CN112472695A - Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals - Google Patents
Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals Download PDFInfo
- Publication number
- CN112472695A CN112472695A CN202011458101.5A CN202011458101A CN112472695A CN 112472695 A CN112472695 A CN 112472695A CN 202011458101 A CN202011458101 A CN 202011458101A CN 112472695 A CN112472695 A CN 112472695A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- spermatogonial stem
- mammals
- regeneration
- lovastatin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
Landscapes
- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Reproductive Health (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Pregnancy & Childbirth (AREA)
- Gynecology & Obstetrics (AREA)
- Epidemiology (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a statin compound in preparation of a medicine for promoting the regeneration function of spermatogonial stem cells of mammals. The statin compound can be applied to the preparation of medicaments for promoting the regeneration function of spermatogonial stem cells of mammals, provides a new medicament choice for preventing and/or treating male infertility, is mature and safe in application due to the new use of old medicaments, can be quickly converted into clinic, and avoids the period of research and development of new medicaments; specifically, the statin compound can promote the regeneration of the spermatogonial stem cells of the mammal through in vitro culture or direct application in vivo, and can promote the continuous generation of sperms through firstly promoting the regeneration of the spermatogonial stem cells of the mammal through in vitro culture and then transplanting the spermatogonial stem cells into the body, thereby providing various choices for prevention and/or treatment modes of male sterility.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a statin compound in preparation of a medicine for promoting the regeneration function of spermatogonial stem cells of mammals.
Background
Currently, infertility accounts for about 15% of women of childbearing age worldwide and is on the rising trend year by year, wherein infertility caused by male factors accounts for about 50% and is mostly caused by gametogenesis disorder. Since healthy mature male gametes are not available and traditional assisted reproductive technologies cannot be applied for treatment, the only solution to this in the clinic at present is to use the sperm bank for spermatogenesis, so they cannot obtain progeny of real biological significance.
Spermatogonial stem cells are germ cells which originate from primordial germ cells and are in an undifferentiated state, and are distributed on a basement membrane in the seminal tubules of the testis of a male animal. When male mammals enter adolescence, spermatogonial stem cells in testis continuously generate sperms and perform self-renewal continuously to maintain a certain number of stem cells, in the cell differentiation process, the spermatogonial stem cells play an extremely important role, and have the function of maintaining self-renewal, and continuously differentiate to form germ cells in each stage and finally form sperms, namely, the spermatogonial stem cells bear the mission of transmitting genetic information to the next generation, and the regeneration function directly influences the spermatogenesis capacity of male mammals, so that the regeneration function of the individual spermatogonial stem cells can be improved to promote the generation of functional sperms, and then the prevention and/or treatment of male infertility can be realized.
In view of the above, there is a need for a drug for preventing and/or treating male infertility by promoting the spermatogonial stem cell regeneration function.
Disclosure of Invention
Problems to be solved by the invention
The invention provides an application of statin compounds in preparing a medicine for promoting the regeneration function of spermatogonial stem cells of mammals, which promotes the continuous generation of sperms by promoting and maintaining the long-term self-renewal of the spermatogonial stem cells of the mammals, thereby providing a new medicine selection for preventing and/or treating male infertility (such as azoospermia, oligospermia, asthenospermia, teratospermia and asthenospermia), and being a new application of old medicines, mature and safe in application, capable of being quickly converted into clinic and avoiding the period of research and development of new medicines.
Means for solving the problems
In a first aspect, the invention provides an application of a statin compound in preparing a medicament for promoting the regeneration function of spermatogonial stem cells of mammals.
Preferably, the medicament is for the prevention and/or treatment of male infertility in mammals.
Further preferably, the male infertility is selected from one or more of azoospermia, oligospermia, asthenospermia, teratospermia and azoospermia, preferably from one or more of azoospermia and oligospermia.
Preferably, the medicament promotes the regeneration of the spermatogonial stem cells of the mammal by means of in vitro culture.
Preferably, the medicament first promotes the regeneration of the spermatogonial stem cells of the mammal by means of in vitro culture, and then transplants the spermatogonial stem cells into the mammal body so as to promote spermatogenesis.
