老年大鼠睾丸间质细胞结构和功能变化的实验研究

目的:研究老年SD大鼠(PADAM动物模型)睾丸间质细胞形态、分泌功能变化,探讨老年大鼠睾丸间质细胞的功能状态。方法:分别取青年SD大鼠和老年各20只,静脉血测定血清总睾酮和游离睾酮的浓度,并通过组织切片和透射电镜观察两个年龄组大鼠睾丸间质细胞形态学变化;此外,分别用hCG、Forskolin刺激体外培养的两个年龄组大鼠的睾丸间质细胞,比较培养基中睾酮和孕酮的浓度。结果:老年大鼠的血清总睾酮[(3.07±0.75)nmol/L]和游离睾酮[(0.71±0.65)nmol/L]均比青年大鼠[(10.89±6.11)nmol/L和(2.42±1.02)nmol/L]显著降低(P<0.05);细胞形态有较显著差异;体外培养的大鼠睾丸间质细胞分泌能力显著降低(P<0.05)。结论:老年SD大鼠血清睾酮和游离睾酮浓度显著低于青年SD大鼠,原因在于睾酮合成酶系统整体功能衰退。     

老年大鼠睾丸间质细胞形态及睾酮合成功能变化的研究

目的 探讨衰老对睾丸间质细胞的形态及功能的影响.方法 青年(3月龄)及老年(24月龄)清洁级雄性SD大鼠各10只,麻醉后取血清检测总睾酮浓度,取睾丸组织用HE染色观察睾丸组织形态学变化,并用电镜观察睾丸间质细胞的超微结构改变.通过密度梯度离心分离原代睾丸间质细胞,并用LH刺激睾酮分泌后用Western blot比较青年组和老年组睾丸间质细胞类固醇合成快速调节蛋白(steroidogenic acute regulatory protein,StAR)表达水平的差异,并用ELISA法检测其睾酮分泌的差异.结果 HE染色显示老年大鼠睾丸呈老年退行性改变,电镜下观察到老年大鼠睾丸间质细胞线粒体水肿,线粒体嵴消失.老年大鼠血清睾酮水平显著低于青年组(P＜ 0.05).原代培养的睾丸间质细胞无论LH刺激与否,老年组细胞上清中睾酮浓度及StAR蛋白表达水平均显著低于青年组(LH刺激时P＜0.01,无LH刺激时P＜0.05).结论 衰老造成的睾丸间质细胞线粒体水肿及LH诱导的StAR蛋白表达水平下降与其睾酮合成能力降低密切相关.     

大鼠睾丸间质细胞体外培养不同时间3β-羟基类固醇脱氢酶的表达

目的探讨通过差速贴壁法获得的7天龄、3周龄大鼠睾丸间质内细胞的细胞群体构成、体外培养不同时间3β-羟基类固醇脱氢酶（3β-HSD）的表达水平变化,以及细胞睾酮分泌功能变化。方法联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得7天龄、3周龄雄性Wistar大鼠睾丸间质组织内细胞,贴壁细胞以DMEM/F12培养液培养,分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞分析,原代培养细胞同时给予人绒毛膜促性腺激素（HCG）刺激,测定HCG刺激组和非刺激组各代细胞培养液上清中睾酮水平及其对HCG刺激的反应。结果7天龄、3周龄鼠差速贴壁法获得的睾丸间质内细胞群经流式细胞鉴定,3β-HSD阳性细胞所占比例分别为（5.4±1.2）%、（59.2±3.2）%;培养4d后,两组3β-HSD阳性细胞比例分别为（93.6±1.2）%、（95.4±3.2）%。两组原代培养细胞均有睾酮生成功能,在HCG刺激下睾酮分泌均明显上升,7天龄组培养细胞睾酮分泌高峰迟于3周龄组。结论差速贴壁法获得的7天龄、3周龄大鼠睾丸间质细胞群中均含有部分Leydig干细胞（Stem Leydig cells,SLCs）,SLCs在体外培养过程中逐渐分化,表达3β-羟基类固醇脱氢酶,并产生睾酮。     

