Preparation method and application of testis repair factor preparation

Abstract

The invention discloses a preparation method and application of a testis repair factor preparation, relates to the field of biological preparations, and aims to solve the problem that existing mesenchymal stem cells have a poor treatment effect on testis aging. The method comprises the following steps: 1, taking P1-generation umbilical cord mesenchymal stem cells, and culturing by adopting a special culture medium; 2, culturing the culture solution supernatant obtained after the culture in the step 1 in a Graviite microgravity simulation control system, then transferring the culture solution supernatant into an ultrafiltration tube, and centrifuging for ultrafiltration concentration to obtain a cell culture supernatant concentrated solution; and 3, adding normal saline into the cell culture supernatant concentrate obtained in the step 2 to prepare the testis repair factor preparation. The repair factor preparation prepared by the method has a good protection effect on testis intermediate cells and supporting cells, and can recover testosterone secretion and improve testis hypofunction by improving testis oxidative stress and immunoregulation.

Description

Technical Field

[0001] The present invention relates to the field of biological agents, and particularly to a preparation method and application of a testicular repair factor preparation. Background Art

[0002] Aging has attracted much attention from researchers due to its universality, progressiveness, and irreversibility, and has always been a research hotspot. Aging is a complex phenomenon, including factors such as development, genetic defects, environment, diseases, and genetics. The accumulation of these factors will increase the risk of diseases. Androgen deficiency is a common manifestation of male aging, affecting 25% of elderly men, and more than 10% of them show clinical symptoms of hypogonadism. Normal spermatogenesis and sperm function are the keys to male fertility. The impact of healthy testicular aging and premature testicular aging on spermatogenesis, sperm function, and the spermatogenic microenvironment cannot be ignored. Compared with young men, the testes of elderly men are prone to problems such as disordered spermatogenesis, sperm malformation, sperm dysfunction, and damage to Sertoli cells and Leydig cells, ultimately leading to male infertility.

[0003] Since mesenchymal stem cells have the potential for multi-directional differentiation, they can differentiate into various mesenchymal cells or non-mesenchymal cells and have a unique cytokine secretion function. Therefore, mesenchymal stem cells are currently used for the treatment of testicular aging and testicular atrophy. However, the current mesenchymal stem cells have an unsatisfactory treatment effect on testicular aging. Summary of the Invention

[0004] The present invention aims to solve the problem that the current mesenchymal stem cells have a poor treatment effect on testicular aging, and provides a preparation method and application of a testicular repair factor preparation.

[0005] The present invention provides a preparation method of a testicular repair factor preparation, including the following steps:

[0006] I. Take passage 1 umbilical cord mesenchymal stem cells and culture them in a special culture medium; the formula of the special culture medium is: MEM nano-bubble culture medium + 10 - 60 μM artemisinin + 5% Helios serum substitute + 2 IU / mL sodium heparin + 1% penicillin-streptomycin;

[0007] II. Take the culture supernatant obtained after culturing in step I, place it in a Gravite microgravity simulation control system for 15 - 20 minutes, then transfer it to an ultrafiltration tube and centrifuge for ultrafiltration concentration to obtain a concentrated cell culture supernatant;

[0008] III. Add physiological saline to the concentrated cell culture supernatant obtained in step II to prepare a testicular repair factor preparation.

[0009] Further, the preparation method of the MEM nano-bubble culture medium in Step 1 is as follows: First, add distilled water to the MEM powder to obtain an MEM solution, filter and sterilize the MEM solution, and then add hydrogen nano-bubble water (H2-NBW).

[0010] Further, the mass-volume ratio of the MEM powder to the distilled water is 1.905 g: 100 mL, and the volume ratio of the MEM solution to the hydrogen nano-bubble water is 1:1.

[0011] Further, the parameter setting of the Gravite microgravity simulation control system in Step 2 is 10 -3 G.

[0012] Further, the centrifugation speed in Step 2 is 4000×g, and the centrifugation time is 10 - 15 min.

