A small molecule compound promoting stem cell proliferation and its application

Abstract

The present invention discloses a small molecule compound for promoting stem cell proliferation activity and its application. The present invention provides the application of PX-478 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for promoting stem cell proliferation activity. The present invention provides an in vitro method for promoting stem cell proliferation activity. The present invention also provides the application of PX-478 or a pharmaceutically acceptable salt thereof in the preparation of a system, a device, and a non-volatile computer storage medium for automatically producing stem cells. The present invention also provides a corresponding system for automatically producing stem cells, a device for automatically producing stem cells, and a computer-readable storage medium for controlling the automated production of stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a small molecule compound that promotes stem cell proliferation and application thereof. Background Art

[0002] Mesenchymal stem cells (MSC) exist in bone marrow, fat, umbilical cord, etc., and have the potential for multidirectional differentiation and self-renewal. Because of their strong paracrine function, immunosuppression, easy isolation and culture, no ethical issues, convenient implantation and other excellent characteristics, MSC is used to treat cardiovascular and cerebrovascular diseases, liver diseases, bone and muscle degeneration diseases, brain and spinal cord nerve damage, Alzheimer's disease and autoimmune diseases, etc., and is widely used in clinical trials. Therefore, MSC has a high prospect for clinical transformation.

[0003] In the field of stem cells and regenerative medicine, the main advantages of MSC in treating diseases are: 1. It can replace damaged or diseased tissues through proliferation and differentiation; 2. It can enhance endogenous repair capacity through various mechanisms such as anti-inflammatory, immune regulation, and anti-apoptosis by paracrine secretion of a large number of cytokines and exosomes. At present, a large number of preclinical studies have shown that MSC is safe and effective in treating diseases, and clinical trials have also found that MSC is safe in treating diseases, but the clinical efficacy of MSC is not uniform. The possible reason for the analysis is that the transplanted MSCs in clinical trials may have aging problems. On the one hand, the donor source of autologous stem cell transplantation is elderly patients, and the isolated and purified MSCs are senescent cells; on the other hand, the number of transplanted cells needs to meet clinical requirements, and continuous expansion in a normoxic environment in vitro can easily lead to replicative senescence of MSCs. After MSC aging, cell growth stagnation, weakened migration ability, reduced paracrine repair factors, and secretion of a large number of inflammatory factors (aging-related secretory phenotype) will affect the survival, colonization, homing and activation of endogenous repair mechanisms of MSCs in the body, resulting in a decrease in the efficacy of treating IHD. Therefore, finding targets to improve the proliferation activity of aging MSC cells is an important research direction in this field. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides the use of PX-478 in preparing a pharmaceutical composition for promoting the proliferation activity of stem cells.

[0006] Furthermore, the stem cells include pluripotent stem cells, oligopotent stem cells, and unipotent stem cells.

[0007] Furthermore, the pluripotent stem cells include embryonic stem cells, induced pluripotent stem cells, umbilical cord stem cells, and mesenchymal stem cells.

[0008] Furthermore, the PX-478 also includes pharmaceutically acceptable salts thereof.

[0009] Furthermore, the concentration of PX-478 is 10 μM-20 μM.

[0010] In some embodiments, the concentration of PX-478 is 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM. In some embodiments, the concentration of PX-478 is specifically determined according to the number and concentration of administered stem cells.

[0011] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0012] Furthermore, the adjuvants include physiological saline, D5W, Ringer's lactate, hyaluronidase, amino acids, solution tension regulators, solution osmotic pressure regulators, freeze-dried powder freeze-protectants, viscosity-reducing adjuvants, buffers or surfactants, stabilizers, diluents, adhesives, humectants, adsorption carriers, lubricants, fillers, and disintegrants.

[0013] The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implanted drug storage devices. Oral administration or injection is preferred. The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, pharmaceutically acceptable acids, bases or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form.

[0014] The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracorporeal, intralesional, intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be given to the receptor by any route.

[0015] In some embodiments, the diluent is such as lactose, sodium chloride, glucose, urea, starch, water, etc.

[0016] In some embodiments, the binder is such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.

[0017] In some embodiments, surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. Surfactants include ionic or nonionic surfactants, such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and corresponding salts (their alkali metal or alkaline rare earth metal salts, such as sodium salts, potassium salts, calcium salts, and magnesium salts).

