WO2019161707A1 - Method for establishing monkey testosterone-deficiency model - Google Patents

Abstract

Disclosed is a method for establishing a monkey testosterone-deficiency model, wherein same adopts the method of injecting dimethyl ethanesulfonic acid into the testicular artery of adult monkeys in combination with blocking the blood flow in the spermatic cord. The method provides a safe and stable testosterone-deficiency animal model.

Description

Method for establishing a model of monkey testosterone deficiency Technical field

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

Background technique

Testosterone deficiency syndrome (TDS) refers to the abnormality of the target organ morphology and function caused by insufficient testosterone levels in various stages of men's life, which leads to the corresponding clinical symptoms. It is closely related to the occurrence and development of major diseases such as fertility, sexual dysfunction, osteoporosis, depression, cardiovascular and cerebrovascular diseases, diabetes and metabolic syndrome. It is a male reproductive endocrine disease that seriously affects men's health, life expectancy and quality of life. TDS mainly includes late onset hypogonadism (late-onset hypogonadisim (LOH)), congenital hypogonadism, testicular injury and other diseases. With the aging process of Chinese society, the incidence of TDS has increased year by year, and the medical market demand is very large, which 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 supplementation therapy, supplementation of exogenous testosterone by oral or injection, although there is a certain effect, but due to obvious defects such as inconvenient use and side effects, only very A small percentage of patients with TDS receive this therapy. Therefore, exploring a new method for treating TDS has become a key scientific and technological problem that urgently needs to be solved in the social development of our country.

At present, exogenous testosterone drug replacement 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, and physical enhancement. However, exogenous testosterone drug replacement therapy has obvious defects: (1) due to individual differences in testosterone levels, the dose of testosterone is difficult to master, and the dose is too small to be effective. If the dose is too large, it is prone to side effects and complications. (2) will cause the body's testosterone level to lose the original circadian rhythm changes, causing a variety of complications; (3) inconvenient to use, long-term frequent use of drugs; (4) the use of exogenous testosterone drugs will inhibit testicular growth Fine function, long-term use will cause infertility such as oligozoospermia or azoospermia. Due to the above defects, only a small number of patients with TDS are currently receiving exogenous testosterone drugs. There is an urgent need to explore a new approach to the treatment of TDS. More than 95% of testosterone in the body is synthesized and secreted by LCs (Leydig cells, Leydig cells). The decrease in the number of Leydig cells or hypofunction is considered to be the core pathogenesis of TDS, suggesting that cell transplantation therapy may achieve better results. . The development of cell transplantation therapy depends on the development of TDS model, so the research of TDS model becomes a key issue in the research field of new methods of TDS therapy.

At present, there are two TDS models, one is the model of delayed type hypogonadism in the elderly, and the other is the damage model of ethane-1,2-dimethyl sulphonate (EDS); Individual differences are large, difficult to obtain and many other shortcomings, and it is difficult to apply. EDS is a cytotoxic alkylating agent that selectively kills LCs from rats and other species, but does not affect the proliferation of spermatogenic cells. After a single intraperitoneal injection of 75 mg/kg body weight of EDS, rats can induce apoptosis and clear mature LCs in the testicular stroma of rats. LCs nuclear pyknosis and nuclear fragmentation occurred gradually within 6 to 18 hours after EDS injection. Most of the LCs showed apoptosis after 24 hours, and all LCs were cleared after 72 hours. Newer LCs can be found in the testicular stroma around 2 to 3 weeks, and the number of LCs is restored to the pre-dose level in about 8 to 10 weeks. Serum LH gradually increased within 14 days after EDS injection, and serum LH began to decrease due to neonatal LC secretion of testosterone within 14 to 21 days. Serum LH decreased to pre-injection level after 21 days. Within 2 to 8 weeks after EDS injection, the rats lost fertility. Other studies have shown that the weight of the genital organs, including the testes, epididymis, vas deferens and seminal vesicles, is also significantly reduced due to a significant decrease in testosterone levels in the body after EDS injection. Therefore, it is a good model for studying TDS cell transplantation therapy.

