CN113186153B - Application of PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury - Google Patents

Abstract

The invention discloses application of a PRMT5 inhibitor in promoting regeneration and proliferation of spermatogonial stem cell injury. Wherein the PRMT5 inhibitor comprises EPZ015666 and GSK3326595, and the PRMT5 inhibitor is used at a concentration of 0.1-10 mu M. The PRMT5 inhibitor has obvious promotion effect on the continuous proliferation of spermatogonial stem cells in the in-vitro culture process, and has the same effect on primate spermatogonial stem cells, so that the PRMT5 inhibitor can be used for regeneration and in-vitro long-term culture of the spermatogonial stem cells, repair of the problems of reduced number of the spermatogonial stem cells caused by testis injury and the like, and provides a new drug choice for clinical treatment of infertility.

Description

Application of PRMT5 inhibitors in promoting the regeneration and proliferation of spermatogonial stem cells after injury

Technical field

The invention belongs to the field of biomedicine, and specifically relates to the application of PRMT5 inhibitors in promoting the regeneration and proliferation of spermatogonial stem cells after damage.

Background technique

Spermatogonia stem cells are a type of undifferentiated germ cells that are present in the testes of all male mammals and are distributed on the basement membrane in the testicular seminiferous tubules. When male mammals enter puberty, the normally functioning spermatogonial stem cells in the testicles will continue to produce sperm and renew themselves to maintain a certain number of stem cells. When spermatogonial stem cells are damaged or have functional defects, sperm production will be affected, leading to infertility.

Infertility is a global problem. About 15% of couples of childbearing age have infertility problems, and this proportion is increasing year by year due to the influence of external factors such as the environment. Among them, infertility caused by male factors accounts for about 50%. Male asthenozoospermia, oligozoospermia, and azoospermia are partly caused by gametogenesis disorders. This situation is mostly caused by the patient not having high-quality spermatogonial stem cells and thus being unable to obtain healthy mature male gametes. Among related technologies, there are no effective drugs or assisted reproductive technology treatments such as surgery. The current clinical solution is to use donor sperm from a sperm bank to reproduce. Therefore, for these patients, it is actually impossible to obtain truly biologically significant offspring.

Therefore, in order to solve the above problems, developing a drug-based method to treat infertility caused by spermatogonial stem cell damage is of extremely important significance to clinical medicine and infertility patients.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the application of PRMT5 inhibitors in promoting the regeneration and proliferation of spermatogonial stem cells after injury, which can achieve the technical effect of regenerating spermatogonial stem cells after injury in vivo and maintaining sustained proliferation in in vitro culture based on drug treatment, and has Extremely high clinical application value.

A first aspect of the present invention provides the use of a PRMT5 inhibitor in preparing a reagent for promoting the regeneration of damaged spermatogonial stem cells.

According to the first aspect of the invention, in some embodiments of the invention, the compound includes at least one of the following (1) to (4):

(1)EPZ015666 or its pharmaceutically acceptable salt;

(2)GSK3326595 and its pharmaceutically acceptable salts;

(3) EPZ015666 homolog;

(4)GSK3326595 homolog.

PRMT5 inhibitors described in the present invention include but are not limited to EPZ015666 and GSK3326595, and their salts and homologues.

Among them, the CAS number of EPZ015666 is: 1616391-65-1, and the compound name is N-[(2S)-3-(3,4-dihydro-2(1H)-isoquinolyl)-2-hydroxypropyl ]-6-(3-oxetanylamino)-4-pyrimidinecarboxamide, its structural formula is shown in formula I:

Among them, the CAS number of GSK3326595 is: 1616392-22-3, and the compound name is 6-[(1-acetylpiperidin-4-yl)amino]-N-[(2S)-3-(3,4-dihydro-1H- isoquinolin-2-yl)-2-hydroxypropyl]pyrimidine-4-carboxamide, its structural formula is shown in Formula II:

According to the first aspect of the present invention, in some embodiments of the present invention, the PRMT5 inhibitor is used at a concentration of 0.1 to 10 μM.

