhancing the fertility of elderly men.

6. The use according to claim 5, characterized in that: The in vitro culture product is a culture reagent, a culture solution, a culture matrix or a culture device.

7. The use according to claim 5, characterized in that: The product is a medicine, a health product or a food.

8. The use according to claim 5, characterized in that: The derivative is nanoparticle-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.

9. 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, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof.

10. A pharmaceutical composition or pharmaceutical preparation, characterized in that: The pharmaceutical composition or pharmaceutical preparation comprises a preventive and / or therapeutically effective amount of uridine or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, crystalline form or derivative thereof.

Images