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WO2019161707A1 - 一种猴睾酮缺乏模型的建立方法 - Google Patents

一种猴睾酮缺乏模型的建立方法 Download PDF

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WO2019161707A1
WO2019161707A1 PCT/CN2018/123563 CN2018123563W WO2019161707A1 WO 2019161707 A1 WO2019161707 A1 WO 2019161707A1 CN 2018123563 W CN2018123563 W CN 2018123563W WO 2019161707 A1 WO2019161707 A1 WO 2019161707A1
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eds
testosterone
injection
testicular
injected
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项鹏
柯琼
夏凯
邓春华
毛富祥
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/106—Primate
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases

Definitions

  • the invention belongs to the field of animal models, and in particular relates to a method for establishing a testosterone deficiency model of monkeys.
  • Testosterone deficiency syndrome 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.
  • LH late onset hypogonadism
  • congenital hypogonadism congenital hypogonadism
  • 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.
  • 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.
  • 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.
  • 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.
  • EDS ethane-1,2-dimethyl sulphonate
  • 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.
  • 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.
  • data from non-human primate models is essential, so the establishment of a primate TDS model is essential.
  • 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 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.
  • 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.
  • EDS dimethyl sulfonate
  • the amount of dimethyl sulfonate injected is at least 50 mg per testicle.
  • the amount of dimethyl sulfonate injected is from 50 to 200 mg per testicle.
  • the amount of dimethyl sulfonate injected is from 50 to 100 mg per testicle.
  • the amount of dimethyl sulfonate injected is from 100 to 200 mg per testicle.
  • 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.
  • EDS injection 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 ⁇ m thick sections, 5 samples per time point. Immunofluorescence staining stained the Leydig cell-specific protein CYP11A1.
  • the time to inject dimethyl sulfonate into the testicular artery is 1-5 minutes.
  • 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.
  • an adult monkey preferably cynomolgus monkey
  • the method can effectively remove LCs from the testes of adult male monkeys and establish a monkey TDS model.
  • 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.
  • 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.
  • 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.
  • 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.
  • EDS injection 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 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.
  • 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 ⁇ m 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.
  • the cynomolgus monkey 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.
  • EDS injection 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.
  • 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.
  • the cynomolgus monkey 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.
  • EDS injection 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 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.