In some preferred embodiments of the present invention, the PRMT5 inhibitor is used at a concentration of 1 to 2 μM.

The present invention uses an in vitro culture system of mouse spermatogonial stem cells to remove the cytokines (GDNF cytokines) that play a key role in maintaining the self-renewal of spermatogonial stem cells from the culture components, thereby establishing a spermatogonial stem cell in vitro that cannot be maintained for a long time. Self-renewal "dilemma" system, and then using this system, it was found that PRMT5 inhibitors such as EPZ015666 and GSK3326595 can effectively maintain the long-term self-renewal of functional spermatogonial stem cells under this system. The inventor further explored this and found that these PRMT5 inhibitors can also effectively maintain the survival of spermatogonial stem cells during in vitro culture in primate experiments, and have extremely high versatility and universal applicability.

According to the first aspect of the present invention, in some embodiments of the present invention, the spermatogonial stem cell damage includes spermatogonial stem cell damage caused by chemotherapy drugs.

In some preferred embodiments of the invention, the chemotherapeutic drug includes busulfan.

A second aspect of the present invention provides the use of a PRMT5 inhibitor in preparing a reagent for promoting spermatogonial stem cell proliferation.

According to the second aspect of the invention, in some embodiments of the invention, the compound includes at least one of the following (1) to (4):

(1)EPZ015666 or its pharmaceutically acceptable salt;

(2)GSK3326595 and its pharmaceutically acceptable salts;

(3) EPZ015666 homolog;

(4)GSK3326595 homolog.

PRMT5 inhibitors described in the present invention include but are not limited to EPZ015666 and GSK3326595, and their salts and homologues.

According to the second aspect of the present invention, in some embodiments of the present invention, the PRMT5 inhibitor is used at a concentration of 0.1 to 10 μM.

In some preferred embodiments of the present invention, the PRMT5 inhibitor is used at a concentration of 1 to 2 μM.

The present invention first utilizes the in vitro culture system of mouse spermatogonial stem cells to remove the cytokines (GDNF cytokines) that play a key role in maintaining the self-renewal of spermatogonial stem cells from the culture components, thereby establishing a spermatogonial stem cell in vitro that cannot be maintained. Long-term self-renewal "dilemma" system, and then using this system, it was found that PRMT5 inhibitors such as EPZ015666 and GSK3326595 can effectively promote the continued proliferation of spermatogonial stem cells in normal or pathological states under this system. The inventor further explored this and found that these PRMT5 inhibitors can also show good proliferation-promoting effects in primate experiments, and have extremely high versatility and universal applicability.

A third aspect of the present invention provides the use of PRMT5 inhibitors in the preparation of male contraceptives.

According to the third aspect of the invention, in some embodiments of the invention, the compound includes at least one of the following (1) to (4):

(1)EPZ015666 or its pharmaceutically acceptable salt;

(2)GSK3326595 and its pharmaceutically acceptable salts;

(3) EPZ015666 homolog;

(4)GSK3326595 homolog.

PRMT5 inhibitors described in the present invention include but are not limited to EPZ015666 and GSK3326595, and their salts and homologues.

According to the third aspect of the present invention, in some embodiments of the present invention, the PRMT5 inhibitor is used at a concentration of 0.1 to 10 μM.

In some preferred embodiments of the present invention, the PRMT5 inhibitor is preferably used at a concentration of 1 to 2 μM.

The PRMT5 inhibitor in the present invention can inhibit the activity of the target Prmt5 enzyme, thereby inhibiting the initiation of differentiation of spermatogonial stem cells. After the use of the drug is removed, the spermatogonial stem cells can regain their ability to initiate differentiation without affecting the recovery of fertility, and thus can be used as potential Male contraceptive use.

The beneficial effects of the present invention are:

The present invention discloses the application of PRMT5 inhibitors in promoting the regeneration and proliferation of spermatogonial stem cells after injury. It has been verified through experiments that the PRMT5 inhibitors (EPZ015666 and GSK3326595) in the present invention have significant effects on promoting the continued proliferation of spermatogonial stem cells during in vitro culture. Promoting effect, and the promoting effect also has the same effect on primate spermatogonial stem cells, so it can be used for the regeneration and long-term culture of spermatogonial stem cells in vitro, as well as repairing problems such as the reduction in the number of spermatogonial stem cells caused by testicular damage, and is a good treatment for infertility. provides a new drug option for clinical treatment.