Preferably, the medicament promotes regeneration of the spermatogonial stem cells by direct administration to the mammal.
Preferably, the drug partially replaces the cytokine GDNF in maintaining the self-renewal of the mammalian spermatogonial stem cells.
Preferably, the statin compound is selected from one or more of lovastatin, pravastatin, fluvastatin, atorvastatin, pitavastatin, mevastatin, rosuvastatin and pharmaceutically acceptable salts thereof.
Further preferably, the statin compound is selected from lovastatin.
Preferably, the mammal is a rodent and/or primate.
Further preferably, the rodent is a rat or a mouse, preferably a mouse.
Further preferably, the primate is a cynomolgus monkey or a human, preferably a human.
In a second aspect, the present invention also provides a use of a composition for preparing a medicament for promoting the regeneration function of spermatogonial stem cells of a mammal, wherein the composition contains a statin compound.
Preferably, the medicament is for the prevention and/or treatment of male infertility in mammals.
Further preferably, the male infertility is selected from one or more of azoospermia, oligospermia, asthenospermia, teratospermia and asthenospermia.
Preferably, the statin compound is selected from one or more of lovastatin, pravastatin, fluvastatin, atorvastatin, pitavastatin, mevastatin, rosuvastatin and pharmaceutically acceptable salts thereof.
Further preferably, the statin compound is selected from lovastatin.
ADVANTAGEOUS EFFECTS OF INVENTION
The invention provides an application of statin compounds in preparing medicaments for promoting the regeneration function of spermatogonial stem cells of mammals, and provides a new medicament choice for preventing/treating male infertility (such as azoospermia, oligospermia, asthenospermia, teratospermia and asthenospermia).
The application provided by the invention can promote the regeneration of the spermatogonial stem cells of mammals by taking lovastatin as an example through an in vitro culture mode or a mode of direct application to the bodies, and can also promote the continuous generation of sperms by firstly promoting the regeneration of the spermatogonial stem cells of the mammals through in vitro culture and then transplanting the spermatogonial stem cells into the bodies of the mammals, thereby providing a plurality of choices for promoting the regeneration of the spermatogonial stem cells.
Research shows that the statin compounds promote the regeneration of spermatogonial stem cells to realize the function of partially replacing glial cell-derived neurotrophic factor (GDNF), regulate cell metabolism, reduce cell inflammation reaction and further promote the continuous self-renewal function of the spermatogonial stem cells.
According to the invention, researches show that the statin compound has a good promoting effect on the regeneration of spermatogonial stem cells of rodent mammals and primate mammals, and can repair the situation of reduced spermatogonial stem cells caused by testicular damage; moreover, the scheme is new for old drugs, statin compounds are existing drugs which are commonly seen in the market, and have been used as drugs for regulating blood fat in the eighties of the 20 th century, the application of the statin compounds is mature and safe, the statin compounds can be quickly converted and applied to clinic, the long period of new drug development is avoided, and the statin compounds have application and popularization significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. The drawings in the following description are some embodiments of the invention, and it is obvious to those skilled in the art that other drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a comparison of the effect of promoting the regeneration function of mouse spermatogonial stem cells by in vitro culture of lovastatin with different concentrations provided in example 1 of the present invention;
FIG. 2 is a comparison of the effect of various statin compounds on promoting the regeneration of mouse spermatogonial stem cells in an in vitro culture manner, according to example 2 of the present invention;
FIG. 3 is the maintenance of mouse spermatogonial stem cell proliferation by lovastatin in the absence of exogenously added GDNF in vitro as provided in example 3 of the present invention;
FIG. 4 is the implementation of the long-term lovastatin-cultured spermatogonial stem cell spermatogenic transplantation in the testes of the recipient mouse according to example 4 of the present invention;
FIG. 5 shows the effect of injecting lovastatin at different concentrations in vivo on the proliferation of spermatogonial stem cells under physiological conditions as provided in example 5 of the present invention;
FIG. 6 shows the effect of lovastatin injection in vivo on the proliferation of spermatogonial stem cells under simulated pathological conditions of busulfan in accordance with example 5 of the present invention;
FIG. 7 shows the maintenance and proliferation of human seminiferous stem cells in seminiferous tubules by lovastatin according to example 6 of the present invention.