PADAM动物模型进行同种异体睾丸间质细胞移植的研究

目的老年SD大鼠进行同种异体睾丸间质细胞移植探讨中老年男性雄激素部分缺乏症(PADAM)动物模型的可行性和疗效。方法选10只符合PADAM模型标准的老年SD大鼠作为受体，用成年SD大鼠的睾丸进行体外分离和培养，将获得的高纯度和高活力的睾丸间质细胞移植到老年SD大鼠的大腿内侧肌群内，定期检查其移植前后血清睾酮和游离睾酮的水平变化，并观察移植部位的情况。结果未应用免疫抑制剂，移植后的睾丸间质细胞保持良好的分泌功能，老年SD大鼠的血清睾酮和游离睾酮水平均显著升高，并大约于移植后的7～12d开始稳定在一定的水平，可持续27d以上。移植部位未见异常。结论睾丸问质细胞同种异体移植安全、有效、无明显排斥反应。     

简易快速获得大量高纯度大鼠Leydig细胞

目的 建立一种简易快速获得高纯度睾丸间质内莱迪希(Leydig)细胞的分离方法.方法 联合应用复合胶原酶消化、400目(30 μm)不锈钢滤网过滤和差速贴壁法获得雄性Wister大鼠睾丸间质内的Leydig细胞.分离细胞经3β-羟类固醇脱氢酶(3β-HSD)特异性酶学染色、免疫细胞化学检测和流式细胞学进行细胞表型鉴定和纯度分析.将分离细胞进行原代及传代培养,部分细胞同时给予人绒毛膜促性腺激素(HCG)刺激,观察传代后细胞生长情况,并测定HCG刺激组和非刺激组各代细胞培养上清睾酮水平,评价分离细胞的增殖能力、睾酮分泌功能及对HCG的反应.有血清和无血清2种条件培养液培养原代分离细胞,倒置相差显微镜及电镜观察细胞老化和凋亡状况,优化Leydig细胞的培养条件.结果 应用差速贴壁方法每克睾丸组织(湿重)可获得Leydig细胞(1.5±0.8)×106个,3β-HSD特异性酶学染色、免疫细胞化学检测均呈阳性反应.流式细胞学检测证实3β-HSD阳性细胞率为(95.2±3.7)%.传代后未见明显的细胞增殖,但仍可检测到3β-HSD表达.原代及传代细胞均具有睾酮生成功能,HCG刺激后睾酮分泌水平均明显上升,但原代细胞一定水平的睾酮分泌可持续6 d以上,传代后细胞仅持续48 h.结论 应用差速贴壁法可以获得大量高纯度有活性的Leydig细胞,并可以进行传代培养,且传代前后短期内均可分泌睾酮.该方法简单易行,细胞得率高.     

人脐带间充质干细胞在大鼠睾丸间质内向Leydig细胞分化的实验研究

目的:研究人脐带间充质干细胞(HUMSCs)在大鼠睾丸间质内向Leydig细胞分化的可行性。方法:贴壁法获得HUMSCs,分别通过流式细胞表面抗原染色与三系分化验证其纯度与多向分化能力,用CM-Dil标记HUMSCs后将其移植入大鼠睾丸间质内,并在移植后4周与8周对大鼠睾丸进行免疫荧光染色观察HUMSCs的存活与分化情况,移植后8周获得大鼠睾丸细胞悬液,通过流式细胞染色检测HUMSCs表达Leydig细胞标志物3β-HSD判断细胞分化的效率,通过流式细胞分选获得悬液内CM-Dil标记的在睾丸内分化后的HUMSCs,并对其进行培养,培养3 d后收集培养液,检测其睾酮水平。结果:Leydig细胞标志物CYP11a1在HUMSCs移植8周后有表达,而在移植4周后未见表达。3β-HSD流式染色显示其分化效率约为14.5%,流式细胞分选后细胞可存活,且其培养液内能检测出睾酮。结论:HUMSCs在大鼠睾丸间质内可向Leydig细胞分化。     

非酶糖基化终末产物抑制大鼠睾丸Leydig细胞睾酮的生成

目的:研究非酶糖基化终末产物受体(RAGE)在大鼠睾丸Leydig细胞上的表达及非酶糖基化终末产物(AGEs)对大鼠睾丸Leydig细胞睾酮合成的抑制作用。方法:原代培养大鼠睾丸Leydig细胞,RT-PCR和免疫荧光技术检测RACE在大鼠Leydig细胞上表达,不同浓度AGEs处理Leydig细胞(25、50、100、200μg/ml),ELISA法测定睾酮分泌量。结果:RT-PCR和免疫荧光结果表明RAGE在大鼠睾丸Leydig细胞上表达,不同浓度AGEs处理后,人绒毛膜促性腺激素(hCG)诱导的Leydig细胞睾酮合成量呈剂量浓度依赖性下降,与对照组相比,50、100、200μg/ml AGEs处理组差异显著(P0.01)。结论:大鼠Leydig细胞上存在RAGE受体,AGEs显著抑制原代培养大鼠Leydig细胞睾酮的分泌。     