[0018] In some embodiments, humectants include glycerin, starch, and the like; adsorption carriers include starch, lactose, bentonite, silica gel, kaolin, and bentonite, and the like.

[0019] In some embodiments, lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0020] In some embodiments, fillers include mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, and sodium bicarbonate.

[0021] In some embodiments, disintegrants include cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides, and the like.

[0022] In some embodiments, stabilizers include human serum albumin, L-amino acids, sugars and cellulose derivatives. L-amino acids may also include any one of glycine, cysteine ​​and glutamic acid. In some embodiments, carbohydrates include monosaccharides such as glucose, mannose, galactose, fructose, etc.; sugar alcohols such as mannitol, inositol, xylitol, etc.; disaccharides such as sucrose, maltose, lactose, etc.; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, etc. and their derivatives. In some embodiments, cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium hydroxymethylcellulose. Isotonic agents include potassium chloride, sodium chloride, sugars and glycerol. Chelating agents include sodium ethylenediaminetetraacetic acid and citric acid.

[0023] Furthermore, the species on which the pharmaceutical composition acts are vertebrates.

[0024] Furthermore, the vertebrate includes mammals, specifically humans or non-human mammals.

[0025] Furthermore, the species on which the pharmaceutical composition acts is human.

[0026] In some embodiments, the vertebrates described herein include mammals, any member of the subphylum Chordata, including primates (e.g., humans, monkeys, and apes, and including monkey species (e.g., genus Macaca (e.g., macaques, such as cynomolgus monkeys (Macaca fasciculus Laris), and / or rhesus monkeys (Macaca mμLatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (S. (species from the genus Saguinus), and species of apes, such as chimpanzees (Pantroglodytes), rodents (e.g., mice, rats, guinea pigs), Leporidae (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish.

[0027] In some embodiments, pharmaceutically acceptable adjuvants may include inert ingredients that do not unduly inhibit the biological activity of the compound. Pharmaceutically acceptable adjuvants should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic, or non-other undesirable reactions or side effects when administered to a subject. Standard pharmaceutical preparation techniques may be used.

[0028] The present invention provides a pharmaceutical composition for promoting stem cell proliferation, wherein the pharmaceutical composition comprises PX-478 or a pharmaceutically acceptable salt thereof.

[0029] Furthermore, the pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0030] Further, the acid addition salt includes, but is not limited to, any one of the hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, argininate, fumarate, nicotinate, phthalate or oxalate of FTY720, or a combination of at least two thereof.

[0031] Furthermore, the base addition salt includes but is not limited to lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt of FTY720, or a salt of FTY720 with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0032] Furthermore, the pharmaceutically acceptable salt is obtained from an acid, which includes an organic acid or an inorganic acid.

[0033] Furthermore, the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

[0034] Furthermore, the organic acid may be formic acid, acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, α-hydroxy acid such as citric acid or tartaric acid, amino acid, aromatic acid, or sulfonic acid.

[0035] Furthermore, the pharmaceutically acceptable salts include base addition salts, specifically alkali metal salts (such as sodium salts, potassium salts, etc.) and alkaline earth metal salts (such as calcium salts, magnesium salts, etc.).

[0036] Furthermore, the pharmaceutical composition also includes other drugs that promote stem cell proliferation.

[0037] The present invention provides a method for promoting stem cell proliferation in vitro, comprising administering PX-478 or a pharmaceutically acceptable salt thereof.

[0038] The present invention provides the use of PX-478 or a pharmaceutically acceptable salt thereof in preparing a system, a device, and a non-volatile computer storage medium for automatically producing stem cells.

[0039] Furthermore, the stem cells include pluripotent stem cells, oligopotent stem cells, and unipotent stem cells.

[0040] Furthermore, the pluripotent stem cells include embryonic stem cells, induced pluripotent stem cells, umbilical cord stem cells, and mesenchymal stem cells.

[0041] The present invention provides a system for automatically producing stem cells, the system comprising:

[0042] A feeding unit, used for adding PX-478 or a pharmaceutically acceptable salt thereof to a stem cell culture medium;

[0043] A culture unit for culturing stem cells in a stem cell culture medium supplemented with PX-478 or a pharmaceutically acceptable salt thereof.