Detailed Description
In order to facilitate an understanding of the invention, embodiments of the invention will be described in detail below with reference to the accompanying drawings, in which preferred embodiments of the invention are shown, but the invention can be practiced in many different ways, which are defined and covered by the claims and not limited to the embodiments described herein.
Reagents, methods and equipment used in the examples are conventional in the art, unless otherwise specified; the reagents and materials used in the following examples are all commercially available unless otherwise specified.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention is realized by the following technical scheme:
the method firstly utilizes an in-vitro culture system of mouse spermatogonial stem cells to remove a cell factor GDNF which plays a key role in maintaining the self-renewal of the spermatogonial stem cells from culture components, thereby establishing a 'predicament' system in which the spermatogonial stem cells can not maintain the long-term self-renewal in vitro, and further discovers that the statins can effectively maintain the long-term self-renewal of the functional spermatogonial stem cells under the system by using the system and through large-scale drug screening and research.
Further, through the experiment of injecting statins in mice, the statins can promote and maintain the regeneration activity of spermatogonial stem cells, and further promote the generation of sperms.
Further, the role of statins in promoting spermatogenesis in primates has been explored. The experimental result shows that in primate experiments, the statin compound taking lovastatin as an example can also effectively maintain the survival of spermatogonial stem cells.
Definition of
The term "statins" is a class of small molecule hypolipidemic drugs having the structure shown in formula (I):
wherein, the part A is a beta, delta-dihydroxyl valeric acid structure similar to the HMG structure in enzyme substrate HMG-CoA, and is a hypolipidemic drug effect group of statin compounds; the R group of the B part is a hydrophobic rigid plane structure combined with a hydrophobic shallow trench generated after the enzyme is destructured; the part C is a connecting part between the parts A and B.
The term "statin compound" is well known in the art and primarily includes lovastatin, pravastatin, fluvastatin, atorvastatin, pitavastatin, mevastatin, rosuvastatin and pharmaceutically acceptable salts thereof; the "pharmaceutically acceptable salt" mainly includes alkali metal salts and alkaline earth metal salts of statins, such as sodium salts and calcium salts of statins.
The term "composition", i.e. mixture, includes: 1) compositions comprising several known substances, such as a compound of Chinese herbs; 2) compositions composed of several unknown substances, such as effective parts of Chinese medicinal materials; 3) compositions of the novel compounds and physiologically active substances known per se, such as combinations of novel drugs; 4) the new compound and the composition of the known matter without physiological activity, such as tablet, injection, etc. of the new medicine. The composition of the present invention mainly refers to a composition which takes a statin compound as a pharmacodynamically active ingredient and forms any form of the above 1) -4) with other ingredients.
The term "mammal" refers to a warm-blooded vertebrate of the class mammalia subphyla of vertebrates, which breathes air with the lungs, which is named for lactation by the secretion of milk through the mammary glands to young adults; a mammal is a warm-blooded, vertebrate animal with hair in the body, mostly fetal, and feeding offspring through the mammary glands; mammals are the highest stage in the animal's history of development and are also a group most closely related to humans. The "mammal" of the present invention mainly refers to rodent mammals and/or primate mammals, wherein, rodent mammals include but are not limited to rats and mice, and primate mammals include but are not limited to macaques and humans.
The term "male infertility" refers to infertility caused by the inability of spermatogonial stem cells to regenerate, such as one or more of azoospermia, oligospermia, asthenospermia, teratospermia, and asthenospermia.
The term "preventing" refers to completely or nearly completely preventing the disease or condition (e.g., infection, ischemia, or reperfusion injury) from occurring, e.g., when a patient or subject is predisposed to, or at risk of, the disease or condition; prevention may also include inhibiting, i.e., arresting the development of the condition.
The term "treating" means: 1) inhibiting the disease; for example, inhibiting a disease, condition, or disorder (i.e., arresting further development of pathology and/or symptomatology) in an individual who is experiencing or exhibiting the pathology or symptomatology of the disease, condition, or disorder; or 2) ameliorating the disease; for example, ameliorating a disease, condition, or disorder (i.e., reversing the pathology and/or symptomatology) in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition, or disorder.