衰老的Leydig细胞中睾酮合成降低的机制

睾酮是男性体内主要的雄激素。老年男性体内睾酮水平下降使机体呈现肌肉松弛、骨质疏松和性欲减退等衰老现象，这与睾酮合成减少及其分泌节律改变密切相关。除了。肾上腺皮质有少量分泌以外，睾酮主要由睾丸间质中的Leydig细胞生成。对于衰老Leydig细胞中睾酮合成减少的机制的研究已有不少报道，影响     

Annexin 5刺激大鼠睾丸Leydig细胞后的差异表达蛋白的研究

目的:研究annexin 5作用于大鼠睾丸Leydig细胞后的差异表达蛋白。方法:原代培养大鼠睾丸Leydig细胞,1 nmol/L的annexin 5处理Leydig细胞24 h,提取细胞蛋白用二维电泳分离蛋白并进行比较。选择与对照组有明显差异表达的蛋白点,进行质谱分析。结果:获得了分辨率和重复性均很好的差异蛋白图谱。筛选出的显著差异表达的50个蛋白点,共有36个蛋白点被成功鉴定,其中annexin 5处理组中高表达的为23个,低表达的为13个。结论:初步建立了annexin 5作用于大鼠睾丸Leydig细胞后的差异蛋白谱,筛选并鉴定出的这些蛋白质可能是影响睾丸Leydig细胞合成睾酮过程中的关键蛋白,研究结果为阐明annexin 5调控大鼠睾丸Leydig细胞分泌睾酮的具体机制奠定了基础。     

干细胞微囊对大鼠Leydig细胞急性氧化应激损伤的保护机制研究

目的:揭示干细胞微囊改善Leydig细胞急性氧化应激损伤的机制。方法:实验组采用单侧睾丸切除的SD大鼠5例建立睾丸缺血-再灌注损伤模型,经尾静脉注射干细胞微囊进行干预。对照组采用单侧睾丸切除的SD大鼠5例经尾静脉注射等量PBS。再灌注2 h后HE染色检测睾丸组织中中性粒细胞浸润程度,TUNEL和caspase 3免疫组化染色检测Leydig细胞凋亡,Ki67免疫组化染色检测Leydig细胞增殖。结果:实验组大鼠睾丸间质血管中中性粒细胞黏附明显少于对照组,且TUNEL及caspase 3免疫组化结果显示Leydig细胞的凋亡数量明显少于对照组,Ki67免疫组化结果显示实验组Leydig细胞增殖数量多于对照组。结论:干细胞微囊可减少中性粒细胞在急性期的黏附,通过减少活性氧自由基的来源改善Leydig细胞的急性氧化应激损伤。     

中老龄雄性大鼠雄激素减低与代谢关系及发病机制的研究

目的:研究中老龄雄性大鼠雄激素减低与代谢的关系,雄激素减低发生的机制,尤其是睾丸间质细胞数量、形态及分泌功能的变化。方法:分别取9月龄(中青龄)和12、15、18、21月龄(中老龄)雄性SD大鼠每月龄组6只,静脉血检测大鼠代谢指标的变化,包括高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、甘油三酯(TG)、总胆固醇(TC)、血糖(Glu)、胰岛素(INS)、胰高血糖素(IRG)、瘦素(LP)含量;检测大鼠血清激素的改变,包括总睾酮(tT)、黄体生成素(LH)、卵泡刺激素(FSH);通过组织切片观察大鼠睾丸间质细胞形态学变化;使用人绒毛膜促性腺激素(hCG)、毛喉素(Forskolin)刺激体外培养的大鼠睾丸间质细胞,比较培养液中睾酮(T)浓度;采用脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测大鼠睾丸间质细胞凋亡率;分离称重内脏脂肪,计算内脏脂肪/体重比及Lee's指数。结果:中老龄大鼠的血清tT[(1.26±0.65)ng/ml]比中青龄大鼠[(3.24±0.38)ng/ml]显著降低(P<0.01);中老龄大鼠睾酮分泌指数(TSI)[(0.07±0.05)ng/mIU]比中青龄大鼠[(0.21±0.01)ng/mIU]显著降低(P<0.01);不同年龄大鼠睾丸间质细胞形态有较显著差异;体外培养的中老龄大鼠睾丸间质细胞在24、48、72 h内T分泌量均显著低于中青龄大鼠(P<0.05);中老龄大鼠睾丸间质细胞TUNEL阳性率[(17.36±1.31)%]比中青龄大鼠[(7.02±1.05)%]显著升高(P<0.01);中老龄组和中青龄组大鼠IRG、HDL、LDL、TG、TC及内脏脂肪含量有显著性差异(P<0.05)。结论:中老龄大鼠血清tT浓度显著低于中青龄大鼠,代谢指标随血清tT降低发生规律改变;中老龄大鼠雄激素减低可能与睾丸间质细胞分泌功能衰退、间质细胞数量减少及垂体功能衰退有关。     