Description of the drawings

Figure 1 is a cell imaging diagram of mouse spermatogonial stem cell clone cells in an embodiment of the present invention, in which the bright field scale is 100 μm and the local magnification scale is 20 μm;

Figure 2 is a graph showing the relationship between the number of culture days and the number of viable cells in mouse spermatogonial stem cell clones in an embodiment of the present invention;

Figure 3 is an immunofluorescence image of mouse spermatogonial stem cell clones in an embodiment of the present invention;

Figure 4 shows the effects of different concentration gradients of EPZ015666 and GSK3326595 on cell activity in the embodiment of the present invention;

Figure 5 is a photo of a mouse after spermatogonial stem cell transplantation in an embodiment of the present invention;

Figure 6 shows the transplantation situation of spermatogonial stem cells in mice in the embodiment of the present invention. A is the distribution of spermatogonial stem cells in the mouse testes, and B is the immunofluorescence image of the transplanted spermatogonial stem cells;

Figure 7 is an immunofluorescence image of spermatogonial stem cell proliferation in mice in an embodiment of the present invention;

Figure 8 is a schematic diagram of an experiment using PRMT5 inhibitors to promote spermatogonial stem cell regeneration in mice under normal physiological conditions;

Figure 9 is a comparison chart of the effects of different concentrations of EPZ015666 on promoting spermatogonial stem cell regeneration in mice under normal physiological conditions in the embodiments of the present invention;

Figure 10 is a schematic diagram of an experiment using PRMT5 inhibitors to promote spermatogonial stem cell regeneration in busulfan-induced chemotherapy model mice;

Figure 11 is a comparison chart of the effect of EPZ015666 on promoting spermatogonial stem cell regeneration in busulfan-induced chemotherapy model mice in the embodiment of the present invention;

Figure 12 is an immunofluorescence image (A) and the corresponding statistical graph (B) of the survival of spermatogonial stem cells in human seminiferous tubules after PRMT5 inhibitor treatment;

Figure 13 is an immunofluorescence image (A) and the corresponding statistical graph (B) of spermatogonial stem cell proliferation in human seminiferous tubules after treatment with a PRMT5 inhibitor in an embodiment of the present invention.

Detailed ways

In order to make the purpose, technical solution and technical effect of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation modes. It should be understood that the specific embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

The experimental materials and reagents used, unless otherwise specified, are all conventional consumables and reagents available from commercial sources.

Experimental Materials

1. Construction of mouse spermatogonial stem cell line:

The mouse spermatogonial stem cell lines used in the embodiments of the present invention include any mouse-derived spermatogonial stem cell lines with spermatogonial stem cell characteristics that have been disclosed in the prior art and can be passaged in vitro for a long time, and there are no restrictions.

Among them, the construction of the mouse spermatogonial stem cell line used in the embodiments of the present invention can be referred to the article titled "Long-Term Proliferation in Culture and Germline Transmission of Mouse" published by Mito Kanatsu-Shinohara et al. in the international journal Biology of Reproduction in 2003. Male GermlineStem Cells" method. Establishment of a sustainably passaged mouse spermatogonial stem cell line for in vitro drug screening.

2. Feeder cells:

The feeder cells used in the embodiments of the present invention include fetal mouse fibroblasts (treated with mitomycin C to inhibit their proliferation characteristics) produced from fetal mouse epidermis in 13.5-day embryos, which belong to mice. A type of co-cultured cell that provides cell nutrition in embryonic stem cell lines and mouse spermatogonial stem cell lines.

3.PRMT5 inhibitors:

PRMT5 inhibitors used in embodiments of the present invention include EPZ015666 and GSK3326595.