Experimental materials and instruments
A petri dish of spermatogonial stem cells (corning); mouse spermatogonial stem cell line (mouse spermatogonial stem cell line with spermatogonial stem cell characteristic which can be established for long-term in vitro passage according to the existing report), feeder layer cell (a kind of cell which inhibits the proliferation characteristic of embryo mouse by mitomycin C treatment in vitro according to embryo mouse fibroblast prepared by embryo mouse epidermis of 13.5 days described in the existing report, and is commonly used for a kind of co-culture cell which provides cell nutrition in mouse embryo stem cell line and mouse spermatogonial stem cell line); dimethylsulfoxide dmso (sigma); StemPro-34 SFM basal medium and StemPro medium additives thereof, non-essential amino acids, sodium pyruvate, L-glutamine, beta-mercaptoethanol, fetal bovine serum, N2 additive (both commercially available from Gibco); lactic acid, vitamins, biotin, vitamin C, progesterone, estradiol, glucose, bovine serum albumin (all commercially available from Sigma); basic fibroblast factor (Gibco); mouse-derived leukemia inhibitory factor (Cell Signaling Technology); mouse-derived epidermal growth factor (Gibco); rat-derived cytokine GDNF (R & D); busulfan (Sigma); agarose (Sigma); 4% paraformaldehyde solution (Sigma); anti-UTF1 (abcam); anti-DDX4 (abcam); hoechst 33342 (Sigma).
Clean bench (Saimei fly); thermostated cell culture chambers (seemer fly); cytation5 cell imaging microplate detection System (Boteng); fluorescence microscopy (zeiss); phosphor mirrors (olympus); confocal laser microscopy (zeiss); a plurality of glass needles, micro-tweezers, sharp-pointed tweezers, etc.;
experimental methods
Example 1 promoting action of different concentrations of lovastatin on mouse spermatogonial stem cell regeneration function by in vitro culture
(1) Preparation of a Medium for in vitro culturing of spermatogonial Stem cells (50 mL as an example)
(2) Preparing culture dish for inoculating spermatogonial stem cells and feeder layer cells
The fetal mouse fibroblasts treated with mitomycin C were seeded in each culture well of a 96-well culture plate as feeder cells for culturing mouse spermatogonial stem cells.
(3) Action of different concentrations of lovastatin on in vitro culture of spermatogonial stem cells
Inoculating mouse spermatogonial stem cells into culture wells prepared with feeder cells at a density of 104Sequentially adding lovastatin with different concentrations of 0-20 mu M into the culture solution per well; cells were placed in 5% CO2A constant temperature cell culture box at 37 ℃; changing the liquid every other day.
(4) Detection of spermatogonial stem cell clonogenic capacity
The number of clones formed by spermatogonial stem cells in each well of the culture plate was detected by high content differentiation 5.
The results are shown in fig. 1, lovastatin can maintain the self-renewal of mouse spermatogonial stem cells under the condition that no cell factor GDNF is added externally in vitro, and has the function of promoting the regeneration of the spermatogonial stem cells; wherein, the effect of promoting the self-renewal of mouse spermatogonial stem cells in vitro and the formation of clones is best when 5 mu M lovastatin is added into the culture medium.
Example 2 promoting Effect of various statin Compounds on the regeneration function of mouse spermatogonial Stem cells by in vitro culture
(1) Preparation of a Medium for in vitro culturing of spermatogonial Stem cells (50 mL as an example)
(2) Preparing culture dish for inoculating spermatogonial stem cells and feeder layer cells
The fetal mouse fibroblasts treated with mitomycin C were seeded in each culture well of a 96-well culture plate as feeder cells for culturing mouse spermatogonial stem cells.
(3) Effect of various statin compounds on in vitro culturing of spermatogonial stem cells
Inoculating mouse spermatogonial stem cells into a cell prepared with a feeder layerCells were plated at a density of 10 in culture wells4Cells/wells, different kinds of statin compounds were added sequentially to the culture: lovastatin, pravastatin sodium, fluvastatin sodium, atorvastatin calcium, pitavastatin calcium, mevastatin, rosuvastatin calcium and rosuvastatin, and a statin compound dilution solvent DMSO with the same volume is added into a control group; cells were placed in 5% CO2A constant temperature cell culture box at 37 ℃; changing the liquid every other day.