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Abstract

一种猴睾酮缺乏模型的建立方法，其采用向成年猴睾丸动脉注射二甲基磺酸乙烷结合阻断精索血流的方式，提供了安全、稳定的睾酮缺乏动物模型。

Description

一种猴睾酮缺乏模型的建立方法 技术领域
本发明属于动物模型领域，具体涉及一种猴睾酮缺乏模型的建立方法。
背景技术
睾酮缺乏综合征(Testosterone deficiency syndrome，TDS)是指在男性一生中的不同时期可能因各种原因导致体内睾酮水平不足而造成其靶器官形态、功能异常，进而引起相应的临床症状，与男性不育症、性功能障碍、骨质疏松、抑郁症、心脑血管疾病、糖尿病和代谢综合症等重大疾病的发生发展密切相关，是严重影响男性健康、预期寿命和生活质量的男性生殖内分泌疾病。TDS主要包括迟发性性腺功能减退症(迟发性性腺功能减退(late-onset hypogonadisim，LOH))、先天性性腺功能减退症、睾丸损伤等疾病。随着中国社会老龄化进程，TDS发病率逐年升高，医疗市场需求非常庞大，已成为严重影响我国人民身心健康的重大疾病。目前，临床上治疗TDS主要使用外源性睾酮药物补充疗法，通过口服、注射等途径补充外源性睾酮，虽然有一定疗效，但是由于存在使用不方便和副作用大等明显缺陷，目前仅有很少一部分TDS患者接受该疗法。因此，探索一种治疗TDS的新方法成为我国社会发展中迫切需要解决的关键科技问题。
目前外源性睾酮药物补充疗法是TDS的常用治疗方法。研究证实，患者可从睾酮替代治疗中获益，如性欲的提高、骨密度的增加、情绪和认知的改善、体质增强等。但是，外源性睾酮药物补充疗法存在明显缺陷：(1)由于患者的睾酮下降水平存在个体差异，造成补充睾酮的剂量难于掌握，剂量过少难于奏效，剂量过大则易于出现副作用和并发症；(2)会造成体内睾酮水平丧失了原有的昼夜节律变化，引起多种并发症；(3)使用不方便，需长期频繁用药；(4)使用外源性睾酮药物会抑制睾丸的生精功能，长期使用会造成少精子症或无精子症等不育症。由于上述缺陷，目前仅有很少一部分TDS患者在接受外源性睾酮药物治疗。迫切需要探索一种治疗TDS的新方法。体内超95％的睾酮是LCs(Leydig cells，睾丸间质细胞)合成和分泌的，睾丸间质细胞数量减少或功能减退被认为是TDS的核心发病机制，这提示细胞移植疗法可能取得较好疗效。而细胞移植疗法的开展有赖于TDS模型的发展，因此TDS模型的研究成为TDS治疗新方法研究领域中的关键问题。
目前TDS模型有两种，一种为老年迟发型性腺功能减退模型，另一种为二甲基磺酸乙烷(ethane-1，2-dimethyl sulphonate，EDS)损伤模型；因老年动物成本高，个体差异大，不易获取等诸多缺点，难以应用。EDS是一种细胞毒性烷化剂，能够选择性杀死大鼠和其他 物种的LCs，但是对生精细胞的增殖并不影响。大鼠单次腹腔注射75mg/kg体重的EDS后，可以诱导凋亡而清除大鼠睾丸间质内成熟的LCs。在注射EDS后6～18小时内逐渐出现LCs核固缩和核碎裂，24小时后大部分LCs出现凋亡，72小时后所有的LCs被清除干净。2～3周左右可以在其睾丸间质内发现新生的LCs，大约8～10周左右LCs数量恢复到给药前水平。EDS注射14天内血清LH逐渐上升，14～21天内由于新生的LC分泌睾酮而导致血清LH开始下降，21天后血清LH降至注射前水平。EDS注射后的2～8周内，大鼠失去生育功能。其他研究显示，由于EDS注射后导致体内睾酮水平明显下降，包括睾丸、附睾、输精管和精囊在内的生殖器官的重量也明显减少。因此是一种较好的研究TDS细胞移植疗法的理想模型。
然而，尽管大鼠、小鼠等啮齿类动物在科学研究中被广泛使用，但与人类存在较多差异。食蟹猴属灵长类动物，在解剖、生理机能及生化代谢等方面与人类相似，是了解人类生理和病理生理知识的较为理想的实验动物，也是临床转化研究中最重要的模型。为了促进人SLCs治疗TDS的临床转化，评估人SLCs治疗TDS的安全性和有效性，非人灵长类模型的数据是必不可少的，因此建立灵长类动物TDS模型至关重要。但非人灵长类模型十分昂贵，如何保证高效安全地建立动物模型，是人SLCs治疗TDS临床转化十分重要的基础。食蟹猴模型建立的关键在EDS的使用，由于EDS的剂量与肝毒性的发生密切相关，寻找既能诱导非人类灵长类动物稳定的TDS模型又能保证动物的高存活率的EDS最佳剂量和使用方式是非常重要的研究。