[0044] Furthermore, the system also includes a separation unit for classifying the cultured stem cells from the stem cell culture medium.

[0045] In some embodiments, the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place or distributed over multiple network units.

[0046] The present invention provides a device for automatically producing stem cells, the device comprising:

[0047] A feeding module is used to add PX-478 or a pharmaceutically acceptable salt thereof and stem cells into a stem cell culture medium.

[0048] A culture module is used for the culture of stem cells.

[0049] The separation module separates stem cells from the culture medium.

[0050] The system, device or computer-readable storage medium provided by the present invention is operated by a computer or artificial intelligence.

[0051] The present invention provides a computer-readable storage medium for automatically controlling the production of stem cells, the computer-readable storage medium comprising: a computer program, the computer program is stored in the computer-readable storage medium, the computer program carries the aforementioned method for promoting stem cell proliferation in vitro, and when the computer program is executed by a processor, the purpose of automatically controlling the production of stem cells is achieved.

[0052] In some embodiments, the device, electronic device, non-volatile computer storage medium and method provided by the present invention correspond to each other, and therefore, the device, electronic device and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding device, electronic device and non-volatile computer storage medium will not be repeated here.

[0053] In some embodiments, the above modules or steps can achieve the effect through a computing device. In some embodiments, the modules or steps can be concentrated on a single computing device. In some embodiments, the modules or steps can be distributed on multiple computing devices, and multiple computing devices are linked by a network or other linking methods. In some embodiments, the computing device stores a program code for executing the above modules or steps, and the program code is stored in a storage device. In some embodiments, the modules or steps can be prepared into integrated circuits respectively, or combined into a single integrated circuit. In the specific embodiments of the present invention, no implementation method is limited, whether it is hardware or software.

[0054] The term "stem cell" used in the present invention may refer to an undifferentiated cell that can proliferate and produce more progenitor cells with the ability to produce a large number of mother cells, which in turn can produce differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce offspring, which subsequently differentiate into one or more mature cell types while also retaining one or more cells with the developmental potential of the parent.

[0055] The term "pluripotent stem cell" used in the present invention refers to a stem cell that has the potential to differentiate into any of the three germ layers (endoderm, mesoderm and ectoderm). Pluripotent stem cells can produce any fetal or adult cell type. However, single cells or aggregates of pluripotent cells cannot develop into fetuses or adult animals because they lack the potential to organize into embryos.

[0056] The term "mesenchymal stem cells" used in the present invention is also called mesenchymal stem cell, MSC, mesenchymal stem cells, mesenchymal stem cells, and generally refers to a group of multipotent stromal cells with certain differentiation potential, which can differentiate into various cell types and have tissue repair functions. In addition to existing in the "bone marrow", they include multipotent cells widely derived from other "non-bone marrow" tissues, such as: placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, and deciduous tooth pulp. Although MSC is a mesenchymal tissue, it does not differentiate into hematopoietic cells, which is opposite to HSC (hematopoietic stem cells) and does not have the ability to rebuild the entire organ.

[0057] The term "comprising / including" as used in the present invention means that the composition or method of one or more named elements or steps is open-ended, meaning that the named elements or steps are required, but other elements or steps may be added within the scope of the composition and method. To avoid redundancy, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes a corresponding more limited composition or method "consisting essentially of the same named elements or steps", meaning that the composition or method includes the named required elements or steps, and may also include other elements or steps that do not substantially affect the basic and novel characteristics of the composition or method.

[0058] The term "PX-478" as used in the present invention is an orally active, selective hypoxia-inducible factor-1α (HIF-1α) inhibitor.

[0059] The term "pharmaceutically acceptable salt" as used in the present invention means salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other components constituting the formulation and / or the mammal to be treated therewith.

[0060] The term "pharmaceutically acceptable acid addition salt" as used in the present invention refers to those pharmaceutically acceptable salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid) and organic acids (selected from aliphatic, cycloaliphatic, aromatic, aromatic aliphatic, heterocyclic, carboxyl, and sulfonic acid organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid / hydroxyacetic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid / maleic acid, malonic acid, succinic acid, fumaric acid / fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid).

[0061] The term "pharmaceutically acceptable base addition salt" as used in the present invention means those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, ethylenediamine, glucosamine, diatrizoate, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins).

