A composition for treating testicular atrophy and its preparation method

Abstract

A composition for treating testicular atrophy and a preparation method thereof, relate to the field of drugs for treating testicular atrophy, and in particular to a composition for treating testicular atrophy and a preparation method thereof. The purpose is to solve the problem that the existing platelet-rich plasma is not effective in treating testicular atrophy. The composition includes platelet lysate and BMSCs exosomes, and the volume ratio of platelet lysate and BMSCs exosomes is 1:1. The present invention prepares a composition for promoting cell regeneration by using platelet lysate sPL and BMSCs exosomes, which is used for improving and treating testicular atrophy in rats. Taking the activation and restoration of spermatogonial stem cell function as the starting point, a method for preparing sPL that can increase the secretion of three growth factors that promote spermatogonial stem cell proliferation, namely EGF, bFGF and IGF-1, and a method for culturing BMSCs with high expression of miR-486-5p exosomes are sought. The present invention is used for the treatment of testicular atrophy.

Description

Technical Field

[0001] The invention relates to the field of medicines for treating testicular atrophy, and in particular to a composition for treating testicular atrophy and a preparation method thereof. Background Art

[0002] As human life expectancy increases and the aging population increases, fertility issues are becoming increasingly important. Studies have found that with age, the number and quality of male sperm and the level of bioavailable or free serum testosterone (T) gradually and steadily decline, leading to an increased incidence of testicular atrophy in men. Age-related differences in the human testis include a decrease in the volume or number of Leydig cells that produce T cells, and changes in the morphology and number of Sertoli cells and germ cells, both of which may lead to age-related degeneration of germ cells during spermatogenesis. These findings suggest that the mechanism of age-related testicular function decline is intrinsic to the testis; however, testicular atrophy is not unique to aging animals and can be caused by a variety of factors, such as environmental factors, endocrine diseases, and genetic factors. Spermatogenesis is a dynamic and synchronous process that occurs in the testicular seminiferous epithelium. During this process, spermatogonial stem cells proliferate and differentiate to produce mature haploid germ cells. This complex process depends on paracrine factors produced by Sertoli cells and androgens produced by Leydig cells. It can be seen that good reproductive function depends on the coordinated action of various cells in the testis.

[0003] The testicle is part of the male reproductive organ, which has the functions of secreting male hormones, producing sperm, and maintaining normal male reproductive function and sexual function. In recent years, due to the influence of factors such as living environment, the symptoms of testicular atrophy have become more and more common, and there is currently no good treatment. Due to the special physiological function of androgens, some people choose to give androgens exogenously to slow down or improve testicular atrophy. However, the irrational use of exogenous androgens may aggravate the occurrence of testicular atrophy and thus affect its normal physiological function. Therefore, it is considered to use exogenous growth factors to activate autologous cells so that the cells can restore their original functions and achieve therapeutic effects. In recent years, many studies have found that platelet-rich plasma (PRP) has a certain improvement effect on male reproductive function diseases. In 2019, Dehghani F et al. evaluated the effect of PRP on the function of rats with testicular structure damage model by stereological methods. The results showed that PRP can significantly increase the number of spermatogenic stem cells, sperm count, and testosterone levels in rats with testicular damage model, indicating that PRP can improve the testicular structure and function damage in rats. In 2020, Samy A et al. found that PRP can significantly reduce torsional degenerative testicular degeneration. In 2017, Sekerci CA et al. found that PRP has a certain protective effect on ischemia / reperfusion injury of testicular tissue by inhibiting neutrophil infiltration and oxidative stress. The above studies have shown that PRP is helpful in improving male reproductive dysfunction. However, the therapeutic effect of PRP on testicular atrophy is not significant. Summary of the invention

[0004] The present invention aims to solve the problem that the existing platelet-rich plasma has a poor effect in treating testicular atrophy, and provides a composition for treating testicular atrophy and a preparation method thereof.

[0005] The composition for treating testicular atrophy of the present invention comprises platelet lysate (sPL) and BMSCs exosomes, and the volume ratio of platelet lysate to BMSCs exosomes is 1:1. The concentration of EGF factor in platelet lysate is 631-674pg / mL, the concentration of bFGF factor is 86-107pg / mL, and the concentration of IGF-1 factor is 26-30ng / mL; the concentration of exosome protein in BMSCs exosomes is 1μg / μL.

[0006] Furthermore, the preparation method of the platelet lysate is specifically as follows:

[0007] The collected rat whole blood was centrifuged at 150-500×g for 15-20 min, the upper plasma was taken, and then centrifuged at 900-1300×g for 20-25 min, and the lower platelet-rich plasma (PRP) was taken to ensure that the volume of the lower layer was 1 / 10 of the whole blood volume;

[0008] CaCl2 and type III collagen were added to the prepared PRP, frozen at -80°C for 8 hours, thawed at 37°C for 5 minutes, and then repeatedly frozen and thawed 10 times. The lysate of the last thaw was centrifuged at 3000×g for 15-20 minutes, and the supernatant was taken as sPL. The concentration of CaCl2 in PRP was 20-30mM, and the concentration of type III collagen in PRP was 15-35μg / mL.

[0009] The repeated freezing and thawing conditions are freezing at -80°C for 1 hour each time and then thawing at 37°C for 5 minutes.

[0010] Furthermore, the preparation method of the BMSCs exosomes is specifically as follows:

[0011] Rat bone marrow mesenchymal stem cells were obtained, resuspended in BMSCs conditioned medium, inoculated into culture bottles, and cultured in a CO2 incubator at 37°C. The medium was changed every 48-72 hours, and the cells were passaged when they adhered to 80%-90% of the wall.

[0012] When rat bone marrow mesenchymal stem cells were cultured to the third generation and the cells adhered to 50%-60%, the medium was replaced with exosome-free medium and cultured continuously. When the cells grew to 90-100%, the supernatant was collected and exosomes were extracted by ultracentrifugation.