[0013] Further, the volume ratio of the cell culture supernatant concentrate to the physiological saline in Step 3 is 1: (10 - 20).

[0014] The present invention provides the application of the testicular repair factor preparation in the preparation of a drug for protecting testicular interstitial cells.

[0015] The present invention provides the application of the testicular repair factor preparation in the preparation of a drug for protecting testicular Sertoli cells.

[0016] The present invention provides the application of the testicular repair factor preparation in the preparation of a drug for improving testicular hypofunction.

[0017] The present invention provides the application of the testicular repair factor preparation in the preparation of a drug for protecting the structural integrity of the blood-testis barrier.

[0018] The beneficial effects of the present invention:

[0019] During the culture process of MSCs in the present invention, adding artemisinin and hydrogen nano-bubbles to treat the cells can promote the cells to secrete abundant testicular repair factors into the culture supernatant, play a good protective role on testicular interstitial cells and Sertoli cells, and restore testosterone secretion and improve testicular hypofunction by improving testicular oxidative stress and immune regulation.

[0020] The testicular repair factor preparation of the present invention is prepared from the culture supernatant rich in various factors released by MSCs treated with artemisinin and hydrogen nanobubbles. The active effects of artemisinin and hydrogen nanobubbles cause specific changes in MSCs, releasing a large number of functional cytokines. After further activation in a microgravity environment, testicular repair factors with specific improvement of testicular function are obtained. By acting on Leydig cells and Sertoli cells, it has a good protective effect, improves testicular oxidative stress and blood-testis barrier immune regulation on testicular germ cells, slows down testicular function decline, realizes testicular reverse aging, and provides new ideas for the repair of male gonadal function diseases such as testicular aging and dysfunction. Description of the Drawings

[0021] Figure 1 is the percentage of SA-β-gal positive cells in TM3 (per 500 cells);

[0022] Figure 2 is the protein expression of Nrf2 in TM3;

[0023] Figure 3 is the protein expression of HO-1 in TM3;

[0024] Figure 4 is the protein expression of SOD in TM3;

[0025] Figure 5 is the percentage of SA-β-gal positive cells in TM4 (per 500 cells);

[0026] Figure 6 is the relative protein expression level of GDNF in TM4;

[0027] Figure 7 is the relative protein expression level of PLZF in TM4;

[0028] Figure 8 is the relative protein expression level of BMP4 in TM4;

[0029] Figure 9 is the relative protein expression level of SCF in TM4;

[0030] Figure 10 is the change in the level of SOD in testicular tissue;

[0031] Figure 11 is the change in the level of 8-OHdG in testicular tissue;

[0032] Figure 12 is the protein expression of Nrf2 in testicular tissue;

[0033] Figure 13 is the protein expression of HO-1 in testicular tissue;

[0034] Figure 14 Protein expression of SOD in testicular tissue;

[0035] Figure 15 Detection of the specific marker protein WT1 of Sertoli cells in the testis by immunofluorescence technique;

[0036] Figure 16 Change in the number of Sertoli cells in testicular tissue;

[0037] Figure 17 Relative protein expression level of GDNF in testicular tissue;

[0038] Figure 18 Relative protein expression level of PLZF in testicular tissue;

[0039] Figure 19 Relative protein expression level of BMP4 in testicular tissue;

[0040] Figure 20 Relative protein expression level of SCF in testicular tissue;

[0041] Figure 21 Relative protein expression level of Occludin in testicular tissue;

[0042] Figure 22 Relative protein expression level of β-Catenin in testicular tissue. Detailed implementation mode

[0043] The following is a detailed description of the embodiments of the present invention. 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. However, the protection scope of the present invention is not limited to the following embodiments.

[0044] Example 1:

[0045] The preparation method of the testicular repair factor preparation in this example includes the following steps:

[0046] I. Isolate Wharton's jelly from umbilical cord tissue, and obtain umbilical cord mesenchymal stem cells (UMSCs) by the method of cell culture on slides.

