雄激素对快速老化小鼠海马神经元凋亡及超微结构的影响

目的:探讨雄激素对快速老化小鼠海马神经元凋亡及超微结构的影响.方法:7月龄雄性快速老化小鼠(SAMP8)随机分为假手术对照组、去势组及去势 雄激素补允治疗组.十一酸睾酮(TU)剂量为37.4 mg·kg-1·15 d-1.雄激素补充治疗45 d后,通过Nissl染色观察海马神经元数量和形态的变化;TUNEL法检测细胞凋亡;流式细胞仪检测细胞的凋亡率;透射电镜观察超微结构的改变.结果:去势组海马神经元数量明显减少,凋亡数量和凋亡率显著增加,超微结构病理变化严重.雄激素补充治疗后,观察结果与假手术对照组相比无统计学意义.结论:去势后雄激素缺乏可导致海马神经元异常凋亡增加,数目减少,结构受损.雄激素补充治疗可减轻神经元损伤,这可能是其改善SAMP8小鼠学习记忆功能作用机制之一.     

雄激素对SAMP8小鼠学习记忆能力及海马神经元的影响

目的探讨雄激素对SAMP8小鼠学习记忆能力及海马CA1区神经元的影响。方法7月龄雄性SAMP8小鼠30只,随机分为假手术组、去势组及去势 雄激素补充治疗组。十一酸睾酮(TU)剂量为37.4mg/(kg.15d)。雄激素补充治疗45d后,通过Morris水迷宫观察小鼠学习记忆能力;用苏木素伊红染色,Aβ免疫组织化学及图像分析观察海马CA1区神经元的变化。结果1.Morris水迷宫中,去势组定位航行试验潜伏期明显长于其他组(P<0.05),探索实验跨越平台次数减少(P<0.05)。TU补充治疗能改善学习记忆能力,与假手术组比较差异无统计学意义(P>0.05)。2.去势组海马CA1区弥漫性空泡变性,细胞排列疏松、紊乱,核深染、固缩;Aβ阳性神经元染色深,其数量及吸光度(A)明显高于其他组(P<0.05)。结论去势后,雄激素缺乏可导致学习记忆能力下降,海马神经元严重损伤,雄激素补充治疗可减轻神经元损伤,改善学习和记忆能力。     

双氢睾酮对快速老化小鼠海马CA1区突触可塑性和N-甲基-D-天冬氨酸受体1的影响

目的 探讨双氢睾酮(DHT)对快速老化小鼠(SAMP8)海马CA1区突触可塑性和N-甲基-D-天冬氨酸受体1(NMDAR1)的影响.方法 6月龄雄性SAMP8小鼠21只随机分为假手术组、去势组及去势+DHT补充治疗组,每组7只.DHT剂量为1mg/(kg·d),皮下注射21d后,通过Golgi染色观察海马CA1区顶树突树突棘的变化;免疫组织化学及图像分析检测突触素和NMDAR1表达的改变.结果 1.Golgi染色,去势组海马CA1区顶树突树突棘个数明显减少;DHT补充治疗后,树突棘个数明显增多.2.去势组海马CA1区突触素和NMDAR1的表达明显减少,平均吸光度值明显低于其他组(P<0.05).DHT补充治疗能明显增加突触素和NMDAR1的表达.结论 DHT可调节海马CA1区突触可塑性,使树突棘密度增多.DHT对突触可塑性的影响可能与其调节锥体细胞的NMDAR1有关.     

快速老化小鼠海马突触体内NMDA受体的表达变化

目的:观察NMDA受体在SAMP8小鼠海马突触体内的表达变化。方法:首先应用生物化学的方法分离海马突触蛋白,并对其进行鉴定。其次,Western Blot检测NMDA受体的主要亚基NR1、NR2A和NR2B在SAMP8小鼠海马突触体内的表达变化。结果:PSD-95和synaptophysin特异性抗体检测显示突触蛋白的分离是成功的。SAMP8小鼠海马内NR1、NR2A和NR2B在突触的表达均显著低于SAMR1小鼠。进一步分析NR1、NR2A和NR2B蛋白在突触的表达量占总表达量的比值,SAMP8小鼠同样显著低于SAMR1小鼠,而SAMR1和CD-1小鼠间没有显著性差异。结论:SAMP8小鼠海马突触体内NR1、NR2A和NR2B的蛋白表达水平均显著性降低,推测NMDA受体在突触表达水平的降低可能是导致受体功能失调,激发突触功能损伤信号途径的原因之一,进而导致SAMP8小鼠学习记忆功能的下降。     

雌激素对SAMP8小鼠学习记忆及海马CA1区神经元的影响

目的 观察雌激素对SAMP8鼠学习记忆及海马神经元的影响. 方法 将6月龄SAMP8鼠45只分为假手术组(sham组)、去卵巢组(OVX组)和去卵巢+雌激素组(OVX+E组)3组,并用同龄正常老化SAMR1小鼠作为同源对照组.采用Morris水迷宫实验检测小鼠学习记忆能力,苏木素-伊红染色和免疫组织化学显示海马CA1区神经元及神经型一氧化氮合酶(nNOS)阳性神经元的变化,流式细胞术检测其nNOS的表达量. 结果 OVX组与sham组相比,逃避潜伏期明显延长(P<0.05),跨越平台次数显著减少(P<0.05).雌激素补充治疗能改善学习记忆能力,与sham组比较差异无统计学意义(P>0.05);OVX组海马CA1区神经元病变严重,且CA1区nNOS阳性神经元的数量、吸光度和海马nNOS荧光指数(FI)值均低于sham组(P<0.05);给予雌激素后,OVX+E组各项实验结果与OVX组相比差异有统计学意义(P<0.05),与sham组相比,差异无统计学意义(P>0.05). 结论 雌激素能够改善小鼠的学习记忆能力,有效保护海马CA1区的神经元,提高海马CA1区nNOS阳性神经元的表达.     