[0013] Furthermore, the BMSCs conditioned medium consists of 92% MSCBM, 5% sPL, 2% seminal vesicle fluid, 1% penicillin-streptomycin and 2 IU / mL heparin sodium by volume.

[0014] The exosome-free culture medium is prepared by collecting 2 / 3 volume of the upper layer of culture medium after BMSCs conditioned culture at 4° C. and 100,000×g ultracentrifugation for 16 h.

[0015] Further, the ultracentrifugation method is specifically:

[0016] 1. Centrifuge the collected supernatant at 300×g for 10 min at 4°C to remove live cells, and take the supernatant for the next step;

[0017] 2. Centrifuge the supernatant obtained in step 1 at 2000×g for 10 min at 4°C to remove dead cells, and take the supernatant for the next step;

[0018] 3. Centrifuge the supernatant obtained in step 2 at 10,000 × g for 30 min at 4°C to remove cell debris;

[0019] 4. Transfer the supernatant to an ultrafiltration centrifuge tube and collect the concentrate by centrifugation;

[0020] 5. Transfer the concentrate to an ultracentrifuge tube and ultracentrifuge at 100,000 × g for 75 min at 4°C, retaining the precipitate at the bottom of the tube;

[0021] 6. Then add PBS to resuspend the precipitate, centrifuge at 100,000 × g for 75 min at 4°C, and retain the precipitate at the bottom of the tube;

[0022] 7. Resuspend the precipitate with pre-cooled PBS to obtain exosomes; add 150 μL of PBS for every 150 μg of exosomes.

[0023] Beneficial effects of the present invention:

[0024] The present invention prepares a composition promoting cell regeneration by using platelet lysate sPL and BMSCs exosomes, which is used for improving and treating testicular atrophy in rats. With the activation and restoration of spermatogonial stem cell function as the starting point, the preparation method of sPL that can increase the secretion of three growth factors that promote the proliferation of spermatogonial stem cells, namely EGF, bFGF and IGF-1, and the BMSCs culture method that highly expresses miR-486-5p exosomes are sought, and the sPL is used in combination with the exosomes to activate spermatogonial stem cells in a targeted manner, so that they proliferate and self-renew, providing technical support and treatment ideas for the clinical study of testicular atrophy using human BMSCs exosomes and sPL.

[0025] 1. The separation of PRP of the present invention is carried out by a two-step centrifugation method. The first step is centrifugation at a speed of 150-500×g, which uses a low speed to separate plasma and blood cells, ensuring that platelets remain in the plasma and do not sink into the blood cells; all the plasma from the first step of centrifugation is collected for a second centrifugation. The second time is a high-speed centrifugation, which allows the platelets to settle as much as possible at the bottom of the tube and in the bottom plasma, so that the platelet content in the upper layer is low, thereby enhancing the platelet concentration efficiency.

[0026] 2. In the present invention, different types and concentrations of activators are added to highly concentrated PRP, and the activation time is adjusted, thereby specifically releasing growth factors related to spermatogonial stem cell proliferation and increasing the concentration of effective factors, thereby screening out the best SPL preparation scheme, thereby enhancing the therapeutic effect of testicular atrophy.

[0027] In the present invention, platelet activation is performed using platelet activators such as CaCl2, bovine thrombin, and type III collagen, and the purpose of activation is more effectively achieved through different types of combinations and different concentration adjustments. CaCl2 can promote the activation of endogenous thrombin, thereby activating the α particles in the platelets, thereby releasing the factors therein; type III collagen can adhere to platelets, and a suitable concentration of type III collagen can aggregate and effectively activate platelets, so that the growth and immunomodulatory factors in the α particles are fully released. In the present invention, an appropriate concentration of CaCl2 is used to activate endogenous thrombin, and a suitable concentration of type III collagen is used to recruit and activate platelets, so that the α particles can release the required factors more fully.

[0028] 3. The cells used in the preparation of exosomes in the present invention are BMSCs, which have multidirectional differentiation potential and high proliferation characteristics, good tissue compatibility, weak rejection reaction, and simple separation. The exosomes contain a variety of growth factors, and in this study, BMSCs were cultured using a specific conditioned medium containing seminal vesicle fluid. The exosomes obtained contain higher levels of related components that promote the proliferation of spermatogonial stem cells, providing a possibility for the treatment of male testicular atrophy.

[0029] In the process of culturing BMSCs in the present invention, sPL and seminal vesicle fluid are added to the basal culture medium. SPL is rich in growth factors required for stem cell proliferation and can effectively maintain the proliferation and secretion characteristics of stem cells. At the same time, the seminal vesicle fluid contains high concentrations of fructose, prostaglandins, proteins, ascorbic acid, inositol, sorbitol, etc., which can provide nutrition and energy for cells. At the same time, the water channel protein AQPs can participate in cell proliferation, migration and apoptosis, change the intracellular DNA transcription spectrum, and then regulate the changes in miRNA types and expression levels. Therefore, the culture medium of group c in the present invention improves the expression of miR-486-5p of BMSCs, thereby obtaining exosomes with a high miR-486-5p content.

[0030] 4. The present invention utilizes the characteristics of sPL that are rich in growth factors and nutrients and adds it to the BMSCs culture system, which is more conducive to stem cell proliferation and characteristic maintenance, and solves the impact of the introduction of xenogeneic and heterologous components on the safety of treatment, avoiding the problem that serum easily causes differentiation and aging and changes its function.