在食蟹猴TDS模型的建立方面，国内尚未有报道，国外仅见一篇文献报道(Ethane dimethylsulphonate selectively destroys Leydigcells in the adult bonnet monkeys(Macaca radiata))。文献中动物选用的是7-8岁的冠毛猕猴，重6-8千克。作者通过向双侧睾丸内注射5、10、20、50mg的EDS，发现前四天各组睾酮水平出现明显的下降，5mg/testis组的睾酮水平处理后第四天下降到62％，并且在EDS后45天恢复到给药前水平。但是灵长类睾丸中由睾丸纵隔发出许多结缔组织小隔，将睾丸实质分成许多锥体形的睾丸小叶，局部注射难以保证EDS的均匀分布及造模效果。另外，EDS具有明显的肝毒性，选择合适的造模剂量、给药方式及评估造模后药物损害也至关重要，但目前尚未见国内外相关报道。因此复制稳定的TDS模型，为研究SLCs治疗TDS有效性、安全性具有重要意义。
发明内容
本发明的目的是针对现有非人灵长类TDS模型的不足，提供一种安全、稳定的睾酮缺乏动物模型，为研究人SLCs治疗TDS有效性、安全性提供良好的基础。
为了实现以上目的，本发明提供了一种猴睾酮缺乏模型的建立方法，其采用向成年猴睾丸动脉注射二甲基磺酸乙烷(EDS)结合阻断精索血流的方式。
根据本发明的方法，注射二甲基磺酸乙烷的量为至少50mg/睾丸。优选地，注射二甲基磺酸乙烷的量为50-200mg/睾丸。优选地，注射二甲基磺酸乙烷的量为50-100mg/睾丸。优选地，注射二甲基磺酸乙烷的量为100-200mg/睾丸。根据本发明的方法，其具体步骤为：将成年食蟹猴麻醉后，消毒其下腹及会阴部，采用腹股沟内环切口暴露双侧精索，用无损伤钳阻断精索血流20-30分钟；阴囊切口暴露睾丸，将二甲基磺酸乙烷注射入睾丸动脉内。
EDS注射后0、1、2、3、4、5、7、9、11、13、15、20、25、30、35、40、45天上午9点-10点，从上肢静脉采血，每次1-2ml血液，室温静置90分钟，2000g离心15分钟，分离血清，-20℃储存，用于检测睾酮及肝功能。
EDS注射后4、14、28、45天分别进行睾丸穿刺取得睾丸组织，用4％多聚甲醛固定后，在10％、20％、30％蔗糖溶液中梯度脱水至组织沉底，利用OCT包埋，冰冻切片机中行6μm厚的切片，每个时间点5个样品。免疫荧光染色对睾丸间质细胞特异性蛋白CYP11A1染色。
优选地，将二甲基磺酸乙烷注射入睾丸动脉内的时间为1-5分钟。
根据本发明的方法，所述成年猴的年龄为至少6岁。
本发明的方法选用成年猴(优选食蟹猴)，采用睾丸动脉注射EDS结合阻断精索血流的方法，建立了安全、稳定的猴TDS模型。通过连续、长期观测肝脏等指标，证实了本方法能够有效清除成年雄性猴睾丸中LCs，建立了猴TDS模型；此外，本发明的方法降低了EDS导致的肝脏损伤等毒副作用，从而为TDS发病机制研究、药效学研究和其他转化医学临床前研究提供稳定的模型载体。
附图说明
图1是睾丸动脉内注射EDS结合阻断精索血流效果。
图2是食蟹猴睾丸内局部注射EDS和睾丸动脉内注射EDS联合阻断精索血流效果比较。
图3是食蟹猴睾丸动脉注射EDS和睾丸动脉内注射EDS联合阻断精索血流效果比较。
图4是睾丸染色图。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步的详述，以便于理解。但本发明的保护范围并不限于以下实施例。
实施例1：睾丸动脉内注射EDS结合阻断精索血流，清除睾丸间质细胞，建立睾酮缺乏模型。
本实验中选用的动物为成年雄性食蟹猴，年龄＞6岁，重量8-10kg，共8只成年雄性食蟹猴，随机分为4组，每组2只。
第一组(也称为“溶媒组”)2只食蟹猴双侧睾丸动脉内注射溶媒(DMSO∶H2O，1∶3，V/V)。EDS溶于溶媒，第二组2只食蟹猴睾丸动脉内注射50mg EDS；第三组2只食蟹猴睾丸动脉内注射100mg的EDS；第四组2只食蟹猴睾丸动脉内注射200mg的EDS。
实验当日，食蟹猴麻醉后，消毒下腹及会阴部，采用腹股沟内环切口暴露双侧精索，用无损伤钳阻断精索血流20-30分钟；阴囊切口暴露睾丸，相应剂量的EDS缓慢注入睾丸动脉内，注射时间为1-5分钟。
EDS注射后0、1、2、3、4、5、7、9、11、13、15、20、25、30、35、40、45天上午9点-10点，从上肢静脉采血，每次1-2ml血液，室温静置90分钟，2000g离心15分钟，分离血清，-20℃储存，用于检测睾酮及肝功能。