[0062] The term "substance" as used herein can be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Where appropriate, it will be clear to those skilled in the art that the term can be used to refer to an entity that is a cell or organism or a fraction, extract or component thereof or that contains a cell or organism or a fraction, extract or component thereof. As an alternative or supplement, as the context will make clear, the term can be used to refer to a natural product because it is found in nature and / or obtained from nature. In some cases, again as will be clear from the context, the term can be used to refer to one or more artificial entities because it is designed, engineered and / or produced by the action of a human hand, and / or cannot be found in nature. In some embodiments, the substance can be used in an isolated form or a pure form; in some embodiments, the substance can be used in a crude form. In some embodiments, potential substances can be provided as a collection or library, for example, which can be screened to identify or characterize active substances therein. In some cases, the term "substance" may refer to a compound or entity that is a polymer or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymer parts. In some embodiments, the term "substance" may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or one or more specific polymer parts. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymer part. In some embodiments, the substance is a compound. In some embodiments, the enhancer may refer to a food-grade compound combined with a salt. The enhancer is combined with the salt by forming electrostatic interactions, hydrogen bonds, ionic interactions, coordination interactions, non-covalent interactions, van der Waals interactions, or any combination thereof.

[0063] The term "unit" used in the present invention refers to functionality, application, software module, function, routine, instruction set or program that can be executed by a programmable processor such as a microprocessor, a central processing unit (CPU) or a specially designed programmable device or controller. The memory contains instructions or programs that, when executed by the CPU, cause the CPU to perform operations corresponding to the unit or function. In the hardware context, the term "unit" refers to a hardware element, circuit, assembly, physical structure, system, module or subsystem. According to a specific embodiment, the term "unit" may include mechanical, optical or electronic components, or any combination thereof. The term "unit" may include active components (such as transistors) or passive components (such as capacitors). The term "unit" may include a semiconductor device having a substrate and other material layers, wherein the other material layers have various concentrations of conductivity. The term "unit" may include a CPU or a programmable processor that can execute a program stored in the memory to perform a specific function. The term "unit" may include a logic element (for example, AND, OR) implemented by a transistor circuit or any other switching circuit.

[0064] The term "module" used in the present invention may refer to, be part of, or include: an application specific integrated circuit (ASIC); a digital, analog, or mixed analog / digital discrete circuit; a digital, analog, or mixed analog / digital integrated circuit; a combinational logic circuit; a field programmable gate array (FPGA); a memory circuit (shared, dedicated, or group) that executes code; a processor circuit (shared, dedicated, or group) that stores code executed by a processor circuit; other suitable hardware components that provide the described functions; or a combination of some or all of the above, such as a system on a chip. In some embodiments, a module may include one or more interface circuits. In some examples, an interface circuit may include a wired or wireless interface connected to a local area network (LAN), the Internet, a wide area network (WAN), or a combination thereof. The functions of any given module of the present disclosure may be distributed among multiple modules connected via an interface circuit. For example, multiple modules may allow load balancing. In another example, a server (also referred to as a remote or cloud) module may perform some functions on behalf of a client module.

[0065] The term "processor" or "processing circuit" used in the present invention may generally refer to any one of the aforementioned logic circuits alone or in combination with other logic circuits or any other equivalent circuits.

[0066] The term "memory" as used herein refers to any type of long-term, short-term, volatile, nonvolatile or other memory, and is not limited to any particular type of memory or amount of memory, or type of medium on which the memory is stored.

[0067] Data stored in memory cannot simply be deleted or overwritten to free up a location in memory for a new write. Some of the data in memory may be static data. Static data is data that is infrequently accessed, and it either does not change over time or does not change as frequently as dynamic data. Static data is also referred to as persistent data. (It should be noted that static data or persistent data is different from data stored in a "persistent" type of storage such as a hard disk or non-volatile storage. Static / persistent data can be located anywhere, such as in cache, RAM, or main memory, and is not exclusively limited to persistent types of storage). Static data can be thought of as the opposite of dynamic data, which is data that can change frequently. One type of static data is read-only data. Another type of static data is data that is rarely read.