[0031] SPL has a high concentration of cell proliferation factors and contains some immunomodulatory factors, such as EGF, bFGF, IGF-1 and TGF-β, which can regulate the inflammatory environment and promote the proliferation of testicular interstitial cells, supporting cells and spermatogonial stem cells, but lacks SCF, Plzf, Sohlh and some miRNAs that promote the differentiation of spermatogonial stem cells; while BMSCs exosomes contain fewer growth factors and do not have a great advantage in promoting proliferation, but after being cultured in seminal vesicle fluid, their exosomes can contain high levels of intrinsic proteins and miRNAs that can regulate the balance between proliferation and differentiation. These substances can maintain the balance of stem cells by regulating signal pathways. When spermatogonial stem cells proliferate weakly, factors secreted by supporting cells (such as GDNF) can promote the proliferation of spermatogonial stem cells and ensure the maintenance of the number of stem cells, but when spermatogonial stem cells proliferate strongly, Plzf can inhibit the transcription of the Kit gene, promote the differentiation of spermatogonial stem cells, maintain the balance of stem cells, and thus ensure reproductive capacity. Therefore, after sPL regulates the inflammatory environment and promotes the proliferation of testicular interstitial cells, supporting cells and spermatogonial stem cells, repairs and supplements defective cells, BMSCs exosomes further regulate the secretion of supporting cells, and miR-486-5p regulates the differentiation balance of spermatogonial stem cells, and finally testicular atrophy is repaired through the synergistic effect of BMSCs exosomes and sPL.

[0032] 5. The BMSCs prepared in the present invention are cryopreserved at ultra-low temperatures after being amplified and purified, and can be revived when used, thus avoiding time constraints on patient use.

[0033] The composition of the present invention can improve the therapeutic effect during the treatment of testicular atrophy, and provide new ideas and methods for clinical research on testicular atrophy and spermatogenesis disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the growth status of bone marrow mesenchymal stem cells that were cultured for 5 days using BMSCs conventional culture medium;

[0035] Figure 2 The growth status of bone marrow mesenchymal stem cells after 5 days of primary cell culture using the BMSCs conditioned medium of formula a;

[0036] Figure 3 The growth status of bone marrow mesenchymal stem cells after 5 days of primary cell culture using the BMSCs conditioned medium of formula b;

[0037] Figure 4 The growth status of bone marrow mesenchymal stem cells after 5 days of primary cell culture using the BMSCs conditioned medium of formula c;

[0038] Figure 5P1 cells cultured using conventional BMSCs culture medium;

[0039] Figure 6 P1 cells cultured with BMSCs conditioned medium of formula a;

[0040] Figure 7 P1 cells cultured with BMSCs conditioned medium of formula b;

[0041] Figure 8 P1 cells cultured with BMSCs conditioned medium of formula c;

[0042] Fig. 9 The morphology of exosomes under transmission electron microscopy;

[0043] Fig.10 This is the result of identification of exosome surface markers;

[0044] Fig.11 The expression of miR-486-5P in exosomes. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0046] Specific embodiment 1: The composition for treating testicular atrophy in this embodiment includes platelet lysate (sPL) and BMSCs exosomes, and the volume ratio of platelet lysate to BMSCs exosomes is 1:1; wherein the concentration of EGF factor in platelet lysate is 631-674pg / mL, the concentration of bFGF factor is 86-107pg / mL, and the concentration of IGF-1 factor is 26-30ng / mL; the concentration of exosome protein in BMSCs exosomes is 1μg / μL.

[0047] Specific embodiment 2: This embodiment is a further explanation of specific embodiment 1: The preparation method of the platelet lysate is specifically as follows:

[0048] The collected rat whole blood was centrifuged at 150-500×g for 15-20 min, the upper plasma was taken, and then centrifuged at 900-1300×g for 20-25 min, and the lower platelet-rich plasma (PRP) was taken to ensure that the volume of the lower layer was 1 / 10 of the whole blood volume;

[0049] CaCl2 and type III collagen were added to the prepared PRP, frozen at -80°C for 8 hours, thawed at 37°C for 5 minutes, and then repeatedly frozen and thawed 10 times. The lysate thawed for the last time was centrifuged at 3000×g for 15-20 minutes, and the supernatant was taken, which was sPL. The concentration of CaCl2 in PRP was 20-30mM, and the concentration of type III collagen in PRP was 15-35μg / mL. The rest was the same as the first embodiment.

[0050] In the preparation method of sPL in this embodiment, CaCl2 can promote the activation of endogenous thrombin, thereby activating the α granules in the platelets, thereby releasing the factors therein; type III collagen can adhere to platelets, and a suitable concentration of type III collagen can aggregate and effectively activate platelets, so that the growth and immunomodulatory factors in the α granules are fully released. In this embodiment, an appropriate concentration of CaCl2 is used to activate endogenous thrombin, and a suitable concentration of type III collagen is used to recruit and activate platelets, so that the α granules can release the required factors more fully. Freezing and thawing can accelerate the lysis of platelets, and the auxiliary activator activates the α granules to release the factors therein.

[0051] Specific implementation method three: This implementation method is a further explanation of specific implementation method two: the conditions of repeated freezing and thawing are freezing at -80°C for 1 hour each time and then thawing at 37°C for 5 minutes. Other conditions are the same as those of specific implementation method two.

[0052] Specific embodiment 4: This embodiment is a further explanation of specific embodiment 1 or 2: the preparation method of BMSCs exosomes is specifically as follows:

[0053] Rat bone marrow mesenchymal stem cells were obtained, resuspended in BMSCs conditioned medium, inoculated into culture bottles, and cultured in a CO2 incubator at 37°C. The medium was changed every 48-72 hours, and the cells were passaged when they adhered to 80%-90% of the wall.

[0054] When rat bone marrow mesenchymal stem cells are cultured to the third generation and the cells adhere to the wall to 50%-60%, the medium without exosomes is replaced and culture is continued. When the cells grow to 90-100%, the supernatant is collected and the exosomes are extracted by ultracentrifugation. The rest is the same as the first or second embodiment.