[0047] Take the P1 generation of umbilical cord mesenchymal stem cells and culture them in a culture medium 1 in an incubator at 37°C and 5% CO2; the formula of the culture medium 1 is: MEM nano-bubble medium + 30 μM artemisinin + 5% Helios serum substitute + 2 IU / mL heparin sodium + 1% penicillin-streptomycin;

[0048] The preparation method of the MEM nanobubble culture medium is as follows: First, add 500 mL of distilled water to 9.526 g of MEM powder, adjust the pH to 7.0 to obtain a MEM solution, filter and sterilize the MEM solution with a 0.2 μm filter, and then add 500 mL of hydrogen nanobubble water (H2-NBW) to obtain the MEM nanobubble culture medium;

[0049] II. Take the culture supernatant obtained in Step I, place it in a Gravite microgravity simulation control system for culturing for 15 min, set the parameters to 10 -3 G, and then transfer it to a 50 mL 100KD ultrafiltration tube, centrifuge at 4000×g for 10 min for ultrafiltration concentration to obtain a concentrated cell culture supernatant;

[0050] III. Add normal saline to the concentrated cell culture supernatant obtained in Step II for dilution, and the volume ratio of the concentrated solution to normal saline is 1:20 to prepare a testicular repair factor preparation.

[0051] Example 2:

[0052] I. Isolate Wharton's jelly from umbilical cord tissue and obtain umbilical cord mesenchymal stem cells (UMSCs) by the cover slip method.

[0053] Take the P1 generation of umbilical cord mesenchymal stem cells and culture them in a 37°C, 5% CO2 incubator using Medium 2; the formula of Medium 2 is: MEM nanobubble culture medium + 5% Helios serum substitute + 2 IU / mL heparin sodium + 1% penicillin-streptomycin;

[0054] The preparation method of the MEM nanobubble culture medium is as follows: First, add 500 mL of distilled water to 9.526 g of MEM powder, adjust the pH to 7.0 to obtain a MEM solution, filter and sterilize the MEM solution with a 0.2 μm filter, and then add 500 mL of hydrogen nanobubble water (H2-NBW) to obtain the MEM nanobubble culture medium;

[0055] II. Take the culture supernatant obtained in Step I, place it in a Gravite microgravity simulation control system for culturing for 15 min, set the parameters to 10 -3 G, and then transfer it to a 50 mL 100KD ultrafiltration tube, centrifuge at 4000×g for 10 min for ultrafiltration concentration to obtain a concentrated cell culture supernatant;

[0056] III. Add normal saline to the concentrated cell culture supernatant obtained in Step II for dilution, and the volume ratio of the concentrated solution to normal saline is 1:20 to prepare a testicular repair factor preparation.

[0057] Example 3:

[0058] 1. Isolate Wharton's jelly from umbilical cord tissue and obtain umbilical cord mesenchymal stem cells (UMSCs) by the method of cell culture on coverslips.

[0059] Take passage 1 UMSCs and culture them in medium 3 in an incubator at 37°C with 5% CO2. The formula of medium 3 is: MEM basal medium + 30 μM artemisinin + 5% Helios serum substitute + 2 IU / mL heparin sodium + 1% penicillin-streptomycin.

[0060] 2. Take the supernatant of the culture solution obtained in step 1 and culture it in a Gravite microgravity simulation control system for 15 min with the parameter set to 10 -3 G, then transfer it to a 50 mL 100 KD ultrafiltration tube and centrifuge at 4000×g for 10 min for ultrafiltration concentration to obtain a concentrated cell culture supernatant.

[0061] 3. Add normal saline to the concentrated cell culture supernatant obtained in step 2 for dilution, with the volume ratio of the concentrated solution to normal saline being 1:20, to prepare a testicular repair factor preparation.

[0062] Example 4:

[0063] 1. Isolate Wharton's jelly from umbilical cord tissue and obtain umbilical cord mesenchymal stem cells (UMSCs) by the method of cell culture on coverslips.