However, although rodents such as rats and mice are widely used in scientific research, they are more different from humans. The cynomolgus primate is similar to humans in terms of anatomy, physiological function and biochemical metabolism. It is an ideal experimental animal for understanding human physiology and pathophysiology, and is the most important model in clinical transformation research. In order to promote the clinical transformation of human SLCs in the treatment of TDS, and to evaluate the safety and efficacy of human SLCs in the treatment of TDS, data from non-human primate models is essential, so the establishment of a primate TDS model is essential. However, the non-human primate model is very expensive. How to ensure the efficient and safe establishment of animal models is the basis for the clinical transformation of human SLCs in the treatment of TDS. The key to the establishment of the cynomolgus monkey model is the use of EDS. Since the dose of EDS is closely related to the occurrence of hepatotoxicity, it is best to find the TDS model that can induce the stability of non-human primates and ensure the high survival rate of the animals. The dosage and mode of use are very important studies. In the establishment of the TDS model of cynomolgus monkeys, there has not been any report in China, and only one foreign literature has been reported (Ethane dimethylsulphonate selective destroys Leydigcells in the adult bonnet monkeys (Macaca radiata)). In the literature, the animals were selected from 7-8 years old, and they weighed 6-8 kg. By injecting 5, 10, 20, 50 mg of EDS into the bilateral testes, the authors found a significant decrease in testosterone levels in the first four days, and testosterone levels in the 5 mg/testis group decreased to 62% on the fourth day after treatment, and The pre-dose level was restored 45 days after EDS. However, in the primate testis, a number of connective tissue septa are issued from the testicular mediastinum, and the testicular parenchyma is divided into a plurality of pyramidal testicular lobules. Local injection is difficult to ensure uniform distribution of EDS and modeling effect. In addition, EDS has obvious hepatotoxicity. It is also important to choose the appropriate dosage, mode of administration and evaluation of drug damage after modeling. However, there are no reports at home and abroad. Therefore, replication of a stable TDS model is of great significance for the study of the efficacy and safety of SLCs in the treatment of TDS.

Summary of the invention

The purpose of the present invention is to provide a safe and stable animal model of testosterone deficiency in view of the deficiencies of existing non-human primate TDS models, and 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 testosterone deficiency model of monkeys by injecting dimethyl sulfonate (EDS) into the testicular arteries of an adult monkey to block the flow of spermatic cord blood.

According to the method of the invention, the amount of dimethyl sulfonate injected is at least 50 mg per testicle. Preferably, the amount of dimethyl sulfonate injected is from 50 to 200 mg per testicle. Preferably, the amount of dimethyl sulfonate injected is from 50 to 100 mg per testicle. Preferably, the amount of dimethyl sulfonate injected is from 100 to 200 mg per testicle. According to the method of the present invention, the specific steps are as follows: after anesthetizing the adult cynomolgus monkey, disinfecting the lower abdomen and the perineum, exposing the bilateral spermatic cord with the inguinal incision, and blocking the spermatic cord blood flow with the non-invasive forceps 20-30 Minutes; the scrotal incision exposes the testes and dimethyl sulfonate is injected into the testicular artery.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, 45 days after EDS injection, from 9:00 am to 10:00 am, blood was collected from the upper extremity vein, each 1-2 ml of blood was allowed to stand at room temperature for 90 minutes, centrifuged at 2000 g for 15 minutes, and serum was separated and stored at -20 ° C for testosterone and liver function.

Testicular tissue was obtained by testicular puncture on 4, 14, 28, and 45 days after EDS injection. After fixation with 4% paraformaldehyde, the sample was dehydrated in 10%, 20%, and 30% sucrose solution to the bottom of the tissue. Buried, frozen slicer 6 μm thick sections, 5 samples per time point. Immunofluorescence staining stained the Leydig cell-specific protein CYP11A1.

Preferably, the time to inject dimethyl sulfonate into the testicular artery is 1-5 minutes.

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

The method of the invention adopts an adult monkey (preferably cynomolgus monkey), and adopts a method of injecting EDS into the testicular artery to block the blood flow of the spermatic cord, and establishes a safe and stable monkey TDS model. Through continuous and long-term observation of liver and other indicators, it was confirmed that the method can effectively remove LCs from the testes of adult male monkeys and establish a monkey TDS model. In addition, the method of the present invention reduces the side effects such as liver damage caused by EDS, thereby causing the onset of TDS. Mechanism studies, pharmacodynamic studies, and other preclinical studies in translational medicine provide stable model vectors.

DRAWINGS

Figure 1 shows the effect of intra-articular injection of EDS in combination with blocking spermatic blood flow.