[0055] Specific embodiment 5: This embodiment is a further explanation of specific embodiment 4: the BMSCs conditioned medium is composed of 92% MSCBM, 5% sPL, 2% seminal vesicle fluid, 1% penicillin-streptomycin and 2 IU / mL heparin sodium by volume. The rest is the same as specific embodiment 4.

[0056] In this embodiment, sPL and seminal vesicle fluid are added to the basal culture medium during the BMSCs culture process. SPL is rich in growth factors required for stem cell proliferation and can effectively maintain the proliferation and secretion characteristics of stem cells. At the same time, the seminal vesicle fluid contains high concentrations of fructose, prostaglandins, proteins, ascorbic acid, inositol, sorbitol, etc., which can provide nutrition and energy for cells. At the same time, the water channel protein AQPs can participate in cell proliferation, migration and apoptosis, change the intracellular DNA transcription spectrum, and then regulate the changes in miRNA types and expression levels. Therefore, the group c culture medium in the present invention increases the expression of miR-486-5p in BMSCs, thereby obtaining exosomes with high miR-486-5p content.

[0057] Specific embodiment 6: This embodiment is a further explanation of specific embodiment 4: the exosome-free culture medium is prepared by collecting 2 / 3 volume of the upper layer of culture medium after BMSCs conditional culture at 4°C and 100,000×g ultracentrifugation for 16 hours. The rest is the same as specific embodiment 4.

[0058] Specific implementation method seven: This implementation method is a further explanation of specific implementation method four: the ultracentrifugation method is specifically as follows:

[0059] 1. Centrifuge the collected supernatant at 300×g for 10 min at 4°C to remove live cells, and take the supernatant for the next step;

[0060] 2. Centrifuge the supernatant obtained in step 1 at 2000×g for 10 min at 4°C to remove dead cells, and take the supernatant for the next step;

[0061] 3. Centrifuge the supernatant obtained in step 2 at 10,000 × g for 30 min at 4°C to remove cell debris;

[0062] 4. Transfer the supernatant to an ultrafiltration centrifuge tube and collect the concentrate by centrifugation;

[0063] 5. Transfer the concentrate to an ultracentrifuge tube and ultracentrifuge at 100,000 × g for 75 min at 4°C, retaining the precipitate at the bottom of the tube;

[0064] 6. Then add PBS to resuspend the precipitate, centrifuge at 100,000 × g for 75 min at 4°C, and retain the precipitate at the bottom of the tube;

[0065] 7. Resuspend the precipitate with pre-cooled PBS to obtain exosomes, wherein 150 μL of PBS is added for every 150 μg of exosomes. The rest is the same as the fourth embodiment.

[0066] Specific embodiment eight: This embodiment is a further explanation of specific embodiment four: the extracted exosomes are frozen and stored at -80°C and revived when used. Other aspects are the same as those of specific embodiment four.

[0067] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0068] Embodiment 1:

[0069] The preparation method of the composition for treating testicular atrophy in this embodiment comprises the following steps:

[0070] 1. High concentration PRP separation

[0071] (1) Blood collection: After the animals were anesthetized, whole blood was collected from the rats using a blood collection tube containing sodium citrate anticoagulant;

[0072] (2) PRP separation: The collected whole blood was transferred to a separation chamber at 2-8°C. After mixing, the whole blood was centrifuged at 150-500×g for 15 min. The upper layer of plasma was collected and then centrifuged at 900-1300×g for 20 min. The upper layer of platelet-poor plasma (PPP) was removed and the lower layer of platelet-rich plasma (PRP) was retained. The volume of the lower layer was ensured to be 1 / 10 of the volume of the whole blood. The platelet precipitate attached to the bottom of the centrifuge tube was fully suspended by blowing the bottom with a pipette.

[0073] 2. Preparation of sPL: The following four schemes were used to prepare sPL.

[0074] Scheme A: Add CaCl2 (final concentration of 20-30 mM) and type III collagen (final concentration of 15-35 μg / mL) to the above PRP, freeze at -80°C for 8 hours, thaw at 37°C for 5 minutes, freeze and thaw repeatedly 5 or 10 times, and centrifuge at 3000×g for 15 minutes; Scheme A1 uses 5 freeze-thaw cycles, and Scheme A2 uses 10 freeze-thaw cycles.

[0075] Scheme B: Add CaCl2 (final concentration of 20-30 mM) and bovine thrombin (final concentration of 75-105 U / mL) to the above PRP, freeze at -80°C for 8 hours, thaw at 37°C for 5 minutes, freeze and thaw repeatedly 5 or 10 times, and centrifuge at 3000×g for 15 minutes; Scheme B1 uses 5 freeze and thaw cycles, and Scheme B2 uses 10 freeze and thaw cycles.

[0076] Scheme C: Add type III collagen (final concentration of 15-35 μg / mL) and bovine thrombin (final concentration of 75-105 U / mL) to the above PRP, freeze at -80°C for 8 hours, thaw at 37°C for 5 minutes, freeze and thaw repeatedly 5 or 10 times, and centrifuge at 3000×g for 15 minutes; Scheme C1 uses 5 freeze-thaw cycles, and Scheme C2 uses 10 freeze-thaw cycles.

[0077] Scheme D: The PRP was frozen at -80℃ for 8 hours, thawed at 37℃ for 5 minutes, and repeatedly frozen and thawed for 5 or 10 times, and centrifuged at 3000×g for 15 minutes. The one with repeated freezing and thawing for 5 times was Scheme D1, and the one with repeated freezing and thawing for 10 times was Scheme D2.

[0078] In the above four schemes, after centrifugation at 3000×g for 15 minutes, the supernatant is sPL, and the precipitate is cell or platelet fragments; the sPL obtained after centrifugation does not need to be diluted, and this is the platelet lysate containing growth factors.