[0064] Take passage 1 UMSCs and culture them in medium 4 in an incubator at 37°C with 5% CO2. The formula of medium 4 is: MEM basal medium + 5% Helios serum substitute + 2 IU / mL heparin sodium + 1% penicillin-streptomycin.

[0065] 2. Take the supernatant of the culture solution obtained in step 1 and culture it in a Gravite microgravity simulation control system for 15 min with the parameter set to 10 -3 G, then transfer it to a 50 mL 100 KD ultrafiltration tube and centrifuge at 4000×g for 10 min for ultrafiltration concentration to obtain a concentrated cell culture supernatant.

[0066] 3. Add normal saline to the concentrated cell culture supernatant obtained in step 2 for dilution, with the volume ratio of the concentrated solution to normal saline being 1:20, to prepare a preparation.

[0067] Use ELISA to detect the cytokine content in the cell culture solutions obtained in step 1 of Examples 1 to 4 respectively. The cytokine secretion conditions are shown in Table 1. Through comparison, it can be seen that the content of each cytokine in the medium 1 group in Example 1 is higher, indicating that UMSCs specially treated with the addition of H2-NBW and artemisinin can secrete more testicular repair-related cytokines.

[0068] Table 1 Changes in the content of cytokines related to testicular repair secreted by cultured UMSCs

[0069]

[0070] Example 5:

[0071] I. Wharton's jelly was isolated from umbilical cord tissue, and umbilical cord mesenchymal stem cells (UMSCs) were obtained by the method of cell spreading on slides.

[0072] The passage 1 umbilical cord mesenchymal stem cells were cultured in medium 1 in an incubator at 37°C and 5% CO2; the formula of medium 1 was: MEM nano-bubble medium + 30 μM artemisinin + 5% Helios serum substitute + 2 IU / mL heparin sodium + 1% penicillin-streptomycin;

[0073] The preparation method of the MEM nano-bubble medium was as follows: first, 500 mL of distilled water was added to 9.526 g of MEM powder, and the pH was adjusted to 7.0 to obtain a MEM solution. The MEM solution was filtered and sterilized with a 0.2 μm filter, and then 500 mL of hydrogen nano-bubble water (H2-NBW) was added to obtain the MEM nano-bubble medium;

[0074] II. The supernatant of the culture solution obtained in step I was transferred to a 50 mL 100 KD ultrafiltration tube and centrifuged at 4000×g for 10 min for ultrafiltration and concentration to obtain a concentrated solution (CCS);

[0075] III. The concentrated solution obtained in step II was diluted with physiological saline, and the volume ratio of the concentrated solution to physiological saline was 1:20 to prepare a CCS preparation.

[0076] The cytokine content of the testicular repair factor preparation prepared in Example 1 and the CCS preparation prepared in Example 5 was detected by ELISA respectively, and the detection results are shown in Table 2. Through comparison, it can be seen that the cytokine content of Example 1 after microgravity treatment is the highest, indicating that microgravity treatment can further activate UMSCs and promote the specific release of factors related to testicular repair.

[0077] Table 2 Changes in the content of cytokines related to testicular repair in UMSCs preparation after microgravity treatment

[0078]

[0079] Example 6: Observation of in vivo effects

[0080] 1. Effects of testicular repair factor preparation on Leydig cells

[0081] Mouse Leydig cell line (TM3) was divided into four groups for culture:

[0082] ① TM3 group: TM3 cells were cultured conventionally using TM3 cell medium and cultured at 37 °C and 5% CO2 until 80% of the cells adhered to the wall, serving as the control group.

[0083] The TM3 cell medium was DMEM / F - 12 medium containing 5% FBS (by volume) and 2.5% HS (by volume).

[0084] ② D - galactose group: TM3 cells were cultured conventionally using TM3 cell medium and cultured at 37 °C and 5% CO2; then TM3 cells (1×10 6 cells / well) were seeded on a 6 - well cell culture plate containing TM3 cell medium and incubated for 24 hours, and then replaced with TM3 cell medium containing 20 mg / mL D - galactose and cultured for 48 hours. An aging model was established.