(4) Detection of spermatogonial stem cell clonogenic capacity
The number of clones formed by spermatogonial stem cells in each well of the culture plate was detected by high content differentiation 5.
The results are shown in fig. 2, and under the condition that various statin compounds are not added with the cell factor GDNF in vitro, the self-renewal of mouse spermatogonial stem cells can be maintained, and the function of promoting the regeneration of the spermatogonial stem cells is realized.
Example 3 statins maintain the long-term culture of mouse spermatogonial stem cells in the absence of exogenously added cytokine GDNF in vitro
(1) Preparation of a Medium for in vitro culturing of spermatogonial Stem cells (50 mL as an example)
(2) Preparing culture dish for inoculating spermatogonial stem cells and feeder layer cells
The embryonic mouse fibroblasts treated with mitomycin C were seeded in each culture well of a 12-well culture plate as feeder cells for culturing mouse spermatogonial stem cells.
(3) Statin compound for culturing and subculturing in vitro cultured spermatogonial stem cells
Mouse spermatogonial stem cells were seeded at a density of 2X 10 in culture wells prepared in advance with feeder cells5Cells/well in cultureAdding lovastatin 5 μ M into the solution; two control groups are simultaneously set, namely lovastatin solvent DMSO with the same volume and cytokine GDNF (10ng/mL) with the same volume which is known to promote the in-vitro proliferation of spermatogonial stem cells are respectively added into a culture solution; cells were placed in 5% CO2A constant temperature cell culture box at 37 ℃; changing the liquid every other day, and carrying out passage once every 6 days.
The number of cells in each group is counted in the passage process, and the result is shown in fig. 3, which shows that the lovastatin-exemplified statin compound can partially replace the function of the cytokine GDNF to continuously maintain the self-renewal of mouse spermatogonial stem cells and realize the function of promoting the regeneration of the spermatogonial stem cells under the condition that the cytokine GDNF is not externally added in vitro.
Example 4 transplantation of spermatogonial stem cells after the promotion of regeneration by statin compounds in example 3 was performed in vivo
(1) Preparation of Busulfan receptor mice
ICR mice mating, the next day of vaginal suppository was checked and recorded as 0.5 days, 12.5 days after mating to pregnant mice intraperitoneal injection of Busulfan at 40mg/kg concentration for molding, and mice birth day 10 after testicular transplantation of spermatogonial stem cells.
(2) After the recipient mice were anesthetized, the glass needle was broken at an inner diameter of about 40 μm, and the glass needle was placed in an oral pipette, 5 μ L of the spermatogonial stem cell suspension after promoting regeneration with the statin compound in example 3 was aspirated, and the needle was slightly aspirated into the position indicated by trypan blue.
(3) Dissecting the lower abdomen of a receptor mouse, finding a fat pad connected with a testis beside a bladder, gently dragging out of the abdominal cavity, finding an output tubule along the direction parallel to the artery of the testis under a body type microscope, holding the output tubule with a pair of microscope forceps by one hand, holding a mouth suction tube by the other hand, poking a needle into the output tubule, inserting the output tubule along the direction of the output tubule to the retest, and then blowing the spermatogonial stem cells which are promoted to be regenerated by the statin compound in example 3 into the seminiferous tubule of the testis.
(4) After the injection is finished, the testis and the fat pad are carefully sent into the abdominal cavity without twisting the testis, then the testis is sutured layer by layer, and finally the alcohol cotton is applied to the abdomen of the mouse to prevent infection; the mice were placed on a 37 ℃ incubator and returned to the feeding room after they were awakened.
(5) After 60 days, taking bilateral testes of a receptor mouse to detect the colonization and differentiation conditions of the exogenously transplanted spermatogonial stem cells.
