CN108524485A - A kind of monkey testosterone lacks the method for building up of model - Google Patents

Abstract

The present invention provides the method for building up that a kind of monkey testosterone lacks model, by the way of combining blocking spermatic cord blood flow to Adult Monkey testicular artery injection dimethyl sulfonic acid ethane.The present invention provides safe and stable testosterones to lack animal model.

Description

A kind of establishment method of monkey testosterone deficiency model

technical field

The invention belongs to the field of animal models, and in particular relates to a method for establishing a monkey testosterone deficiency model.

Background technique

Testosterone deficiency syndrome (TDS) refers to the lack of testosterone levels in the body due to various reasons in different periods of a man's life, resulting in abnormal morphology and function of his target organs, which in turn causes corresponding clinical symptoms. It is a male reproductive endocrine disease that seriously affects male health, life expectancy and quality of life. TDS mainly includes late-onset hypogonadism (late-onset hypogonadism (LOH)), congenital hypogonadism, testicular damage and other diseases. With the aging process of Chinese society, the incidence of TDS is increasing year by year, and the medical market demand is very huge. It has become a major disease that seriously affects the physical and mental health of our people. At present, the clinical treatment of TDS mainly uses exogenous testosterone drug supplement therapy, which supplements exogenous testosterone through oral administration, injection, etc. Although it has a certain curative effect, due to obvious defects such as inconvenient use and large side effects, there are only a few at present. A small number of TDS patients receive this therapy. Therefore, exploring a new method of treating TDS has become a key scientific and technological problem that needs to be solved urgently in the social development of our country.

Currently exogenous testosterone drug supplementation therapy is a common treatment for TDS. Studies have confirmed that patients can benefit from testosterone replacement therapy, such as increased libido, increased bone density, improved mood and cognition, enhanced physical fitness, and more. However, there are obvious defects in exogenous testosterone drug supplement therapy: (1) Due to individual differences in the level of testosterone decline in patients, it is difficult to control the dose of testosterone supplementation, and it is difficult to be effective if the dose is too small, and side effects and complications are prone to occur if the dose is too large (2) will cause the testosterone level in the body to lose the original circadian rhythm changes, causing multiple complications; (3) inconvenient to use, need long-term frequent medication; (4) use of exogenous testosterone drugs will inhibit the growth of testis Sperm function, long-term use can cause infertility such as oligospermia or azoospermia. Due to the above defects, only a small number of TDS patients are currently receiving exogenous testosterone drugs. There is an urgent need to explore a new approach to treat TDS. More than 95% of testosterone in the body is synthesized and secreted by LCs (Leydig cells, Leydig cells). The reduction in the number or function of Leydig cells is considered to be the core pathogenesis of TDS, which suggests that cell transplantation therapy may achieve better results . The development of cell transplantation therapy depends on the development of TDS models, so the study of TDS models has become a key issue in the field of new methods of TDS treatment.

Currently, there are two TDS models, one is the elderly delayed-onset hypogonadism model, and the other is the ethylene-1,2-dimethyl sulphonate (EDS) injury model; due to the high cost of elderly animals, Large individual differences, difficult to obtain and many other shortcomings, difficult to apply. EDS is a cytotoxic alkylating agent that selectively kills LCs in rats and other species, but has no effect on the proliferation of spermatogenic cells. After a single intraperitoneal injection of 75mg/kg body weight of EDS in rats, it can induce apoptosis and clear the mature LCs in the interstitium of rat testis. LCs nuclear pyknosis and nuclear fragmentation gradually appeared within 6-18 hours after EDS injection, most LCs appeared apoptosis after 24 hours, and all LCs were cleared after 72 hours. Newborn LCs can be found in the interstitium of the testes in about 2 to 3 weeks, and the number of LCs returns to the level before administration in about 8 to 10 weeks. Serum LH gradually increased within 14 days of EDS injection, and serum LH began to decrease within 14 to 21 days due to the secretion of testosterone by newborn LC, and serum LH decreased to the level before injection after 21 days. Within 2-8 weeks after EDS injection, the rats lost their reproductive function. Other studies have shown that the weight of the reproductive organs, including the testes, epididymis, vas deferens, and seminal vesicles, is also significantly reduced due to a significant drop in testosterone levels in the body following EDS injections. Therefore, it is an ideal model for studying TDS cell transplantation therapy.