[0085] ③ D - galactose + CCS group: TM3 cells were cultured with the CCS preparation of Example 5 using TM3 cell medium added with the CCS preparation and cultured at 37 °C and 5% CO2; then TM3 cells (1×10 6 cells / well) were seeded on a 6 - well cell culture plate containing TM3 cell medium added with the CCS preparation and incubated for 24 hours, and then replaced with TM3 cell medium added with the CCS preparation containing 20 mg / mL D - galactose and cultured for 48 hours.

[0086] The TM3 cell medium added with the CCS preparation was DMEM / F - 12 medium containing 1% CCS (by volume), 5% FBS (by volume) and 2.5% HS (by volume).

[0087] ④ D - galactose + AF group: TM3 cells were cultured with the testicular repair factor preparation (AF) of Example 1 using TM3 cell medium added with the AF preparation and cultured at 37 °C and 5% CO2; then TM3 cells (1×10 6 cells / well) were seeded on a 6 - well cell culture plate containing TM3 cell medium added with the AF preparation and incubated for 24 hours, and then replaced with TM3 cell medium added with the AF preparation containing 20 mg / mL D - galactose and cultured for 48 hours.

[0088] The TM3 cell medium added with the AF preparation was DMEM / F - 12 medium containing 1% AF (by volume), 5% FBS (by volume) and 2.5% HS (by volume).

[0089] Collect the proteins and culture medium supernatants of the above 4 groups of cells, detect the senescence of cells by β-galactosidase (SA-β-gal) kit, detect the expressions of β-actin, Nrf2, HO-1 and SOD by Western blot, and detect the expression level of VEGF by ELISA kit. Evaluate the protective effect of the testicular repair factor preparation (AF) of the present invention on oxidative stress after treating interstitial cells with senescence.

[0090] Interstitial cells are the main source of male testosterone or androgen, and play a crucial role in many important physiological processes of men, including sperm production or spermatogenesis, controlling sexual development, and maintaining secondary sexual characteristics and behaviors. The establishment and maintenance of the male testicular microenvironment both depend on interstitial cells, which are the main sites for androgen synthesis and secretion. Therefore, interstitial cell damage is related to the decline of fertility and erectile function, and is an important factor in the occurrence of testicular dysfunction. Oxidative stress is a potential cause of interstitial cell damage, leading to a decrease in testosterone levels, thereby impairing fertility. Therefore, inhibiting oxidative stress can prevent interstitial cell damage, thus avoiding the occurrence and development of testicular failure.

[0091] The aging process is accompanied by a large accumulation of testicular ROS, which causes oxidative stress and further exacerbates reproductive aging. β-galactosidase (SA-β-gal) is a widely used cell senescence marker. Since the lysosomal content in senescent cells usually increases, the activity of the lysosomal enzyme β-galactosidase increases. By detecting the activity level of SA-β-Gal in cells, the degree of cell senescence can be evaluated. The percentage of SA-β-gal positive cells (per 500 cells) in TM3 is as Figure 1 shown. The results show that compared with the D-galactose group and the D-galactose + CCS group, the percentage of SA-β-gal positive cells in the D-galactose + AF group is significantly down-regulated, indicating that the testicular repair factor preparation of the present invention can inhibit the cell senescence caused by treating TM3 with D-galactose.

[0092] The transcription factor Nrf2 is a redox-sensitive transcription factor. It regulates the expression of HO-1 and confers cell protection against oxidative stress. The activation of the Nrf2 / HO-1 pathway can lead to an increase in the SOD level. It has been found that the expression level of Nrf2 / HO-1 in TM3 cells is significantly decreased after oxidative stress. The protein expression results of Nrf2, HO-1 and SOD in TM3 are as Figures 2 - 4 shown. The results show that compared with normal TM3 cells, the expressions of Nrf2, HO-1 and SOD in the D-galactose group cells are significantly decreased after D-galactose treatment, while the expression levels in the D-galactose + AF group are significantly increased, indicating that the testicular repair factor preparation can inhibit the oxidative stress of senescent TM3.