Figure 2 is a comparison of the effects of local injection of EDS in the testes of cynomolgus monkeys and intra-articular injection of EDS to block spermatic cord blood flow.

Figure 3 is a comparison of the effects of injection of EDS into the testicular arteries of the cynomolgus monkey and intra-articular injection of EDS to block the spermatic blood flow.

Figure 4 is a testicular staining diagram.

Detailed ways

The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments for ease of understanding. However, the scope of protection of the present invention is not limited to the following embodiments.

Example 1: Intra-arterial injection of EDS in the testis blocked the spermatic cord blood flow, cleared the Leydig cells, and established a testosterone deficiency model.

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

The first group (also referred to as "solvent group") 2 cynomolgus monkeys were injected intraarticularly with vehicle (DMSO: H2O, 1:3, V/V). EDS was dissolved in vehicle, the second group of 2 cynomolgus monkeys were intra-articularly injected with 50 mg EDS; the third group of 2 cynomolgus monkeys were intra-articularly injected with 100 mg of EDS; the fourth group of 2 cynomolgus monkeys were injected with 200 mg of testicular artery. EDS.

On the day of the experiment, the cynomolgus monkey was anesthetized, the lower abdomen and the perineum were disinfected, and the bilateral spermatic cords were exposed by the inguinal incision. The spermatic cord blood flow was blocked with a non-invasive forceps for 20-30 minutes; the scrotal incision exposed the testes, and the corresponding dose of EDS Slowly inject into the testicular artery for an injection time of 1-5 minutes.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, 45 days after EDS injection, from 9:00 am to 10:00 am, blood was collected from the upper extremity vein, each 1-2 ml of blood was allowed to stand at room temperature for 90 minutes, centrifuged at 2000 g for 15 minutes, and serum was separated and stored at -20 ° C for testosterone and liver function.

See Figure 1 for the results. Among them, on the 4th day after A: EDS, the testosterone level in the vehicle group was not significantly affected. Testicular intra-arterial injection of 50, 100, 200mg/testis EDS combined with blocking spermatic cord blood flow can significantly reduce serum testosterone levels in cynomolgus monkeys. Testosterone levels in the 50 mg/testis group were reduced to 30% of normal levels, and testosterone levels in the 100 and 200 mg/testis groups were below the lower limit of detection of 0.13 ng/ml. Testosterone levels in the 50, 100, 200 mg/testis group returned to pre-dose levels 45 days after EDS injection. B, C: After EDS injection, the ALT and AST of the liver group showed no significant changes in liver damage, and were at normal levels; 1 day after EDS injection, ALT and AST increased in 50, 100, 200 mg/testis group, and the dose The increase was increasing and returned to pre-dose levels within 7 days.

Testicular tissue was obtained by testicular puncture on 4, 14, 28, and 45 days after EDS injection. After fixation with 4% paraformaldehyde, the sample was dehydrated in 10%, 20%, and 30% sucrose solution to the bottom of the tissue. Buried, frozen slicer 6 μm thick sections, 5 samples per time point. Immunofluorescence staining stained the Leydig cell-specific protein CYP11A1.

Figure 4 is a testicular staining diagram. Among them, A: testicular staining on the 4th day after EDS injection, the test shows that the Leydig cells were completely cleared; B: Testicular staining on the 14th day after EDS injection, the test shows that the Leydig cells began to recover; C: EDS Testicular staining on the 28th day after injection, the figure shows that the number of Leydig cells was restored by about 50%; D: Testicular staining on the 45th day after EDS injection, the figure showed that the number of Leydig cells was restored to normal. Testicular sections show that EDS only affects Leydig cells, but does not affect other cells.

Example 2: Comparison of local injection of EDS in the testes of cynomolgus monkeys and intra-articular injection of EDS to block spermatic blood flow.

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

On the day of the experiment, the cynomolgus monkey was anesthetized and disinfected in the lower abdomen and perineum. The first group (also known as the "local group") was injected with 100 mg/testis EDS in two testes of the two cynomolgus monkeys; the second group (also called " The two groups of cynomolgus monkeys exposed the bilateral spermatic cords with an inguinal incision, and the spermatic cord blood flow was blocked with a non-invasive forceps for 20-30 minutes; the scrotal incision exposed the testicles, and an equal amount of EDS was slowly injected 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, 45 days after EDS injection, from 9:00 am to 10:00 am, blood was collected from the upper extremity vein, each 1-2 ml of blood was allowed to stand at room temperature for 90 minutes, centrifuged at 2000 g for 15 minutes, and serum was separated and stored at -20 ° C for testosterone and liver function.