However, although rodents such as rats and mice are widely used in scientific research, there are many differences from humans. Cynomolgus monkeys are primates, which are similar to humans in terms of anatomy, physiological functions, and biochemical metabolism. They are ideal experimental animals for understanding human physiology and pathophysiology, and are also the most important models for clinical translational research. In order to promote the clinical translation of human SLCs in the treatment of TDS and evaluate the safety and efficacy of human SLCs in the treatment of TDS, the data of non-human primate models are indispensable, so the establishment of a primate TDS model is crucial. However, non-human primate models are very expensive. How to ensure efficient and safe establishment of animal models is an important basis for the clinical transformation of human SLCs in the treatment of TDS. The key to establishing the cynomolgus monkey model is the use of EDS. Since the dose of EDS is closely related to the occurrence of liver toxicity, it is best to find the best EDS that can induce a stable TDS model in non-human primates and ensure a high survival rate of animals. Dosage and mode of use are very important to research. There is no domestic report on the establishment of TDS model in cynomolgus monkeys, and there is only one report abroad (Ethane dimethylsulphonate selectively destroys Leydig cells in the adultbonnet monkeys (Macaca radiata)). The animal used in the literature is a 7-8-year-old crested macaque weighing 6-8 kg. By injecting 5, 10, 20, and 50 mg of EDS into the bilateral testes, the author found that the testosterone levels in each group decreased significantly in the first four days, and the testosterone level in the 5mg/testis group dropped to 62% on the fourth day after treatment, and in 45 days after EDS, it returned to the pre-administration level. However, in primate testes, there are many connective tissue septa emanating from the testicular mediastinum, which divide the testicular parenchyma into many cone-shaped testicular lobules, so local injection is difficult to ensure the uniform distribution of EDS and the effect of modeling. In addition, EDS has obvious hepatotoxicity, and it is also very important to choose the appropriate dosage and administration method for modeling and evaluate the drug damage after modeling, but there are no related reports at home and abroad so far. Therefore, replicating a stable TDS model is of great significance for studying the effectiveness and safety of SLCs in treating TDS.

Contents of the invention

The purpose of the present invention is to provide a safe and stable testosterone-deficient animal model aiming at the deficiency of the existing non-human primate TDS model, and to provide a good basis for studying the effectiveness and safety of human SLCs in treating TDS.

In order to achieve the above object, the present invention provides a method for establishing a monkey testosterone deficiency model, which adopts the method of injecting ethylene dimethylsulfonate (EDS) into the testicular artery of an adult monkey and blocking the blood flow of the spermatic cord.

According to the method of the present invention, the amount of dimethylsulfonate injected is at least 50 mg/testis. Preferably, the injection amount of ethane dimethylsulfonate is 50-200 mg/testis. Preferably, the injection amount of ethane dimethylsulfonate is 50-100 mg/testis. Preferably, the injection amount of ethane dimethylsulfonate is 100-200 mg/testis. According to the method of the present invention, the specific steps are: after the adult cynomolgus monkey is anesthetized, disinfect its lower abdomen and perineum, expose the bilateral spermatic cords by using an inguinal inner ring incision, and block the blood flow of the spermatic cords for 20-30 minutes with non-invasive forceps. Minutes; the testis was exposed through a scrotal incision, and dimethylsulfonate was injected into the testicular artery.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, and 45 days after EDS injection, blood was collected from the upper limb vein at 9:00 a.m. to 10:00 a.m. 1-2ml blood at a time, let stand at room temperature for 90 minutes, centrifuge at 2000g for 15 minutes, separate serum, store at -20°C, and use to test testosterone and liver function.

4, 14, 28, and 45 days after EDS injection, testicular puncture was performed to obtain testicular tissue, which was fixed with 4% paraformaldehyde, dehydrated in 10%, 20%, and 30% sucrose solution until the tissue sank to the bottom, and was packed with OCT. Buried, 6 μm thick sections were made in a cryostat, with 5 samples per time point. Immunofluorescence staining for Leydig cell-specific protein CYP11A1.

Preferably, the injection time of ethanedimethylsulfonate into the testicular artery is 1-5 minutes.

According to the method of the invention, said adult monkey is at least 6 years old.

The method of the present invention selects adult monkeys (preferably cynomolgus monkeys), adopts the method of injecting EDS into the testicular artery and blocking the blood flow of the spermatic cord, and establishes a safe and stable monkey TDS model. Through continuous and long-term observation of indicators such as the liver, it has been confirmed that the method can effectively remove LCs in the testes of adult male monkeys, and a monkey TDS model has been established; in addition, the method of the present invention reduces the toxic side effects such as liver damage caused by EDS, thereby preventing the onset of TDS Mechanism studies, pharmacodynamic studies and other translational preclinical studies provide stable model carriers.

Description of drawings

Figure 1 shows the effect of injecting EDS into the testicular artery combined with blocking the blood flow of the spermatic cord.