[0093] 2. Effects of testicular repair factor preparation on Sertoli cells

[0094] The mouse Sertoli cell line (TM4) was divided into four groups for culture:

[0095] ① TM4 group: TM4 cells were cultured conventionally using TM4 cell medium and cultured at 37 °C and 5% CO2 until 80% of the cells adhered to the wall, serving as the control group.

[0096] The TM4 cell medium was DMEM / F-12 medium containing 5% FBS (by volume) and 2.5% HS (by volume).

[0097] ② D-galactose group: TM4 cells were cultured conventionally using TM4 cell medium and cultured at 37 °C and 5% CO2; then TM4 cells (5×10 5 cells / well) were seeded on a 6-well cell culture plate containing TM4 cell medium and incubated for 24 hours, and then replaced with TM4 cell medium containing 20 mg / mL D-galactose and cultured for 48 hours. An aging model was established.

[0098] ③ D-galactose + CCS group: TM4 cells were cultured with the CCS preparation of Example 5 using TM3 cell medium supplemented with the CCS preparation and cultured at 37 °C and 5% CO2; then TM4 cells (5×10 5 cells / well) were seeded on a 6-well cell culture plate containing TM4 cell medium supplemented with the CCS preparation and incubated for 24 hours, and then replaced with TM4 cell medium containing 20 mg / mL D-galactose and supplemented with the CCS preparation and cultured for 48 hours.

[0099] The TM4 cell medium supplemented with the CCS preparation was DMEM / F-12 medium containing 1% CCS (by volume), 5% FBS (by volume), and 2.5% HS (by volume).

[0100] ④ D-galactose + AF group: TM4 cells were cultured with the testicular repair factor preparation (AF) of Example 1 using TM4 cell medium supplemented with the AF preparation and cultured at 37 °C and 5% CO2; then TM4 cells (5×10 5 cells / well) were seeded on a 6-well cell culture plate containing TM4 cell medium supplemented with the AF preparation and incubated for 24 hours, and then replaced with TM4 cell medium containing 20 mg / mL D-galactose and supplemented with the AF preparation and cultured for 48 hours.

[0101] The TM4 cell medium supplemented with the AF preparation was DMEM / F-12 medium containing 1% AF (by volume), 5% FBS (by volume), and 2.5% HS (by volume).

[0102] Collect the proteins and culture supernatants of the above 4 groups of cells, and detect the senescence of cells by β-galactosidase kit, and detect the protein expression levels of GDNF, PLZF, BMP4 and SCF by Western blot.

[0103] Sertoli cells are key somatic cells in the testis. It promotes testicular formation and spermatogenesis by forming the blood-testis barrier and producing various cytokines, including glial cell line-derived neurotrophic factor (GDNF), promyelocytic leukemia zinc finger protein (PLZF), bone morphogenetic protein 4 (BMP4) and stem cell factor (SCF). These factors promote the self-renewal and differentiation of spermatogonial stem cells (SSCs) to ensure the development of spermatogonia into mature sperm. The blood-testis barrier plays a role in preventing autoimmune reactions and blocking harmful substances from entering the seminiferous tubules during spermatogenesis, providing a special stable internal environment for the spermatogenic process. Some studies have found that the damage of the blood-testis barrier is directly related to testicular senescence. During the aging process, the expressions of the junction proteins Occludin and β-Catenin in the rat testis decrease, and the integrity of the blood-testis barrier is damaged, resulting in reduced spermatogenic function. There are also clinical studies showing that with age, the morphology of Sertoli cells changes abnormally, accompanied by a significant decrease in the level of secreted proteins of Sertoli cells, loss of germ cells, and increased apoptosis of germ cells. The decline of Sertoli cell function is an important cause of reproductive dysfunction in the aging testis. Therefore, protecting Sertoli cells from damage during the aging process is the key to treating testicular dysfunction.