Among them, the CAS number of EPZ015666 is: 1616391-65-1, and the compound name is N-[(2S)-3-(3,4-dihydro-2(1H)-isoquinolyl)-2-hydroxypropyl ]-6-(3-oxetanylamino)-4-pyrimidinecarboxamide, its structural formula is shown in Formula I:

Among them, the CAS number of GSK3326595 is: 1616392-22-3, and the compound name is 6-[(1-acetylpiperidin-4-yl)amino]-N-[(2S)-3-(3,4-dihydro-1H- isoquinolin-2-yl)-2-hydroxypropyl]pyrimidine-4-carboxamide, its structural formula is shown in Formula II:

PRMT5 inhibitor promotes the self-renewal ability of mouse spermatogonial stem cells cultured in vitro

(1) EPZ015666 promotes the self-renewal ability of mouse spermatogonial stem cells cultured in vitro:

In this example, EPZ015666 is used as an example to demonstrate the effect of PRMT5 inhibitors on promoting the self-renewal ability of mouse spermatogonial stem cells cultured in vitro. The specific steps are:

① Construct an in vitro culture system (culture medium) for spermatogonial stem cells. The specific formula of the culture medium (50mL system) is shown in Table 1:

Table 1 Components and contents of spermatogonial stem cell in vitro culture medium

Among them, the non-essential amino acids were purchased from Gibco and contained 750 mg/L glycine, 890 mg/L alanine, 1320 mg/L L-asparagine, 1330 mg/L L-aspartic acid, 1470 mg/L glutamic acid, 1150mg/L proline, 1050mg/LL-serine. The vitamins were purchased from Gibco and contained 100 mg/L choline chloride, 100 mg/L D-calcium pantothenate, 100 mg/L folic acid, 100 mg/L nicotinamide, 100 mg/L pyridoxal hydrochloride, 10 mg/L riboflavin, 100mg/L thiamine hydrochloride, 200mg/L inositol, 8500mg/L sodium chloride.

② Preparation of spermatogonial stem cells and feeder cells:

Mouse spermatogonial stem cells and feeder cells were inoculated in a 96-well culture plate. The feeder cells were used as co-culture cells to culture mouse spermatogonial stem cells and provide them with cell nutrition.

②Detection of spermatogonial stem cell clonogenic ability:

High-content Cytation 5 cell imaging was used to detect the number of clones formed by mouse spermatogonial stem cells in culture plates.

The addition of GDNF cytokine (also called glial cell-derived neurotrophic factor) to the culture medium was used as a positive control, and the removal of GDNF cytokines from the culture medium and the addition of the small molecule solvent DMSO was used as a negative control.

The results are shown in Figures 1 and 2.

As shown in Figure 1, EPZ015666 can maintain the clonal formation of significantly more mouse spermatogonial stem cells (compared to the negative control group) without exogenous addition of GDNF cytokines, which further illustrates that it promotes and maintains mouse spermatogonial stem cells. Self-renewal of spermatogonial stem cells. Furthermore, as shown in Figure 2, cell growth curves were drawn for spermatogonial stem cells in different media through continuous subculture, confirming that EPZ015666 can continuously maintain mouse spermatozoa without the addition of exogenous GDNF cytokines. Stem cell self-renewal.

The cloned cells were further tested using Ddx4 gene markers (germ cell-specific genes, showing purple fluorescence), Plzf gene markers (spermatogonia stem cell-specific genes, showing red fluorescence), and cell-derived markers (used to display living cells). , showing green fluorescence), DNA dye (used to display DNA, showing blue fluorescence), and observing the fluorescence reaction.

The results are shown in Figure 3.

After long-term in vitro culture, the cloned cells in this example can respectively display purple, red, green and purple colors at specific wavelengths through markers of Ddx4 gene markers, Plzf gene markers, cell source markers, and DNA dyes. Blue fluorescence indicates that the cloned cells in this example are germ cells, spermatogonial stem cells, living cells, and their DNA is intact. It shows that EPZ015666 in this example can well maintain the stem cell identity of mouse spermatogonial stem cells without adding external GDNF cytokines.