Figure 2 is a comparison of the effects of local injection of EDS into the testis of cynomolgus monkeys and injection of EDS into the testicular artery combined with blocking the blood flow of the spermatic cord.

Figure 3 is a comparison of the effects of injecting EDS into the testicular artery of cynomolgus monkeys and injecting EDS into the testicular artery combined with blocking the blood flow of the spermatic cord.

Figure 4 is a testis staining diagram.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments, so as to facilitate understanding. But the protection scope of the present invention is not limited to the following examples.

Example 1: Injecting EDS into the testicular artery combined with blocking the blood flow of the spermatic cord, clearing the Leydig cells of the testis, and establishing a model of testosterone deficiency.

The animals used in this experiment were adult male cynomolgus monkeys, aged >6 years old, weighing 8-10 kg, a total of 8 adult male cynomolgus monkeys were randomly divided into 4 groups, 2 in each group.

In the first group (also called "vehicle group"), two cynomolgus monkeys were intra-arterially injected with vehicle (DMSO:H2O, 1:3, V/V) in both testes. EDS was dissolved in vehicle, and 2 cynomolgus monkeys in the second group were injected with 50 mg of EDS into the testicular artery; 2 cynomolgus monkeys in the third group were injected with 100 mg of EDS into the testicular artery; EDS.

On the day of the experiment, after cynomolgus monkeys were anesthetized, the lower abdomen and perineum were disinfected, and the bilateral spermatic cords were exposed by incisions made in the inner groin ring, and the blood flow of the spermatic cords was blocked by non-invasive forceps for 20-30 minutes; the testes were exposed by scrotal incisions, and corresponding doses of EDS Slowly inject into the testicular artery, the injection time is 1-5 minutes.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, and 45 days after EDS injection, blood was collected from the upper limb vein at 9:00 a.m. to 10:00 a.m. 1-2ml blood at a time, let stand at room temperature for 90 minutes, centrifuge at 2000g for 15 minutes, separate serum, store at -20°C, and use to test testosterone and liver function.

See Figure 1 for the results. Among them, A: On the 4th day after EDS, the testosterone level of the vehicle group was not significantly affected. Intra-arterial injection of EDS at 50, 100, and 200 mg/testis combined with blocking the blood flow of the spermatic cord could significantly reduce the serum testosterone level of cynomolgus monkeys. The testosterone levels in the 50mg/testis group dropped to 30% of the normal level, and the testosterone levels in the 100 and 200mg/testis groups were lower than the detection limit of 0.13ng/ml. The testosterone levels in the 50, 100, and 200 mg/testis groups returned to the pre-administration level 45 days after EDS injection. B, C: After EDS injection, ALT and AST, the indicators of liver damage in the vehicle group, did not change significantly, and were at normal levels; 1 day after EDS injection, ALT and AST increased in the 50, 100, and 200 mg/testis groups, and the levels increased with the dose The increase of increasing constantly increases, and recovers to the pre-administration level within 7 days.

4, 14, 28, and 45 days after EDS injection, testicular puncture was performed to obtain testicular tissue, which was fixed with 4% paraformaldehyde, dehydrated in 10%, 20%, and 30% sucrose solution until the tissue sank to the bottom, and was packed with OCT. Buried, 6 μm thick sections were made in a cryostat, with 5 samples per time point. Immunofluorescence staining for Leydig cell-specific protein CYP11A1.

Figure 4 is a testis staining diagram. Among them, A: the testis staining picture on the 4th day after EDS injection, the picture shows that the Leydig cells are all cleared; B: the testis staining picture on the 14th day after the EDS injection, the picture shows that the Leydig cells begin to recover; C: EDS The testis staining picture on the 28th day after injection, the picture shows that the number of Leydig cells recovered to about 50%; D: the testis staining picture on the 45th day after EDS injection, the picture shows that the number of Leydig cells returned to normal. Testicular sections showed that EDS only affected Leydig cells but not other cells.

Example 2: Comparison of the effects of local injection of EDS into the testis of cynomolgus monkeys and injection of EDS into the testicular artery combined with blocking the blood flow of the spermatic cord.

In addition, 4 adult male cynomolgus monkeys, aged >6 years old and weighing 8-10 kg were selected and randomly divided into 2 groups with 2 monkeys in each group.