See Figure 2 for the results. Among them, on the 4th day after A: EDS, testicular local injection of EDS group (local group) testosterone level was not affected, testicular intra-arterial injection of EDS + block spermatic cord blood flow group (combination group) cynomolgus monkey serum testosterone level is lower than detection The lower limit was 0.13 ng/ml and returned to pre-dose levels 45 days after EDS. B, C: After EDS injection, the local group showed no significant changes in liver damage index ALT, AST, at normal level; 1 day after EDS injection, the combined group ALT, AST mildly increased, and returned to administration within 4 days Pre-level.

Example 3: Comparison of the effect of injection of EDS and testicular intra-articular injection of EDS on the spermatic cord blood flow in cynomolgus monkey testicular arteries.

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

On the day of the experiment, the cynomolgus monkey was anesthetized and disinfected in the lower abdomen and perineum. The first group of 2 cynomolgus monkeys were injected with 100 mg/testis EDS in both testicular arteries; the second group (also called “combined group”) was treated with the inguinal inner ring. The incision exposes the bilateral spermatic cord, and the spermatic cord blood flow is blocked with a non-invasive forceps for 20-30 minutes; the testicular is exposed by the scrotal incision, and an equal amount of EDS is slowly injected into the testicular artery for an injection time of 1-5 minutes.

0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 20, 25, 30, 35, 40, 45 days after EDS injection, from 9:00 am to 10:00 am, blood was collected from the upper extremity vein, each 1-2 ml of blood was allowed to stand at room temperature for 90 minutes, centrifuged at 2000 g for 15 minutes, and serum was separated and stored at -20 ° C for testosterone and liver function.

The results are shown in Figure 3: A, EDS injection test group cynomolgus monkey testosterone levels showed a downward trend, and died on the third day due to liver failure. On the 4th day after EDS injection, the testosterone level of the combined group was lower than the lower limit of detection of 0.13 ng/ml, and returned to the pre-dose level 45 days after EDS. B, C: On the second day after EDS, simple testicular intra-arterial injection of EDS seriously impaired the liver function of cynomolgus monkeys. Two cynomolgus monkeys showed obvious hepatic coma symptoms and died on the third day after EDS. The combined groups had mild elevations in ALT and AST and returned to pre-dose levels within 4 days.

It can be seen from the above experimental results that the method of the present invention blocks the spermatic blood flow by EDS injection in the testicular arteries of cynomolgus monkeys, and continuously and long-term observation of liver and other indicators, and proves that the method can effectively remove LCs from the testes of adult male cynomolgus monkeys. Established a TDS model for cynomolgus monkeys; and reduced toxic side effects such as liver damage caused by EDS, providing a stable model vector for TDS pathogenesis research, pharmacodynamic studies and other pre-clinical studies of translational medicine.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Therefore, any of the above embodiments can be made according to the technical essence of the present invention without departing from the technical solution of the present invention. Simple modifications, equivalent changes and modifications are still within the scope of the technical solutions of the present invention.

Claims (8)

1. A method for establishing a testosterone deficiency model of monkeys, characterized in that a method of injecting dimethyl sulfonate into an adult monkey testicular artery to block sperm blood flow is used.
2. The method of claim 1 wherein the amount of dimethyl sulfonate injected is at least 50 mg per testicle.
3. The method of claim 1 wherein the amount of dimethyl sulfonate injected is from 50 to 200 mg per testicle.
4. The method of claim 1 wherein the amount of dimethyl sulfonate injected is from 50 to 100 mg per testicle.
5. The method of claim 1 wherein the amount of dimethyl sulfonate injected is from 100 to 200 mg per testicle.
6. The method according to claim 1, wherein the specific step is: after anesthetizing the adult monkey, disinfecting the lower abdomen and the perineum, and exposing the bilateral spermatic cord by using the inguinal inner ring incision to block the spermatic cord blood flow. The scrotal incision exposes the testes and the dimethyl sulfonate is injected into the testicular artery.
7. The method according to any one of claims 1 to 6, wherein the injection of the dimethyl sulfonate into the testicular artery is performed for 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.

Images