The result is shown in fig. 4, the exogenous implanted spermatogonial stem cell marked with green fluorescent protein colonizes in the seminal seminiferous tubule of recipient mouse (the cell of spermatogonial stem cell marker gene PLZF is marked together with green fluorescent protein in the figure), and performs the function of spermatogonial stem cell differentiation into sperm, i.e. the meiosis process (the cell marked with DNA double-strand break marker gamma H2AX and synaptonemal complex protein SYCP3 in the figure) can be started, and finally the functional sperm (the cell marked with germ cell marker protein DDX4 and sperm cell marker protamine PNA in the figure) is generated. The above experimental results show that when mouse spermatogonial stem cells are cultured in vitro and then transplanted into the body, the spermatogonial stem cells can perform normal spermatogenic function, which indicates that the statin compounds exemplified by lovastatin can promote the proliferation of the spermatogonial stem cells, and simultaneously, can effectively maintain the identity and function of the spermatogonial stem cells and promote the generation of sperms.
Example 5 direct administration to mice demonstrates that statins can treat male infertility
(1) Experiment of mouse intraperitoneal injection of lovastatin under physiological state
Under the physiological state, 1mg/kg body weight, 5mg/kg body weight and 10mg/kg body weight of lovastatin are respectively injected into the abdominal cavity of a mouse in an experimental group every day, and an equal dose of a small molecular solvent DMSO is injected into the same position of a mouse in a control group; injecting the same dose of drug or control solvent DMSO into the same group of mice for 5 consecutive days; on the 6 th day, testis tissues of each group of mice are taken, and the proliferation condition and the Spermatogenic Stem Cell (SSC) recovery condition in the seminal tubules of the mice are detected through immunofluorescence staining.
The results are shown in fig. 5, which shows that the statin compound exemplified by lovastatin can be directly applied to the body of a mouse in a physiological state to promote the regeneration of spermatogonial stem cells, and the prevention of male sterility can be realized; wherein, the injection of lovastatin with the concentration of 5mg/kg body weight in a mouse body under the physiological state has the optimal effect of promoting the proliferation of spermatogonial stem cells.
(2) Experiment for mouse abdominal cavity injecting lovastatin under simulated pathological state of busulfan treatment
Preparing a pathological model mouse induced by Busulfan, namely injecting Busulfan at 10mg/kg body weight into the abdominal cavity of a mouse, injecting lovastatin into the abdominal cavity of an experimental group every day from 10 days after injection, and simultaneously injecting a small molecular solvent DMSO with equal dosage into the same position of a control group mouse; taking testis tissues of each group of mice every 5 days, and detecting and counting the proliferation condition and Spermatogenic Stem Cell (SSC) recovery condition of seminal plasma in a seminal tubule by immunofluorescence staining.
The results are shown in fig. 6, the number of spermatogonial stem cells in the seminal tubules of the mouse testis was observed after continuous injection of lovastatin for 5 days, and compared with the control group, the number of spermatogonial stem cells of the lovastatin-treated mouse was significantly recovered after continuous injection for 5 days; after continuously injecting lovastatin for 10 days, the number of spermatogonial stem cells in mouse testicular seminiferous tubules is observed, compared with the treatment for 5 days, the number of spermatogonial stem cells is still recovered continuously, and the control group is also recovered gradually in 10 days, but the recovery speed and the recovery quantity are not the same as those of the lovastatin treatment group. This suggests that statins, exemplified by lovastatin, can be administered directly into pathological mice to promote spermatogonial stem cell regeneration, thereby achieving treatment of male infertility.
Example 6 promotion of Protaminic Stem cell maintenance and proliferation in human seminiferous tubules by Tatin Compounds
(1) In vitro culture of human testicular seminiferous tubule tissue
After a human testis tissue sample obtained by biopsy is washed by PBS, a sharp-pointed forceps is used for separating a curved and fine tubule in the tissue to form a lumen sample with the length of about 2 mm/section; placing the tube cavity on a 1% agarose gel block, adding a spermatogonial stem cell culture solution, adding lovastatin 5 mu M into a culture solution of an experimental group, adding an isovolume of lovastatin solvent DMSO into a culture solution of a control group, placing in an incubator at 35 ℃ for culture, and changing the solution every other day.