[0068] Advantages and beneficial effects of the present invention:

[0069] The present invention protects a small molecule compound PX-478 2HCl, which has a significant effect on promoting the proliferation of bone marrow MSCs; wherein 10 μM and 20 μM PX-478 can increase the proliferation activity of bone marrow MSCs. The present invention protects the application of PX-478 2HCl in increasing MSC production and proliferation in the field of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The CCK8 experiment results of the effects of PX-478 and KC7F2 on the proliferation of bone marrow mesenchymal stem cells; (AB) PX-478 promotes the proliferation of senescent MSCs: the effects of 10μM and 20μM PX-478 on the proliferation of bone marrow mesenchymal stem cells at the P16 generation; (CD) KC7F2 (another HIF1α inhibitor) inhibits the activity of senescent MSCs: the effects of 10μM and 20μM KC7F2 on the proliferation of bone marrow mesenchymal stem cells at the P16 generation. All data are mean±SD. ****p<0.0001. DETAILED DESCRIPTION

[0071] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those skilled in the art after reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be easily separated or combined with features of any of the other several aspects without departing from the scope or essence of this disclosure.

[0072] It should also be understood that the terms used herein are only used for the purpose of describing specific aspects and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and related art, and should not be interpreted as idealized or overly formal unless explicitly defined herein.

[0073] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0074] Example PX-478 promotes the proliferation of bone marrow mesenchymal stem cells

[0075] 1. Experimental Materials

[0076] 1) Bone marrow mesenchymal stem cells (regular ones are acceptable);

[0077] 2) Bone marrow mesenchymal stem cell basal medium (regular medium is acceptable);

[0078] 3) Complete medium for bone marrow mesenchymal stem cells (regular medium will do);

[0079] 4) PX-478 2HCl (Sellek, accession number: S7612);

[0080] 5) KC7F2 (Sellek, registration number: S7946);

[0081] 6) DMSO: solvent (conventional reagent);

[0082] 7) CCK-8 reagent (conventional reagent).

[0083] 2. Experimental methods

[0084] 1) Bone marrow mesenchymal stem cells (replicative senescence) at passage P16 were resuspended in complete medium for bone marrow mesenchymal stem cells at a concentration of 1 × 10 4 / ml was inoculated into a 96-well plate and placed in a cell culture incubator to grow the cells overnight at 37°C and 5% CO2.

[0085] 2) When the cell confluence reaches 60-70%, wash the cells three times with PBS and replace with bone marrow mesenchymal stem cell basal medium to continue culturing for 12 hours to eliminate the effect of serum on MSC proliferation.

[0086] 3) After 12 hours of incubation, discard the above basal culture medium, wash with PBS 2-3 times, add fresh basal culture medium, add DMSO to the control group, and add PX-478 2HCl and KC7F2 to the treatment group according to the concentration gradient, and set the final concentrations to 10μM and 20μM, respectively.

[0087] 4) Continue to culture in the cell culture incubator for 24 hours, add 10 μL CCK-8 to each well, incubate for 2-4 hours, and use a microplate reader to detect the absorbance (450 nm).

[0088] 3. Experimental results

[0089] The experimental results are as follows Figure 1 As shown, Figure 1 A and Figure 1 B The results showed that PX-478 could promote the proliferation of aged mesenchymal stem cells. Different concentrations of PX-478 did not affect its effect on the proliferation of bone marrow mesenchymal stem cells. PX-478 at 10μM and 20μM could provide significant promotion effect on the proliferation of mesenchymal stem cells. Compared with the control group, the p value was <0.0001. Figure 1 C and Figure 1 D The results showed that the addition of another HIF1α inhibitor, KC7F2, inhibited the activity of aged mesenchymal stem cells. Regardless of the concentration of KC7F2, the degree of inhibition on mesenchymal stem cells was extremely significant. Compared with the control group, the proliferation of bone marrow mesenchymal stem cells was significantly inhibited after the addition of KC7F2, and the p value of the significant degree of inhibition was <0.0001.

[0090] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of PX-478 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for promoting the proliferation activity of bone marrow mesenchymal stem cells, wherein the concentration of PX-478 is 10 μM-20 μM.

2. The use according to claim 1, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

3. A method for promoting the proliferation activity of bone marrow mesenchymal stem cells in vitro for non-therapeutic purposes, characterized in that: The method comprises administering PX-478 or a pharmaceutically acceptable salt thereof, wherein the concentration of PX-478 is 10 μM to 20 μM.

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