结果参见图1。其中，A：EDS后第4天，溶媒组睾酮水平未受明显影响睾丸动脉内注射50、100、200mg/testis的EDS联合阻断精索血流均能显著降低食蟹猴血清睾酮水平，其中50mg/testis组睾酮水平降至正常水平的30％，100和200mg/testis组睾酮水平低于检测下限0.13ng/ml。50、100、200mg/testis组的睾酮水平在EDS注射后45天恢复至给药前水平。B、C：EDS注射后，溶媒组反映肝脏损伤的指标ALT、AST没有明显变化，处于正常水平；EDS注射后1天，50、100、200mg/testis组ALT、AST升高，且随着剂量的加大不断增高，并于7天内恢复至给药前水平。
EDS注射后4、14、28、45天分别进行睾丸穿刺取得睾丸组织，用4％多聚甲醛固定后，在10％、20％、30％蔗糖溶液中梯度脱水至组织沉底，利用OCT包埋，冰冻切片机中行6μm厚的切片，每个时间点5个样品。免疫荧光染色对睾丸间质细胞特异性蛋白CYP11A1染色。
图4是睾丸染色图。其中，A：EDS注射后第4天睾丸染色图，图中显示睾丸间质细胞被全部清除；B：EDS注射后第14天睾丸染色图，图中显示睾丸间质细胞开始恢复；C：EDS注射后第28天睾丸染色图，图中显示睾丸间质细胞数量恢复约50％；D：EDS注射后第45天睾丸染色图，图中显示睾丸间质细胞数量恢复至正常。睾丸切片显示EDS只影响睾丸间质细胞，但不影响其他细胞。
实施例2：食蟹猴睾丸内局部注射EDS和睾丸动脉内注射EDS联合阻断精索血流效果比较。
另外选用4只成年雄性食蟹猴，年龄＞6岁，重量8-10kg，随机分为2组，每组2只。
实验当日，食蟹猴麻醉后，消毒下腹及会阴部，第一组(也称为“局部组”)2只食蟹 猴双侧睾丸局部注射100mg/testis EDS；第二组(也称为“联合组”)2只食蟹猴采用腹股沟内环切口暴露双侧精索，用无损伤钳阻断精索血流20-30分钟；阴囊切口暴露睾丸，将等量EDS缓慢注入睾丸动脉内，注射时间为1-5分钟。
EDS注射后0、1、2、3、4、5、7、9、11、13、15、20、25、30、35、40、45天上午9点-10点，从上肢静脉采血，每次1-2ml血液，室温静置90分钟，2000g离心15分钟，分离血清，-20℃储存，用于检测睾酮及肝功能。
结果参见图2。其中，A：EDS后第4天，睾丸局部注射EDS组(局部组)睾酮水平未受影响，睾丸动脉内注射EDS+阻断精索血流组(联合组)食蟹猴血清睾酮水平低于检测下限0.13ng/ml，并于EDS后45天恢复至给药前水平。B、C：EDS注射后，局部组反映肝脏损伤的指标ALT、AST没有明显变化，处于正常水平；EDS注射后1天，联合组ALT、AST轻度升高，并于4天内恢复至给药前水平。
实施例3：食蟹猴睾丸动脉注射EDS和睾丸动脉内注射EDS联合阻断精索血流效果比较。
另外选择成年雄性食蟹猴4只，年龄＞6岁，重量8-10kg，4只成年雄性食蟹猴，随机分为2组，每组2只。
实验当日，食蟹猴麻醉后，消毒下腹及会阴部，第一组2只食蟹猴双侧睾丸动脉内注射100mg/testis EDS；第二组(也称为“联合组”)采用腹股沟内环切口暴露双侧精索，用无损伤钳阻断精索血流20-30分钟；阴囊切口暴露睾丸，将等量EDS缓慢注入睾丸动脉内，注射时间为1-5分钟。
EDS注射后0、1、2、3、4、5、7、9、11、13、15、20、25、30、35、40、45天上午9点-10点，从上肢静脉采血，每次1-2ml血液，室温静置90分钟，2000g离心15分钟，分离血清，-20℃储存，用于检测睾酮及肝功能。
结果参见图3：A、EDS注射后动脉组食蟹猴睾酮水平呈现下降趋势，并于第3天因肝脏衰竭死亡。EDS注射后第4天，联合组睾酮水平低于检测下限0.13ng/ml，并于EDS后45天恢复至给药前水平。B、C：EDS后第2天，单纯睾丸动脉内注射EDS严重损害食蟹猴肝脏功能，2只食蟹猴均出现明显的肝昏迷症状，并于EDS后第3天死亡。联合组ALT、AST轻度升高，并于4天内恢复至给药前水平。
由以上实验效果可见，本发明的方法通过食蟹猴睾丸动脉内EDS注射联合阻断精索血流，并且连续、长期观测肝脏等指标，证实本方法能够有效清除成年雄性食蟹猴睾丸中LCs，建立了食蟹猴TDS模型；并且降低了EDS导致的肝脏损伤等毒副作用从而为TDS发病机制研究、药效学研究和其他转化医学临床前研究提供稳定的模型载体
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (8)