[0079] The platelet lysates obtained from the above four groups were subjected to ELISA testing to evaluate the content of growth factors such as EGF, bFGF and IGF-1 that promote the proliferation of testicular supporting cells, interstitial cells and spermatogonial stem cells and compare the results. The results are shown in Table 1. The comparison of the number of freeze-thaw times within the four groups A, B, C, and D showed that the content of growth factors after 10 freeze-thaws was significantly increased compared with that after 5 freeze-thaws; and the inter-group comparison results showed that the activation effect of the three groups A, B, and C with the addition of activators was better than that of the group D with simple freeze-thaw treatment. The original two activators had the best activation effect. Based on the above results, the present invention selected the A2 scheme that can fully activate platelets and release more growth factors. The specific method is to add CaCl2 (final concentration of 20-30mM) and type III collagen (final concentration of 15-35μg / mL) to the previously prepared PRP, freeze it at -80℃ for 8h, thaw it at 37℃ for 5min, repeat freezing and thawing 10 times, and centrifuge the lysate dissolved for the last time at 3000×g for 15min to obtain platelet lysate rich in specific growth factors.

[0080] Table 1 Contents of factors related to spermatogonial stem cell proliferation under different freezing and thawing conditions (n=8)

[0081]

[0082]

[0083] Note: There were significant differences among the four groups A, B, C, and D (P < 0.05). There were significant differences between the three groups A, B, and C and group D (P < 0.05). Among the three groups with added activators, there were significant differences between group A and groups B and C (P < 0.05), but there was no statistical significance between group B and group C (P > 0.05).

[0084] 3. Sampling of seminal vesicle and femur

[0085] (1) Sampling of seminal vesicles and preparation of seminal vesicle fluid: The rats were killed and placed on the operating table. The abdominal skin was cut open and the seminal vesicles were taken out. The seminal vesicles were cut into pieces of about 1 mm. 3 The tissue blocks were then centrifuged at 2000×g for 10 min, and the supernatant was collected to prepare the seminal vesicle fluid.

[0086] (2) Femoral bone sampling and cell preparation: After the rat is killed, it is placed on the operating table, the skin of the hind legs is cut open, the thigh joint is accurately identified, the hind legs are separated from the body, the claws are cut off, the skin and muscles are removed, the femurs on both sides are removed, and placed in physiological saline with double antibodies, and the mouth of the sample bottle is sealed.

[0087] 4. BMSCs Isolation, Culture and Cryopreservation

[0088] (1) Disinfection and rinsing

[0089] The obtained rat femur samples were transferred to 75% alcohol and completely immersed for 5 minutes to fully disinfect and remove surface bacteria. After disinfection, the femur samples were transferred to physiological saline containing 1% double antibody for immersion and cleaning, and the cleaning was repeated 3 times.

[0090] (2) Preparation of solution:

[0091] Reagents: basal medium MSCBM, serum (FBS), heparin sodium, penicillin-streptomycin, dimethyl sulfoxide (DMSO);

[0092] BMSCs conventional culture medium preparation: 89% MSCBM + 10% FBS + 1% penicillin-streptomycin (the content of each component is volume percentage);

[0093] Preparation of BMSCs conditioned medium: (the content of each component is volume percentage)

[0094] Formula A: 97% MSCBM + 2% seminal vesicle fluid + 1% penicillin-streptomycin,

[0095] Formula b: 94% MSCBM + 5% sPL + 2IU / mL heparin sodium + 1% penicillin streptomycin,

[0096] Formula c: 92% MSCBM + 5% sPL + 2% seminal vesicle fluid + 2IU / mL heparin sodium + 1% penicillin-streptomycin;

[0097] Preparation of cell freezing solution: 85% MSCBM+5% sPL+10% DMSO (the content of each component is volume percentage).

[0098] (3) Cell isolation and culture:

[0099] Use bone scissors to cut open both ends of the femur to expose the bone marrow cavity, flush the bone marrow cavity with culture medium until the bone marrow cavity turns white, transfer the culture medium filled with cells to a centrifuge tube, centrifuge at 1800rpm for 5 minutes, discard the supernatant, collect the precipitate at the bottom of the tube, resuspend it with conventional culture medium and three different formulations of BMSCs conditioned culture medium, mix well, and inoculate it into culture bottles, and culture them in a CO2 incubator at 37°C.

[0100] The above steps were all carried out in a biological safety cabinet, and all instruments used were sterilized by high-pressure steam.

[0101] (4) Cell passaging

[0102] After primary cells were isolated and extracted, the medium was changed every three days. The dead cells and cell debris were removed and then replaced with a new BMSCs conditioned medium with the corresponding formula. The growth of bone marrow mesenchymal stem cells after 5 days of primary cell culture using BMSCs conventional medium was as follows: Figure 1 As shown in Figure 2, the growth of bone marrow mesenchymal stem cells after primary cell culture for 5 days using the BMSCs conditioned medium of formula a is shown in Figure 2. Figure 2 As shown in Figure 2, the growth of bone marrow mesenchymal stem cells after 5 days of primary cell culture using the BMSCs conditioned medium of formula b is as follows: Figure 3 As shown in Figure 2, the growth of bone marrow mesenchymal stem cells after primary cell culture for 5 days using the BMSCs conditioned medium of formula c is shown in Figure 2. Figure 4 shown. Figure 1-Figure 4 It can be shown that there is no significant difference in cell adhesion and morphology between conventional culture medium and three BMSCs conditioned medium during primary culture.