(2) The effect of GSK3326595 on promoting the self-renewal ability of mouse spermatogonial stem cells cultured in vitro:

In this example, GSK3326595 is used as an example to demonstrate the effect of PRMT5 inhibitors on promoting the self-renewal ability of mouse spermatogonial stem cells cultured in vitro. The specific steps are as in EPZ015666.

When testing the clone-forming ability of spermatogonial stem cells, mouse spermatogonial stem cells were inoculated into the wells of a 96-well culture plate prepared with feeder cells at a seeding density of 2 × 10 ⁵ cells/well. They were divided into two groups and added respectively. Different concentration gradients of EPZ015666 and GSK3326595 (the concentrations are: 0, 0.1, 0.5, 1, 2, 5, 10 μM, the diluting solution is DMSO). At the same time, set up a negative control, that is, add an equal volume of DMSO to the culture medium. The cells in the test group and the control group were placed in a 5% CO ₂ , 37°C constant-temperature cell culture incubator, and the medium was changed every other day. Use MTT chromogenic reagent to label the cells cultured for 6 days, use a microplate reader to detect the activity of spermatogonial stem cells in each well of the culture plate at 570 nm, and draw a cell activity curve.

The results are shown in Figure 4.

As shown in the cell activity detection line chart, both EPZ015666 and GSK3326595 can promote the self-renewal of mouse spermatogonial stem cells without the addition of external GDNF cytokines. Among them, when the added concentration is 1 μM or less, GSK3326595 promotes the self-renewal and colony formation of mouse spermatogonial stem cells in vitro more strongly than EPZ015666. When the concentration rises to 2 μM, EPZ015666 promotes the self-renewal and colony formation of mouse spermatogonial stem cells in vitro. The promotion effect of updating and forming clones is stronger than that of GSK3326595. Within the concentration range of 0 to 10 μM, adding 2 μM EPZ015666 or 1 μM GSK3326595 to the culture medium has the best effect on promoting self-renewal and cloning of mouse spermatogonial stem cells in vitro.

Stability experiment of PRMT5 inhibitor on the self-renewal ability of mouse spermatogonial stem cells cultured in vitro

In this example, EPZ015666 is used as an example to demonstrate the effect of PRMT5 inhibitors on promoting the self-renewal ability of mouse spermatogonial stem cells cultured in vitro. The specific steps and reaction system are as in the above examples.

Among them, the specific operation of using EPZ015666 to culture spermatogonial stem cells in vitro and passaging is as follows: inoculate mouse spermatogonial stem cells into the wells of a 96-well culture plate with feeder cells prepared in advance, and the inoculation density is 2×10 ⁵ cells/well. The experimental group added 1 μM of EPZ015666 (the diluting solution was DMSO). At the same time, a control group was established, that is, an equal volume of DMSO (negative control) or mouse GDNF cytokine (positive control, concentration: 10 mg/mL) was added to the culture medium. The cells in the test group and the control group were placed in a 5% CO ₂ , 37°C constant-temperature cell culture incubator, and the medium was changed every other day. Passage every 6 days. During the passaging process, the number of cells in each group was counted.

The cells obtained by passage are transplanted in vivo. The specific operations are as follows:

①Construction of busulfan receptor mouse model:

Prepare ICR mice. After the ICR mice mate, check the vaginal plug on the next day (recorded as 0.5 days after mating). On 12.5 days after mating, intraperitoneally inject busulfan (also known as busulfan) into the pregnant mice at a concentration of 40 mg/kg. Maryland) for modeling. On the 10th day after the mice were born, the spermatogonial stem cells obtained by the above passage were transplanted into the testes of newborn mice.