[0104] The percentage of SA-β-gal positive cells (per 500 cells) in TM4 was as Figure 5 shown. The results showed that compared with the D-galactose group and the D-galactose + CCS group, the percentage of SA-β-gal positive cells in the D-galactose + AF group was significantly down-regulated, indicating that the testicular repair factor preparation of the present invention can inhibit the cell senescence caused by D-galactose-treated TM4.

[0105] The relative protein expression levels of GDNF, PLZF, BMP4, and SCF in TM4 were as Figures 6 - 9 shown. The results showed that compared with the TM4 control group, the expressions of GDNF, PLZF, BMP4, and SCF in the cells of the D-galactose group were significantly decreased after D-galactose treatment, while the expression levels in the D-galactose + AF group were significantly increased, indicating that the testicular repair factor preparation of the present invention can significantly improve the function of TM4 cells after D-galactose treatment and protect Sertoli cells from damage caused by senescence.

[0106] Example 7: Observation on the therapeutic effect of the senile testicular hypofunction model in mice

[0107] The mice were intraperitoneally injected with 100 mg / kg of D-galactose once a day for 30 days to establish an aging model of mice, and the mice were grouped as follows.

[0108] The first group was the healthy control group (Control), which consisted of healthy mice and was injected with normal saline during the treatment phase;

[0109] The second group was the model control group (D-galactose), which consisted of aging model mice and was injected with normal saline during the treatment phase;

[0110] The third group was the CCS preparation treatment group (CCS), which consisted of aging model mice and was injected with the CCS preparation of Example 5 during the treatment phase;

[0111] The fourth group was the testicular repair factor preparation treatment group (AF), which consisted of aging model mice and was injected with the testicular repair factor preparation of Example 1 during the treatment phase.

[0112] During the treatment phase, the injection dose, time, and frequency of the four groups of mice were unified. The injection was administered into the testicular parenchyma of the mice (50 μL / side), once every 7 days, for 3 treatments, which was considered one course of treatment. The mice were sacrificed 7 days after the last treatment for whole blood collection and bilateral testicular sampling. The collected whole blood was centrifuged to separate the serum, and the serum testosterone expression level was detected by ELISA. The fresh testes were weighed and then stored frozen at -80 °C for nucleic acid and protein detection to compare the treatment effects.

[0113] A model of testicular function decline caused by mouse aging was established. After treatment, the testicular weight and serum testosterone level were measured. As shown in Table 3, the testicular weight and serum testosterone concentration in the model control group decreased significantly, indicating that aging can lead to testicular dysfunction. Compared with the model control group and the CCS preparation treatment group, the testicular weight and serum testosterone concentration in the testicular repair factor preparation treatment group increased significantly, indicating that the testicular repair factor preparation can regulate the function of Leydig cells, restore testosterone secretion, and thus improve testicular function.

[0114] Table 3 Changes in testicular parameters

[0115]

[0116]

[0117] The levels of SOD and the oxidative damage marker 8-OHdG in testicular tissues were detected using an ELISA kit, and the results were as Figure 10 and Figure 11As shown, the expression level of SOD in the testicular dysfunction rats of the model control group decreased significantly, while the expression level of 8-OHdG increased significantly. Compared with the model control group and the CCS preparation treatment group, the expression level of SOD in the testicular repair factor preparation treatment group was significantly up-regulated, and the expression level of 8-OHdG was significantly down-regulated. The protein expression levels of Nrf2, HO-1 and SOD were detected by Western blot technology, and the results were as Figures 12 - 14 shown. Compared with the control group, the expression levels of Nrf2, HO-1 and SOD in the testicular dysfunction rats of the model control group decreased significantly, while the expression levels of Nrf2, HO-1 and SOD in the testicular repair factor preparation treatment group increased significantly, indicating that the testicular repair factor preparation can improve testicular oxidative stress caused by aging.