(2) Immunofluorescence staining assay
Human testicular luminal tissue cultured in vitro for 14 days was collected, fixed with 4% paraformaldehyde solution at room temperature for 24 hours, and then paraffin-embedded and tissue sectioned. The method for carrying out immunofluorescence staining on the tissue section comprises the following steps: adding primary antibodies (anti-UTF1 and anti-DDX4) at the antibody dilution ratio of 1:300, and placing in a refrigerator at 4 ℃ for overnight; adding corresponding fluorescent reaction secondary antibody (such as 488/594/647anti-Ms/Rb/Rt) at the antibody dilution ratio of 1:400, and standing at room temperature for 1 h; staining nuclei by Hoechst 33342 with a dilution ratio of l:1000 at room temperature for 10 min; and (5) observing the slices and carrying out photographing statistics.
As shown in fig. 7, compared to the control group, the human testicular seminiferous tubule tissue cultured with the statin compound, such as lovastatin, can maintain the survival of the spermatogonial stem cells well, which indicates that the statin compound can promote the survival and proliferation of the human spermatogonial stem cells, i.e., the statin compound can not only promote the regeneration function of the spermatogonial stem cells of rodent mammals, such as mice, but also promote the regeneration function of the spermatogonial stem cells of primate mammals, such as macaques, humans, etc., thereby achieving the prevention and/or treatment of male infertility of mammals.
The above examples are intended only to illustrate several embodiments of the present invention, which are described in more detail and detail, but are not to be construed as imposing any limitation on the scope of the present invention. It should be clear that a person skilled in the art can make several variations and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention.
Claims (13)
1. The application of statin compounds in preparing medicaments for promoting the regeneration function of spermatogonial stem cells of mammals.
2. The use according to claim 1, wherein the medicament is for the prevention and/or treatment of male infertility in mammals.
3. Use according to claim 2, wherein the male infertility is selected from one or more of azoospermia, oligospermia, asthenospermia, teratospermia and asthenospermia.
4. The use of claim 1, wherein the medicament promotes regeneration of mammalian spermatogonial stem cells by in vitro culture.
5. The use of claim 1, wherein the medicament first promotes the regeneration of spermatogonial stem cells from said mammal by means of in vitro culture, and then transplants said spermatogonial stem cells into said mammal to promote spermatogenesis.
6. The use of claim 1, wherein said medicament promotes regeneration of said spermatogonial stem cells by direct administration to said mammal.
7. The use of claim 1, wherein the medicament partially replaces the cytokine GDNF in maintaining the self-renewal of spermatogonial stem cells in said mammal.
8. Use according to claim 1, wherein the statin compound is selected from one or more of lovastatin, pravastatin, fluvastatin, atorvastatin, pitavastatin, mevastatin, rosuvastatin and pharmaceutically acceptable salts thereof, preferably lovastatin.
9. Use according to claim 1, wherein the mammal is a rodent and/or a primate.
10. The application of a composition in preparing a medicament for promoting the regeneration function of spermatogonial stem cells of mammals, wherein the composition contains a statin compound.
11. The use according to claim 10, wherein the medicament is for the prevention and/or treatment of male infertility in mammals.
12. The use according to claim 11, wherein the male infertility is selected from one or more of azoospermia, oligospermia, asthenospermia, teratospermia and asthenospermia.