[0103] The cells were placed in a CO2 incubator and continued to be cultured. When the cells adhered to 80-90%, they were subcultured. Subculture process: After digestion with 500 μL 0.3×TrypLE Express, the cells became round, and 1 mL of culture medium was added to neutralize and terminate the digestion. The bottom wall of the culture flask was blown to completely digest the cells. The cells were transferred to a centrifuge tube and centrifuged at 1800 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in complete culture medium and 1×10 5 / mL was inoculated into a T75 cell culture flask and placed in a CO2 incubator for culture. After 3 days, the growth of P1 cells was observed. Figure 5-8 As shown, Figure 5 The P1 cells were cultured using conventional BMSCs culture medium. Figure 6 The P1 cells were cultured using the BMSCs conditioned medium of formula a. Figure 7 The P1 cells were cultured using the BMSCs conditioned medium of formula b. Figure 8 These are P1 cells cultured using BMSCs conditioned medium of formula c. Figure 5-Figure 8 The results showed that the growth rate of cells cultured in conventional medium after passage was slower than that of the three conditioned mediums, and the morphology did not extend into the fibrous shape unique to mesenchyme. At the same time, there was no significant difference in the growth rate and morphology of BMSCs cultured in the three conditioned mediums, indicating that each conditioned medium was beneficial to cell growth and did not affect cell morphology. After the cells were further passaged to P3, the P3 cells were digested and their phenotypes were detected by flow cytometry, including CD73, CD90, CD105, CD34, and CD45. The results showed that the cells expressed low or no CD34 and CD45, but the expression levels of CD73, CD90, and CD105 were all higher than 98%.

[0104] (5) Cell cryopreservation

[0105] When the cells were subcultured to P1 and P3 generations, they were cryopreserved. The cryopreservation solution was prepared in advance according to the number of cell cryopreservation tubes and placed in a 4°C refrigerator for precooling. After the cells were digested and centrifuged, the precipitate was resuspended in the cryopreservation solution and the precipitate was resuspended at 1×10 6 / mL was frozen and placed in a programmed cooling box at -80°C refrigerator overnight, and transferred to liquid nitrogen for long-term storage the next day.

[0106] 5. Extraction and identification of BMSCs exosomes

[0107] (1) Extraction of exosomes

[0108] When rat bone marrow mesenchymal stem cells (BMSCs) were cultured to the third generation, when the cells adhered to 50%-60%, they were replaced with the corresponding exosome-free medium (the four groups of culture media were ultracentrifuged at 4°C 100000×g for 16h, and the upper 2 / 3 volume of the culture medium was collected, which was the corresponding exosome-free medium) and continued to be cultured. When the cells grew to 90%-100%, the four groups of culture supernatants were collected into 50mL centrifuge tubes (the number of cells in the supernatant was 3×10 7 ), and the exosomes were extracted by ultracentrifugation. The specific steps are as follows:

[0109] a. Centrifuge the collected supernatant at 4°C, 300×g for 10 min to remove live cells, and take the supernatant for the next step;

[0110] b. Centrifuge the supernatant at 2000×g for 10 min at 4°C to remove dead cells, and then take the supernatant for the next step;

[0111] c. Centrifuge at 10,000 × g for 30 min at 4°C to remove cell debris;

[0112] d. Transfer the supernatant to a 100KD 50ml ultrafiltration centrifuge tube and collect the concentrate by centrifugation;

[0113] e. Transfer the concentrate to an ultracentrifuge tube and ultracentrifuge at 100,000 × g for 75 min at 4°C, retaining the precipitate at the bottom of the tube;

[0114] f. Then add PBS to resuspend the precipitate, centrifuge at 100,000 × g for 75 min at 4°C, and retain the precipitate at the bottom of the tube;

[0115] g. Then resuspend the precipitate with 150 μL of pre-cooled PBS to obtain exosomes, and store at -80°C for later use.

[0116] (2) Morphological identification of exosomes

[0117] Observation of exosome morphology using transmission electron microscopy (TEM):

[0118] a. Use a pipette to draw 20 μL of sample and drop it on the copper grid, and wait for 15 minutes;

[0119] b. Gently remove excess liquid and negatively stain with uranyl acetate at 25°C for 10 minutes;

[0120] c. Aspirate the negative staining solution, rinse carefully once with PBS, and let it dry naturally;

[0121] d. Place the copper mesh under a transmission electron microscope, observe the morphology of exosomes at 80kv-120kv and take photos.

[0122] Results Fig. 9 , vesicle-like structures can be seen, which are typical saucer-shaped, consistent with the morphology of exosomes.

[0123] (3) Western Blot detection of exosome marker proteins

[0124] a. Mix the cells and four groups of exosome protein samples with 5× buffer at a ratio of 4:1 and place in a 90°C water bath for 8-10 min;

[0125] b. Prepare gel according to the instructions of SDS-PAGE gel preparation kit;

[0126] c. After the gel solidifies, add the protein marker and exosome protein sample into the spotting well;

[0127] d. Install the electrophoresis tank, observe whether the concentrated gel leaks at 70V, adjust to 120V after 30 minutes, and end the electrophoresis after about 60-90 minutes;

[0128] e. Remove the gel after cutting off the concentrated gel and useless areas;

[0129] f. Prepare a PVDF membrane of the same size as the separation gel and activate it with methanol for 1 min;

[0130] g. Install the transfer device in order, from negative electrode to positive electrode, which are fiber mat, three-layer filter paper, gel, PVDF membrane, three-layer filter paper, fiber mat, put the transfer device into the transfer tank, and operate at 400mA for 30min;

[0131] h. Take out the PVDF membrane and block it with 5% skim milk at 25°C for 2h, then rinse it twice with TBST, 5min each time;

[0132] i. Incubate with TSG101 and CD9 antibodies at 4°C overnight (about 10 hours), and rinse thoroughly with TBST five times every other day, 10 minutes each time;

[0133] j. Incubate with corresponding secondary antibody at room temperature for 1 h, and rinse with TBST 5 times, 10 min each time;

[0134] k. Cover the PVDF membrane with 1:1 ECL luminescent developer and observe the experimental results using a gel imaging system.