Effect of unilateral cryptorchidism on the intertubular tissue of the adult rat testis: evidence for intracellular changes within the Leydig cells

Adult male rats were made unilaterally cryptorchid for 1, 2 or 4 weeks, and the morphological response of the Leydig cells was then studied using morphometric assessment of total Leydig cell volume and number per testis in abdominal and scrotal testes. Serum hormone levels were measured and the steroidogenic properties of isolated Leydig cells were evaluated by in-vitro stimulation with hCG and interstitial fluid (IF) obtained from normal rat testes. Total Leydig cell volume and number per testis were not altered in abdominal vs scrotal testes, although the volume of the abdominal testis was 46, 29 and 21%, respectively, of the volume of the contralateral scrotal testis after 1, 2 and 4 weeks. This reduction was accompanied by significant (P less than 0.05) elevation of the serum levels of FSH and LH, although serum testosterone levels were unchanged from the normal range. Despite the lack of quantitative alterations in Leydig cell morphology, hCG- and IF-stimulated testosterone production was significantly (P less than 0.01) greater by abdominal Leydig cells when compared with scrotal Leydig cells derived from the same animals. Ultrastructural examination of Leydig cells in situ suggested an increase in volumetric density of mitochondria in abdominal Leydig cells. Together with the enhanced steroidogenic responses of these cells, these findings suggest that disruption of spermatogenesis in the cryptorchid testis is accompanied by intracellular activation of Leydig cells. Since these effects were not exhibited by Leydig cells from the scrotal testis it is concluded that local factors within the cryptorchid testis are responsible, at least in part, for regulation of Leydig cell activity.     

衰老雄性大鼠睾丸组织脂质过氧化与睾酮水平和bcl-2基因表达的研究

目的:探讨衰老雄性大鼠睾丸组织脂质过氧化对血清睾酮(T)水平和睾丸间质(Leyd ig)细胞bcl-2基因表达的影响。方法:由D-半乳糖建立亚急性衰老模型,SD大鼠20只随机均分为对照(C)组和D-半乳糖(D)组,用分光光度计检测睾丸组织超氧化物歧化酶(SOD)和丙二醛(MDA)的含量、放免法检测血清中T水平以及免疫组化检测Leyd ig细胞bcl-2基因表达情况。结果:①SOD活性:D组(116±18.09)U/mg.prot显著低于C组(156±31.02)U/mg.prot(P<0.01);②MDA含量:D组(1.77±0.41)nmol/mg.prot明显高于C组(1.19±0.15)nmol/mg.prot(P<0.05);③血清T:D组(2.39±0.90)nmol/L显著低于C组(8.95±2.53)nmol/L(P<0.01);④bcl-2基因表达:D组(35.1±3.6)%明显低于C组(49.6±7.4)%(P<0.01)。结论:衰老大鼠睾丸组织脂质过氧化的变化影响T水平和Leyd ig细胞bcl-2基因表达。     

Morphometric study on leydig cells in capsulotomized testis of rats

AIM: To further clarify the changes occurred in the testicular capsulotomized rats. METHODS: In testicular capsulotomized and sham-operated rats, the cross sectional area, the nucleus diameter and the number of Leydig cells were morphologically analyzed by the Vidas Image Processing System connected to a microscope. RESULTS: In the capsulotomized animals, the cross sectional area of Leydig cells was gradually increased from 30 days onwards. There was no obvious change in the nucleus diameter of Leydig cells. However, The Leydig cell number was significantly increased from day 30 onwards. CONCLUSION: In rats, testicular capsulotomy may induce hyperplasia/hypertrophy of Leydig cells in the testis.     