13. Use according to claim 10, wherein the statin compound is selected from one or more of lovastatin, pravastatin, fluvastatin, atorvastatin, pitavastatin, mevastatin, rosuvastatin and pharmaceutically acceptable salts thereof, preferably lovastatin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011458101.5A CN112472695A (en) | 2020-12-10 | 2020-12-10 | Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011458101.5A CN112472695A (en) | 2020-12-10 | 2020-12-10 | Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112472695A true CN112472695A (en) | 2021-03-12 |
Family
ID=74916461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011458101.5A Pending CN112472695A (en) | 2020-12-10 | 2020-12-10 | Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112472695A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186153A (en) * | 2021-04-15 | 2021-07-30 | 南方医科大学 | Application of PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070190651A1 (en) * | 2006-02-10 | 2007-08-16 | Board Of Regents Of The University Of Texas System | Myogenic Development and Protection of Stem Cells Against Inflammation and Apoptosis By Statins and Isoprenoid Pathway Inhibitors |
-
2020
- 2020-12-10 CN CN202011458101.5A patent/CN112472695A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070190651A1 (en) * | 2006-02-10 | 2007-08-16 | Board Of Regents Of The University Of Texas System | Myogenic Development and Protection of Stem Cells Against Inflammation and Apoptosis By Statins and Isoprenoid Pathway Inhibitors |
Non-Patent Citations (2)
| Title |
|---|
| SALEEM ALI BANIHANI: "effect of statin on semem quality characteristics", 《ANDROLOGIA》 * |
| XIANGRONG CUI等: "Protective Effects of Fluvastatin on Reproductive Function in Obese Male Rats Induced by High-Fat Diet through Enhanced Signaling of mTOR", 《CELL PHYSIOL BIOCHEM》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186153A (en) * | 2021-04-15 | 2021-07-30 | 南方医科大学 | Application of PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury |
| CN113186153B (en) * | 2021-04-15 | 2023-09-08 | 南方医科大学 | Application of PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10493109B2 (en) | Lithium stimulation of cord blood stem cell proliferation and growth factor production | |
| US8343479B2 (en) | Methods and compositions for the repair and/or regeneration of damaged myocardium | |
| US8945919B2 (en) | Methods and composition for treating neural degeneration | |
| US8685724B2 (en) | In vitro techniques for use with stem cells | |
| US20030091547A1 (en) | Platelet-derived growth factor protection of cardiac myocardium | |
| US20050186182A1 (en) | Methods of using G-CSF mobilized C-Kit+ cells in the production of embryoid body-like cell clusters for tissue repair and in the treatment of cardiac myopathy | |
| JP7486328B2 (en) | Neurite outgrowth promoter, neuronal dendrite expression promoter, and neurotrophic factor-like substance | |
| US20120263685A1 (en) | Anti-tumor composition comprising human-derived adult stem cells | |
| CN106659741A (en) | Hair growth-promoting function of small-sized stem cells and use thereof | |
| US12110509B2 (en) | Stem cell-derived Sertoli-like cell, preparation method therefor, and use thereof | |
| CN112472695A (en) | Application of statin compound in preparation of medicine for promoting regeneration function of spermatogonial stem cells of mammals | |
| CN105055450A (en) | Applications of human endometrium stem cells in preparation of drugs for premature ovarian failure treatment | |
| EP1272203B1 (en) | Treatment of neurodegenerative gastrointestinal disorders by implanting neural stem cells and/or progeny thereof into gastrointestinal organs | |
| WO2006117237A2 (en) | Regeneration system, its production and use | |
| EP1690929A1 (en) | Regeneration treatment system | |
| CN113186153B (en) | Application of PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury | |
| KR20130085863A (en) | Composition for inducing migration of mesenchymal stem cells | |
| CN107243006B (en) | Use of AMD3100 in the manufacture of a medicament for the treatment and/or prevention of cachexia | |
| US20120142596A1 (en) | Agent for stimulating mobilization of endothelial progenitor cells | |
| WO2014042292A1 (en) | Composition comprising protein kinase c activator for promoting stem cell adhesion, and method for promoting stem cell adhesion | |
| US20200215029A1 (en) | Application of amd3100 in preparation of drug for treating and/or preventing cachexia and pharmaceutical composition thereof | |
| WO2024085651A1 (en) | Medium composition for inducing differentiation from stem cells to macrophages and differentiation method using same | |
| WO2022114730A2 (en) | Composition for treating infectious diseases, comprising exosomes derived from thrombin-treated stem cells | |
| AU2001282436B8 (en) | Osteogenic growth oligopeptides as stimulants of hematopoiesis | |
| Zerbst | An investigation into the role of autophagy in mediating leukaemic cell location in the bone marrow niche |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210622 Address after: 510000 room 604, G12, 31 Kefeng Road, Huangpu District, Guangzhou, Guangdong Province Applicant after: SHOUXI (GUANGZHOU) MEDICAL TECHNOLOGY Co.,Ltd. Address before: 510515 Southern Medical University, 1838 Guangzhou Avenue North, Baiyun District, Guangzhou City, Guangdong Province Applicant before: SOUTHERN MEDICAL University |