②Spermatogonia stem cell transplantation:

After anesthetizing the recipient mouse (neonatal mouse), break the glass needle at an inner diameter of about 40 μm, put it into an oral straw, and inhale 5 μL of the spermatogonial stem cell suspension obtained by the above passage. Cut open the lower abdomen of the recipient mouse, look for the fat pad connected to the testicles next to the bladder, gently drag it out of the abdominal cavity, and find the output canaliculus along the direction parallel to the testicular artery under a stereomicroscope, and pick up the output canaliculus with microscopic forceps in one hand. , hold the oral straw in the other hand, poke the needle into the output tubule, and insert it along the output tubule toward the rete testis, and blow the spermatogonial stem cells obtained by the above passage into the seminiferous tubules of the testis. After the injection, the testicles and fat pad were carefully delivered into the abdominal cavity without twisting the testicles, and then sutured layer by layer. Finally, alcohol cotton was applied to the abdomen of the mouse to prevent infection. Place the mice on a 37°C incubator and return them to the breeding room after they wake up (the transplanted mice are shown in Figure 5). Sixty days after the transplantation, the mice were sacrificed, and both testicles of the recipient mice were harvested to detect the colonization and differentiation of spermatogonia stem cells transplanted at the outer edge of the testicles.

The results are shown in Figures 6 and 7.

Through fluorescent labeling, it can be clearly observed that EPZ015666 and spermatogonial stem cells have successfully colonized the basement membrane of the seminiferous tubules of the testicles of the recipient mice. By observing the bilateral testicular tissue taken from the recipient mice, it can be found that some of these cells can show fluorescence corresponding to cell-derived markers, Plzf gene markers, and DNA dyes, indicating that there is no exogenous addition of GDNF cells. Factors and cells cultured long-term in vitro using EPZ015666. After transplantation into the body, spermatogonial stem cells can perform normal spermatogenic functions and mate with female mice to produce fertile offspring. This shows that EPZ015666 not only promotes the proliferation of spermatogonial stem cells, but also effectively maintains spermatogenesis. Identity and function of spermatogonial stem cells. At the same time, as shown in Figure 7, by observing the slices of bilateral testicular tissue taken from the recipient mice, it can be found that the cells implanted in vitro can show signs corresponding to cell-derived markers, synaptonemal complex markers SYCP3, Fluorescence of DNA double-strand break repair protein γH2AX and DNA dye. This shows that cells cultured in vitro without external addition of GDNF cytokines and using EPZ015666 for a long time have the ability to initiate differentiation into the meiotic spermatogenesis process.

Verification of the effect of PRMT5 inhibitors on promoting spermatogonial stem cell regeneration

In this example, EPZ015666 is used as an example to demonstrate the effect of PRMT5 inhibitors on promoting spermatogonial stem cell regeneration.

(1) The effect of PRMT5 inhibitors on promoting spermatogonial stem cell regeneration in mice under normal physiological conditions:

Mice (8 weeks old) with normal physiological status indicators were taken, and EPZ015666 with a mass ratio of 10 mg/kg mouse body weight and 20 mg/kg mouse body weight was injected intraperitoneally into the experimental group of mice every day. At the same time, the mice in the control group were injected Equal doses of DMSO were injected at the same location for 5 consecutive days. On the 6th day of the experiment, the testicular tissues of mice in each group were collected, and the proliferation of spermatogonial stem cells (SSC) and spermatogenesis recovery in the seminiferous tubules were detected by immunofluorescence staining.

The experimental schematic is shown in Figure 8.

The experimental results are shown in Figure 9.

From the results, it can be found that PRMT5 inhibitor compounds, such as EPZ015666, can be directly administered to mice under normal physiological conditions to promote spermatogonial stem cell regeneration. Among them, injection of EPZ015666 at a concentration of 20 mg/kg body weight in mice under normal physiological conditions has the best effect on promoting the proliferation of spermatogonial stem cells.

(2) The effect of PRMT5 inhibitors on promoting spermatogonial stem cell regeneration in busulfan-induced chemotherapy model mice:

Mice (8 weeks old) with normal physiological status indicators were taken, intraperitoneally injected with busulfan 10 mg/kg mouse body weight, and injected for a total of 10 days to construct busulfan-induced chemotherapy model mice. Ten days after the injection, mice in the experimental group were intraperitoneally injected with EPZ015666 (concentration: 10 mg/kg mouse body weight, injection volume: 20 μL/dose) every day, and equal doses of DMSO were injected into the same position of the mice in the control group for 10 consecutive days. . After the 10th day, the testicular tissues of the mice in each group were collected, and the proliferation of spermatogonial stem cells (SSC) and spermatogenesis recovery in the seminiferous tubules were detected and counted through immunofluorescence staining.