[0135] Results Fig.10 ,In the four groups of exosome samples, the surface marker proteins TSG101 and CD9 were positive, but were basically not expressed in BMSCs, proving that the exosomes were ,successfully extracted.

[0136] (4) Detection of miR-486-5P expression

[0137] ①Extraction of total RNA from BMSCs exosomes

[0138] Take an appropriate amount of exosomes into a new EP tube without RNAse, add Trizol to fully lyse the exosomes; centrifuge at 12000rpm for 10min, take the supernatant and transfer it to a new EP tube; add chloroform to each EP tube, shake vigorously and let it stand at room temperature for 10min; centrifuge at 12000rpm for 15min, transfer the upper aqueous phase to a new EP tube and mix it thoroughly with isopropanol, let it stand for 10min; centrifuge at 12000rpm for 15min, discard the residual liquid in the tube, add an appropriate amount of 75% ethanol solution prepared with DEPC water, and gently turn over to wash the precipitate; centrifuge at 12000rpm for 5min, then discard the supernatant, add an appropriate amount of deionized water when the RNA is colorless and transparent, and measure the RNA sample concentration (the above steps are all performed at 4℃).

[0139] ②Reverse transcription of total RNA from BMSCs exosomes

[0140] Use the miRNA tailing reverse transcription kit Mir-XTM miRNA First-Strand Synthesis Kit to reverse transcribe miRNA. Perform specific operations according to the instructions of the kit. The reverse transcription system is shown in Table 2:

[0141] Table 2 miRNA reverse transcription system

[0142]

[0143] Note: Incubate at 37℃ for 1h and 85℃ for 5min in a water bath. Add 90μl DEPC water to each reaction system and store at -20℃ for later use.

[0144] ③ Detection of miR-486-5P expression by fluorescence quantitative PCR (qRT-PCR)

[0145] Using U6 in the miRNA reverse transcription kit as an internal reference, the expression levels of the target miRNA in the exosomes of mesenchymal stem cells under two conditioned media were detected and compared; the quantitative primers of miR-486-5P (F: GCAGTCCTGTACTGAGCTG, R: GTCCAGTTTTTTTTTTTTTTTTCTCG) were synthesized and purified by Shanghai Bioengineering; the expression level of miRNA was calculated using the relative quantitative method, and all samples were analyzed in triplicate; the fluorescence quantitative reaction system and procedure are shown in Tables 3 and 4 below:

[0146] Table 3 miRNA fluorescence quantitative amplification system

[0147]

[0148] Table 4 miRNA fluorescence quantitative amplification procedure

[0149]

[0150] Results Fig.11 The results of fluorescence quantitative PCR showed that compared with the conventional culture medium group, the expression of miR-486-5p in the exosomes of mesenchymal stem cells in the three conditioned culture medium groups was significantly increased, among which the expression of miR-486-5p in the exosomes of mesenchymal stem cells in the BMSCs conditioned culture medium group of formula c (i.e., the sPL and seminal vesicle fluid co-culture group) was the highest, and the difference was significant compared with groups a and b (P < 0.05). The present invention uses the BMSCs conditioned medium of formula c to culture mesenchymal stem cells to promote the differentiation of spermatogonial stem cells.

[0151] MicroRNA is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in animals and plants. MiR-486-5p is expressed by adipose stem cells, bone marrow mesenchymal stem cells, testicular supporting cells, tumor cells, etc. Overexpression of miR-486-5 downregulates the expression of the PTEN gene, upregulates the expression of the Stra8 gene and the synaptonemal complex 3 (SYCP3) gene, and promotes the conversion of spermatogonial stem cells from mitosis to meiosis, thereby regulating differentiation. In the present invention, bone marrow mesenchymal stem cells are cultured with a culture medium containing seminal vesicle fluid, and by further improving the expression of miR-486-5p, exosomes containing a higher content of miR-486-5p are obtained, thereby regulating the differentiation of spermatogonial stem cells.

[0152] VI. Preparation of Composition for Treating Testicular Atrophy

[0153] The sPL obtained above was mixed with exosomes at a volume ratio of 1:1 to prepare an injection preparation, which was divided into 1.5 mL EP tubes and frozen in a -80°C refrigerator for later use.

[0154] VII. Observation of therapeutic effects on rat testicular atrophy model

[0155] Rats were fed with 1% 2,5-hexanedione in drinking water for 35 days to establish a testicular atrophy model. The model rats were divided into four groups: the first group was an untreated control group, which was treated with testicular injection of saline for sham treatment; the second group was treated with previously prepared sPL for testicular injection; the third group was treated with prepared BMSCs exosomes for testicular injection; the fourth group was treated with a mixed preparation of sPL and exosomes (volume ratio 1:1) for testicular injection. The injection dose and time of the four groups of animals were unified, once every 7 days, 6 times of treatment, designed as a course of treatment, and the rats were killed 7 days after the last treatment for whole blood collection and bilateral testicular sampling. The collected whole blood was centrifuged to separate the serum for ELISA to detect the expression levels of serum testosterone and luteinizing hormone, and the weight and length of the fresh testicles were measured. The testicles were frozen in a -80℃ refrigerator for nucleic acid and protein detection to compare the treatment effects.