大鼠睾丸Leydig细胞的培养和鉴定

目的:研究体外培养大鼠睾丸Leyd ig细胞的有效方法。方法:原代培养大鼠睾丸Leyd ig细胞,用4 U/m l人绒毛膜促性腺激素(hCG)作用细胞,对照组未用hCG,放射免疫法测定培养液中睾酮浓度,3β羟类固醇脱氢酶(3β-HSD)免疫组化染色观察睾丸Leyd ig细胞形态和生物学特性。结果:培养细胞成分均一、增殖旺盛、分化率高。接种72 h后大鼠睾丸Leyd ig细胞纯度达95%。接种后24 h内,hCG刺激组较对照组睾酮分泌量明显提高(P<0.05)。结论:体外培养的睾丸Leyd ig细胞可分泌高浓度的睾酮;睾丸Leyd ig细胞的纯化和培养方法的建立,可为中老年男性雄激素部分缺乏综合征睾酮替代治疗的基础和临床研究提供一条可行的思路。     

Experimental cryptorchidism in the adult mouse. III. Qualitative and quantitative electron microscopic morphology of Leydig cells.

The morphology of Leydig cells of control and 28-day-old cryptorchid mice was studied by electron microscopy and stereologic techniques. Leydig cell profiles of control mice were larger in section when compared to cryptorchid mice, but no differences were observed in the distribution of organelles in Leydig cells in the two groups. Quantitatively, the absolute volumes of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), mitochondria, lysosomes, multivesicular bodies, peroxisomes, cytoplasmic matrix, nucleus, lipid droplets, membrane whorls, ribosomal aggregates, and annulate lamellae per Leydig cell were reduced significantly after 28 days of cryptorchidism. However, the absolute volumes of these organelles per testis were not significantly different between control and cryptorchid mice, due to the increase in Leydig cell number per testis in the cryptorchid testis, compared to the controls, except that the absolute volume of Golgi per Leydig cell was not significantly different between control and cryptorchid rats, but the absolute volume of Leydig cell Golgi was significantly lower in control rats. Based on these results, we conclude that, morphologically, a 28-day cryptorchid mouse Leydig cell clearly approximates a "half unit" of a control Leydig cell.     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.     

Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens,or with a GnRH antagonist,on Leydig cell development and function through puberty into adulthood

This study addressed whether reduced Sertoli cell number or manipulation of the neonatal hormone environment has an influence on final Leydig cell number per testis in the rat, by applying neonatal treatments known to affect these parameters, namely administration of a GnRH antagonist (GnRHa) or diethylstilboestrol (DES, in doses of 10, 1 or 0.1 microg per injection). The effect of treatment with either of two 'environmental oestrogens', bisphenol-A (Bis-A) or octylphenol (OP), was also evaluated. Leydig (3beta-hydroxysteroid dehydrogenase immunopositive) cell development and function (plasma testosterone levels) were studied through puberty into adulthood. Treatment with GnRHa impaired testis growth, Leydig cell (nuclear) volume per testis and testosterone levels during puberty, when compared with controls, but final Leydig cell volume/number in adulthood was comparable with controls. As adult testis weight was reduced by 45% in GnRHa-treated rats, the percentage Leydig cell volume per testis was approximately double (p < 0.01) that in controls, and also at day 35. Testosterone levels in adulthood in GnRHa-treated rats were lower (p < 0.01) than in controls but were within the lower end of the normal range. Treatment with DES caused largely dose-dependent suppression of testis growth, Leydig cell (nuclear) volume per testis and testosterone levels up to day 35. Although by adulthood, Leydig cell volume/number per testis was comparable with controls in DES-treated rats, testosterone levels remained grossly subnormal. Neonatal treatment with either Bis-A or OP had little consistent effect on any of the parameters studied except that both treatments significantly elevated testosterone levels on day 18, as did treatment with DES-0.1 microg. The present findings are interpreted in the context of what is known about the hormonal regulation of Leydig cell development. These lead to the conclusion that final Leydig cell number per testis is not determined by the number of Sertoli cells per testis and appears not to be influenced in any major way by gonadotrophins, androgens or oestrogens in the first 2 weeks of postnatal life. This implies that adult Leydig cell number may be determined prior to birth.