On the day of the experiment, after the cynomolgus monkeys were anesthetized, the lower abdomen and perineum were disinfected, and 2 cynomolgus monkeys in the first group (also called "local group") were locally injected with 100 mg/testis EDS in bilateral testes; Combined group") Two cynomolgus monkeys were exposed to both sides of the spermatic cord by an incision in the inner groin ring, and the blood flow of the spermatic cord was blocked for 20-30 minutes with non-injured forceps; The injection time is 1-5 minutes.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, and 45 days after EDS injection, blood was collected from the upper limb vein at 9:00 a.m. to 10:00 a.m. 1-2ml blood at a time, let stand at room temperature for 90 minutes, centrifuge at 2000g for 15 minutes, separate serum, store at -20°C, and use to test testosterone and liver function.

See Figure 2 for the results. Among them, A: On the 4th day after EDS, the testosterone level of the testicular local injection of EDS group (local group) was not affected, and the serum testosterone level of cynomolgus monkeys in the testicular artery injection of EDS+blocking spermatic cord blood flow group (combined group) was lower than the detection level. The lower limit was 0.13ng/ml, and it returned to the level before administration 45 days after EDS. B, C: After EDS injection, ALT and AST, which reflect liver damage indicators in the local group, did not change significantly, and were at normal levels; 1 day after EDS injection, ALT and AST in the combination group increased slightly, and returned to the level of administration within 4 days front level.

Example 3: Comparison of the effects of injecting EDS into the testicular artery of cynomolgus monkeys and injecting EDS into the testicular artery combined with blocking the blood flow of the spermatic cord.

In addition, 4 adult male cynomolgus monkeys were selected, aged >6 years old, weighing 8-10kg, and the 4 adult male cynomolgus monkeys were randomly divided into 2 groups, 2 in each group.

On the day of the experiment, after the cynomolgus monkeys were anesthetized, the lower abdomen and perineum were disinfected, and 2 cynomolgus monkeys in the first group were injected with 100mg/testis EDS in the bilateral testicular artery; The incision was made to expose the bilateral spermatic cords, and the blood flow of the spermatic cords was blocked for 20-30 minutes with non-invasive forceps; the testis was exposed by an incision in the scrotum, and an equal amount of EDS was slowly injected into the testicular artery for 1-5 minutes.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, and 45 days after EDS injection, blood was collected from the upper limb vein at 9:00 a.m. to 10:00 a.m. 1-2ml blood at a time, let stand at room temperature for 90 minutes, centrifuge at 2000g for 15 minutes, separate serum, store at -20°C, and use to test testosterone and liver function.

The results are shown in Figure 3: A. After EDS injection, the testosterone level of cynomolgus monkeys in the arterial group showed a downward trend, and died of liver failure on the third day. On the 4th day after EDS injection, the testosterone level in the combined group was lower than the detection limit of 0.13 ng/ml, and returned to the level before administration 45 days after EDS. B, C: On the 2nd day after EDS, simple intra-testicular injection of EDS severely damaged the liver function of cynomolgus monkeys. Both cynomolgus monkeys had obvious symptoms of hepatic coma and died on the 3rd day after EDS. In the combination group, ALT and AST increased slightly, and returned to the level before administration within 4 days.

From the above experimental results, it can be seen that the method of the present invention combines EDS injection into the testicular artery of cynomolgus monkeys to block the blood flow of the spermatic cord, and continuous and long-term observation of liver and other indicators proves that this method can effectively remove LCs in the testes of adult male cynomolgus monkeys , established a cynomolgus monkey TDS model; and reduced the toxic side effects such as liver damage caused by EDS to provide a stable model carrier for TDS pathogenesis research, pharmacodynamics research and other preclinical research on translational medicine.

Claims (8)

1. A method for establishing a monkey testosterone deficiency model, characterized in that: the method of injecting dimethyl sulfonate into the adult monkey testicular artery in combination with blocking the blood flow of the spermatic cord. 2. The method according to claim 1, characterized in that the amount of dimethyl sulfonate injected is at least 50 mg/testis. 3. The method according to claim 1, characterized in that the amount of ethane dimethylsulfonate injected is 50-200 mg/testis. 4. The method according to claim 1, characterized in that the amount of ethane dimethylsulfonate injected is 50-100 mg/testis. 5. The method according to claim 1, characterized in that the amount of ethane dimethylsulfonate injected is 100-200 mg/testis. 6. The method according to claim 1, characterized in that, the specific steps are: after the adult monkey is anesthetized, disinfect its lower abdomen and perineum, use an inguinal incision to expose bilateral spermatic cords, and block the spermatic cords Blood flow; the testis was exposed by a scrotal incision and the dimethylsulfonate was injected into the testicular artery. 7. The method according to any one of claims 1 to 6, characterized in that the time for injecting the dimethyl sulfonate into the testicular artery is 1-5 minutes. 8. The method according to any one of claims 1 to 6, wherein the adult monkey is at least 6 years old.