[0156] Treatment results:

[0157] The synthesis of testosterone is related to male reproductive function and aging, and also plays an important physiological role in spermatogenesis and maturation. It is an important androgen in the body. The production of sperm is carried out under many factors such as endocrine, and the process is relatively complicated. If the testicles are damaged, it often manifests as a decrease in testosterone and an increase in luteinizing hormone. Therefore, the levels of serum sex hormones T and LH can reflect the occurrence of sperm. The results of ELISA test (Table 5) showed that serum testosterone levels in the three treatment groups were significantly higher than those in the model group, among which the sPL and exosome mixed preparation treatment group was the most significant. After treatment, the testosterone level increased, while the luteinizing hormone level decreased, indicating that testicular function was improved. Testicular weight and testicular to body weight ratio are shown in Table 6. It can be seen that the testicular wet weight and index of the testicles in the treatment group were significantly increased compared with the model group, among which the sPL and exosome mixed preparation group was the most significant, indicating that the degree of atrophy was restored and the mixed preparation group had the best effect. Fluorescence quantitative PCR detected the expression of C-kit mRNA (Table 7). It can be seen that the expression levels of the three treatment groups were significantly higher than those of the model group, among which the mixed preparation group had the most significant improvement effect.

[0158] Table 5 Serum T and LH expression (n = 8)

[0159]

[0160] Note: Compared with the model group, the serum T and LH levels of the three treatment groups were improved, and the differences were significant (P < 0.05); the serum T level showed that there was no statistical significance between the sPL treatment group and the exosome treatment group, and the two groups were significantly different from the sPL and exosome mixed treatment group (P < 0.05), while the serum LH level showed no statistical significance among the three treatment groups (P > 0.05).

[0161] Table 6 Comparison of body weight, testicular wet weight and testicular index (n=8)

[0162]

[0163] Note: Compared with the model group, the testicular wet weight and testicular index of the three treatment groups were significantly increased, and the difference was significant (P < 0.01); the comparison of the main index testicular wet weight of the three treatment groups showed that there was no statistically significant difference between the sPL treatment group and the exosome treatment group (P > 0.05), and the difference between these two groups and the sPL and exosome mixed treatment group was significant (P < 0.05).

[0164] Table 7 Changes in relative expression of C-kit (n=8)

[0165]

[0166]

[0167] Note: There were significant differences between the three treatment groups and the model group (P < 0.01); there was no statistically significant difference between the sPL treatment group and the exosome treatment group (P > 0.05), but there were significant differences between these two groups and the sPL and exosome mixed treatment group (P < 0.05).

Claims

1. A composition for treating testicular atrophy, characterized in that The composition comprises platelet lysate and BMSCs exosomes, wherein the volume ratio of the platelet lysate to the BMSCs exosomes is 1:1; wherein the concentration of EGF factor in the platelet lysate is 631-674 pg / mL, the concentration of bFGF factor is 86-107 pg / mL, and the concentration of IGF-1 factor is 26-30 ng / mL; and the concentration of exosome protein in the BMSCs exosomes is 1 μg / μL; The preparation method of the BMSCs exosomes is specifically as follows: Rat bone marrow mesenchymal stem cells were obtained, resuspended in BMSCs conditioned medium, inoculated into culture flasks, and cultured in a CO2 incubator at 37°C. The medium was changed every 48-72 hours, and the cells were passaged when they adhered to 80%-90% of the wall. When rat bone marrow mesenchymal stem cells were cultured to the third generation and the cells adhered to 50%-60%, the medium was replaced with an exosome-free medium and cultured continuously. When the cells grew to 90-100%, the supernatant was collected and the exosomes were extracted by ultracentrifugation. The BMSCs conditioned medium consisted of 92% MSCBM, 5% sPL, 2% seminal vesicle fluid, 1% penicillin-streptomycin and 2 IU / mL heparin sodium by volume.

2. A composition for treating testicular atrophy according to claim 1, characterized in that The preparation method of the platelet lysate is specifically as follows: The collected rat whole blood was centrifuged at 150-500×g for 15-20 min, the upper plasma was taken, and then centrifuged at 900-1300×g for 20-25 min, and the lower platelet-rich plasma (PRP) was taken to ensure that the volume of the lower layer was 1 / 10 of the whole blood volume; CaCl2 and type III collagen were added to the prepared PRP, which was frozen at -80°C for 8 h, thawed at 37°C for 5 min, and then frozen and thawed repeatedly 10 times. The lysate thawed for the last time was centrifuged at 3000×g for 15-20 min, and the supernatant was taken as sPL; the concentration of CaCl2 in PRP was 20-30 mM, and the concentration of type III collagen in PRP was 15-35 μg / mL.

3. A composition for treating testicular atrophy according to claim 2, characterized in that The repeated freezing and thawing conditions are freezing at -80°C for 1 h and then thawing at 37°C for 5 min each time.

4. A composition for treating testicular atrophy according to claim 1, characterized in that The exosome-free culture medium is prepared by collecting 2 / 3 volume of the upper layer of culture medium after BMSCs conditioned culture at 4° C. and 100,000×g ultracentrifugation for 16 h.

5. A composition for treating testicular atrophy according to claim 1, characterized in that The ultracentrifugation method is specifically:

1. Centrifuge the collected supernatant at 300 × g for 10 min at 4°C to remove live cells, and take the supernatant for the next step; 2. Centrifuge the supernatant obtained in step 1 at 2000×g for 10 min at 4°C to remove dead cells, and take the supernatant for the next step; 3. Centrifuge the supernatant obtained in step 2 at 10,000 × g for 30 min at 4°C to remove cell debris; 4. Transfer the supernatant to an ultrafiltration centrifuge tube and collect the concentrate by centrifugation; 5. Transfer the concentrate to an ultracentrifuge tube and ultracentrifuge at 100,000 × g for 75 min at 4°C, retaining the precipitate at the bottom of the tube; 6. Then add PBS to resuspend the precipitate, centrifuge at 100,000 × g for 75 min at 4°C, and retain the precipitate at the bottom of the tube; 7. Resuspend the precipitate with pre-cooled PBS to obtain exosomes; add 150 μL of PBS for every 150 μg of exosomes.

6. A composition for treating testicular atrophy according to claim 4 or 5, characterized in that The extracted exosomes were frozen and stored at -80°C and revived before use.

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