  1. 一种猴睾酮缺乏模型的建立方法，其特征在于：采用向成年猴睾丸动脉注射二甲基磺酸乙烷结合阻断精索血流的方式。
  2. 根据权利要求1所述的方法，其特征在于，注射所述二甲基磺酸乙烷的量为至少50mg/睾丸。
  3. 根据权利要求1所述的方法，其特征在于，注射所述二甲基磺酸乙烷的量为50-200mg/睾丸。
  4. 根据权利要求1所述的方法，其特征在于，注射所述二甲基磺酸乙烷的量为50-100mg/睾丸。
  5. 根据权利要求1所述的方法，其特征在于，注射所述二甲基磺酸乙烷的量为100-200mg/睾丸。
  6. 根据权利要求1所述的方法，其特征在于，其具体步骤为：将所述成年猴麻醉后，消毒其下腹及会阴部，采用腹股沟内环切口暴露双侧精索，阻断精索血流；阴囊切口暴露睾丸，将所述二甲基磺酸乙烷注射入睾丸动脉内。
  7. 根据权利要求1至6任一项所述的方法，其特征在于，将所述二甲基磺酸乙烷注射入睾丸动脉内的时间为1-5分钟。
  8. 根据权利要求1至6任一项所述的方法，其特征在于，所述成年猴的年龄为至少6岁。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102883710A (zh) * 2010-04-12 2013-01-16 克劳拉斯医疗有限公司 口服睾酮酯制剂以及包含其的治疗睾酮缺乏症的方法
CN103118684A (zh) * 2010-08-16 2013-05-22 英诺泰斯托比利时公司 用于治疗睾酮缺乏的睾酮溶液
CN105255826A (zh) * 2015-11-27 2016-01-20 中山大学 人诱导多功能干细胞向睾丸间质细胞的诱导分化方法及其用途
CN108524485A (zh) * 2018-02-24 2018-09-14 中山大学 一种猴睾酮缺乏模型的建立方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102883710A (zh) * 2010-04-12 2013-01-16 克劳拉斯医疗有限公司 口服睾酮酯制剂以及包含其的治疗睾酮缺乏症的方法
CN103118684A (zh) * 2010-08-16 2013-05-22 英诺泰斯托比利时公司 用于治疗睾酮缺乏的睾酮溶液
CN105255826A (zh) * 2015-11-27 2016-01-20 中山大学 人诱导多功能干细胞向睾丸间质细胞的诱导分化方法及其用途
CN108524485A (zh) * 2018-02-24 2018-09-14 中山大学 一种猴睾酮缺乏模型的建立方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MA, XUE ET AL.: "Effects of Ethane Dimethane Sulfonate on Fetal Leydig Cells in Neonatal Rats", JOURNAL OF PEKING UNIVERSITY (HEALTH SCIENCES), vol. 45, no. 5, 31 October 2013 (2013-10-31), pages 770 - 773, XP055633422, ISSN: 1671-167X *
SUMATHI, R.: "Ethane dimethylsulphonate selectively destroys Leydig cells in the adult bonnet monkeys (Macaca radiata", ASIAN JOURNAL OF ANDROLOGY, vol. 1, no. 3, 31 January 1999 (1999-01-31), pages 115 - 120, XP055633415, ISSN: 1008-682X, Retrieved from the Internet <URL:http://www.asiaandro.com/archive/1008-682X/1/115.htm> *

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