[0118] The positive expression of the testicular Sertoli cell-specific marker protein WT1 was detected by immunofluorescence technology to detect the number of Sertoli cells in the rat testis. The results were as Figure 15 and Figure 16 shown. Compared with the healthy control group, the number of Sertoli cells in each cross-section of the seminiferous tubules in the model control group decreased significantly. In contrast, the number of Sertoli cells in the testicular repair factor preparation treatment group increased significantly. The protein expression levels of GDNF, PLZF, BMP4, SCF, Occludin and β-Catenin in the testicular tissue were detected by Western blot, as Figures 17 - 20 shown. The results showed that the relative protein expression levels of GDNF, PLZF, BMP4 and SCF in the testicular repair factor preparation treatment group increased significantly, indicating the protective effect of the testicular repair factor preparation on testicular Sertoli cells and its ability to restore Sertoli cell proliferation. Figure 21 and Figure 22 showed the expression levels of blood-testis barrier-related junction proteins in the testicular tissue. The results showed that the protein expression levels of Occludin and β-Catenin in the model control group decreased significantly, while the expression levels of the two proteins in the testicular repair factor preparation treatment group increased significantly, indicating that the testicular repair factor preparation has a certain protective effect on the structural integrity of the blood-testis barrier.

[0119] In summary, the above results indicate that the testicular repair factor preparation can protect interstitial cells from damage by regulating oxidative stress and restore testosterone secretion; by repairing the function of testicular Sertoli cells, promoting the production of cytokines, improving the damaged blood-testis barrier and reproductive function, thereby slowing down testicular function decline and achieving testicular reverse aging.

Claims

1. A preparation method of a testicular repair factor preparation, characterized in that The method comprises the following steps:

1. Take P1 cord mesenchymal stem cells and culture them in a special culture medium; the formula of the special culture medium is: MEM nano-bubble medium + 10 - 60 μM artemisinin + 5% Helios serum substitute + 2 IU / mL sodium heparin + 1% penicillin-streptomycin; 2. Take the supernatant of the culture solution obtained in step 1 and culture it in a Gravite microgravity simulation control system for 15 - 20 min, then transfer it to an ultrafiltration tube and centrifuge for ultrafiltration concentration to obtain a concentrated cell culture supernatant; 3. Add physiological saline to the concentrated cell culture supernatant obtained in step 2 to prepare a testicular repair factor preparation.

2. The preparation method of a testicular repair factor preparation according to claim 1, wherein the preparation method of the MEM nano-bubble medium in step 1 is: first add distilled water to MEM powder to obtain a MEM solution, filter and sterilize the MEM solution, and then add hydrogen nano-bubble water.

3. The preparation method of a testicular repair factor preparation according to claim 2, characterized in that, The mass ratio of MEM powder to the volume of distilled water is 1.905 g:100 mL, and the volume ratio of the MEM solution to hydrogen nano-bubble water is 1:

1.

4. The preparation method of a testicular repair factor preparation according to claim 1, 2 or 3, characterized in that, In Step 2, the parameters of the Gravite microgravity simulation control system are set to 10 -3 G.

5. The preparation method of a testicular repair factor preparation according to claim 4, characterized in that, In step 2, the centrifugation speed is 4000×g and the centrifugation time is 10 - 15 min.

6. The preparation method of a testicular repair factor preparation according to claim 1, characterized in that, In step 3, the volume ratio of the concentrated cell culture supernatant to physiological saline is 1:(10 - 20).

7. Use of the testicular repair factor preparation prepared by the method according to any one of claims 1 to 6 in the preparation of a drug for protecting testicular interstitial cells.

8. Use of the testicular repair factor preparation prepared by the method according to any one of claims 1 to 6 in the preparation of a drug for protecting testicular Sertoli cells.

9. Use of the testicular repair factor preparation prepared by the method according to any one of claims 1 to 6 in the preparation of a drug for improving testicular hypofunction.

10. Use of the testicular repair factor preparation prepared by the method according to any one of claims 1 to 6 in the preparation of a drug for protecting the structural integrity of the blood-testis barrier.

Images