The experimental schematic is shown in Figure 10.

The experimental results are shown in Figure 11.

After 10 days of continuous injection of EPZ015666, by observing the number of spermatogonial stem cells in the testicular seminiferous tubules of mice in the experimental group and the control group, it was found that compared with the control group, after 5 days of continuous injection of EPZ015666, the number of spermatogonial stem cells The numbers recovered significantly. This shows that PRMT5 inhibitor compounds such as EPZ015666 can promote the regeneration of spermatogonial stem cells in mice under pathological conditions.

PRMT5 inhibitor promotes the maintenance and proliferation of spermatogonial stem cells in human seminiferous tubules

In this example, EPZ015666 is used as an example to demonstrate the promoting effect of PRMT5 inhibitors on the maintenance and proliferation of spermatogonial stem cells in human seminiferous tubules.

Specific steps are as follows:

(1) In vitro culture of human testicular seminiferous tubule tissue:

After the human testicular tissue samples taken from the biopsy were washed with PBS, the seminiferous tubules in the tissue were separated with pointed forceps to form a lumen sample of approximately 2 mm/segment. The tube lumen was placed on a 1% agarose gel block, and spermatogonial stem cell culture medium was added. 1 μM EPZ015666 was added to the culture medium of the experimental group, and an equal dose of DMSO was added to the control group. Each group was cultured in a 35°C incubator, and the medium was changed every other day.

(2) Immunofluorescence staining detection:

Human testicular tissue after 14 days of in vitro culture was collected, fixed with 4% paraformaldehyde solution at room temperature for 24 hours, embedded in paraffin, and sectioned. Perform immunofluorescence staining on tissue sections. The target antibodies are specifically: add primary antibody (anti-UTF1 or anti-DDX4 or anti-KI67) with an antibody dilution ratio of 1:400 and place it in a 4°C refrigerator overnight; add secondary antibody. The dilution ratio is 1:400, let it stand at room temperature for 1 hour; use DNA dye (Hoechst 33342) to stain the nuclei, the dilution ratio is 1:1000, and let it stand at room temperature for 10 minutes; observe the sections and take pictures for statistics.

The results are shown in Figures 12 and 13.

By counting the cells co-labeled with UTF1 and DDX4 markers, the proportion of spermatogonial stem cells can be obtained. Furthermore, based on DNA dyes, the survival of spermatogonial stem cells can be effectively obtained. According to the statistical results, it can be seen that human testicular seminiferous tubule tissue cultured with EPZ015666 can well maintain the survival of spermatogonial stem cells. By counting the cells co-labeled with KI67 and DDX4 markers, the proportion of spermatogonial stem cells in the proliferating state can be obtained. Furthermore, based on DNA dyes, the survival of spermatogonial stem cells in the proliferating state can be effectively obtained. According to the statistical results, it can be seen that EPZ015666 can promote the proliferation of human spermatogonial stem cells in human testicular seminiferous tubules.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (4)

1. The application of PRMT5 inhibitor in preparing in vitro culture reagent for promoting regeneration of mouse or primate spermatogonial stem cell injury is characterized in that the PRMT5 inhibitor is EPZ015666 with the concentration of 0.1-5 mu M or GSK3326595 with the concentration of 0.1-1 mu M.

2. The application of PRMT5 inhibitor in preparing in vitro culture reagent for promoting proliferation of mouse or primate spermatogonial stem cells is characterized in that the PRMT5 inhibitor is EPZ015666 with a concentration of 0.1-5 mu M or GSK3326595 with a concentration of 0.1-1 mu M.

3. The use of claim 1, wherein the mouse or primate spermatogonial stem cell injury comprises a spermatogonial stem cell injury caused by a chemotherapeutic agent.

4. The use according to claim 3, wherein the chemotherapeutic agent comprises busulfan.