SAMP8小鼠记忆能力与海马CA1区神经元脱失相关性研究

目的探讨快速老化小鼠(SAMP8)学习记忆能力改变与海马CA1区神经元脱失间的关系。方法随机选取6月龄雄性P8和R1小鼠各15只,使用水迷宫实验分别检测两组小鼠的逃避潜伏期和跨越平台次数,尼氏染色观测两组小鼠海马CA1区神经元形态和数量变化。结果与R1小鼠比较,P8小鼠水迷宫实验逃避潜伏期明显较长,空间探索实验跨越平台次数P8小鼠明显减少,且P8小鼠游泳轨迹呈现无目的性环游,而R1小鼠游泳轨迹多集中于原隐匿平台象限;海马CA1区神经元层次排列紊乱,数量减少;逃避潜伏期与海马CA1区神经元数量呈负相关,跨越平台次数与海马CA1区神经元神经元数量呈正相关。结论快速老化P8小鼠的学习记忆能力明显下降,且与海马CA1区神经元的结构和数量变化有密切关系,该品系小鼠为阿尔茨海默病的研究提供了较为理想的动物模型。     

睾酮对β-淀粉样蛋白1-42寡聚体联合去势大鼠海马内突触素表达的影响

目的 :研究睾酮对β-淀粉样蛋白1-42(Aβ1-42)寡聚体CA1区注射联合去势大鼠脑内突触素表达的影响。方法 :双侧海马CA1区注射Aβ1-42寡聚体同时去势建立动物模型。动物分为空白对照组、模型组、氟他胺组、睾酮组及氟他胺+睾酮组。采用H-E染色法检测海马锥体细胞数量。采用免疫组织化学法及免疫印迹检测各组大鼠脑内突触素的表达量。结果:锥体细胞数量和突触素表达量在睾酮组和空白对照组比较高,两组间差异无统计学意义,但睾酮组与其他各组间差异有统计学意义。睾酮组,氟他胺+睾酮组的锥体细胞数量和突触素表达量均高于模型组。结论 :睾酮可以通过雄激素受体途径增加锥体细胞的生存率,可以增加突触素在模型动物海马中的表达。     

快速老化小鼠海马中细胞外基质金属蛋白酶诱导因子的年龄相关性变化

目的:观察细胞外基质金属蛋白酶诱导因子(extracellular matrix metalloproteinase inducer,CD147)在快速老化小鼠(senescence-accelerated-prone mouse 8,SAMP8)海马中随年龄变化的趋势。方法:应用免疫组织化学和Western blot的方法,观察CD147在SAMP8小鼠海马中的年龄相关性变化。结果:免疫组织化学结果显示SAMP8小鼠海马区CD147免疫反应呈阳性,且随年龄增加无明显的细胞分布变化。Western blot分析数据表明随年龄增加CD147蛋白水平逐渐下降;相对于3月龄小鼠,12月龄小鼠的CD147蛋白水平下降了约53%(P<0.05)。结论:随年龄的增加,海马区CD147蛋白水平逐渐下降,推测其与阿尔茨海默病(Alzheimer’s disease,AD)相关。     

睾酮对可溶性Aβ1-42寡聚体注射联合去势诱导的空间学习记忆损害的改善作用

目的:探讨睾酮改善复合模型大鼠空间学习记忆损害的作用机制.方法:采用双侧海马CA1区分别注射Aβ1-42寡聚体10 μg(2 μg/μl)联合去势,建立阿尔茨海默病(AD)样动物模型(复合模型).在睾酮时效与量效实验的基础上复合模型大鼠随机分为4组,即复合模型组、睾酮组(皮下注射睾酮0.75mg/d,连续8 d)、氟他胺组(双侧海马分别注射氟他胺5 μg/d,连续8 d),氟他胺+睾酮组(联合给药).结果:血清睾酮浓度给药48 h后达高峰,与给药剂量呈高度依赖性.0.75mg组对空间学习记忆的改善作用最明显,而氟他胺可阻断睾酮的作用,但其对血清睾酮浓度无影响.结论:复合模型大鼠更适合用于老年期AD发病机制的研究,睾酮对复合模型大鼠空间学习记忆的改善作用可能是通过雄激素受体途径完成.     

大鼠空间辨别性学习记忆活动对海马结构Ephrin-A3表达的影响

为了探讨大鼠获得空间辨别性学习记忆活动对导向分子ephrin-A3表达的影响,本研究采用免疫组织化学染色和Western blot方法对模型组、游水组和对照组大鼠海马内ephrin-A3的表达部位和表达量进行对比研究。免疫组织化学染色结果显示模型组、游水组和对照组海马结构内各亚区和各层内均可见ephrin-A3样免疫阳性产物分布,模型组大鼠海马CA3区辐射层和腔隙层尤为明显;Western blot方法检测模型组大鼠海马结构内ephrin-A3阳性产物平均积分光密度值明显高于游水组和对照组。模型组内训练14d的大鼠海马结构内ephrin-A3阳性产物平均积分光密度值高于训练7d和21d。以上研究结果提示eph-rin-A3的表达与大鼠空间辨别性学习记忆活动有密切关系。     

Novel molecular defects in the androgen receptor gene of Mexican patients with androgen insensitivity

The androgen insensitivity syndrome (AIS) is an X-linked form of male pseudohermaphroditism caused by mutations in the androgen receptor (AR) gene. In the present study, we analyzed the AR gene in 8 patients, 4 sporadic and 2 familial cases with the syndrome, using exon-specific polymerase chain reaction, single-stranded conformational polymorphism and sequencing analysis and identified six new single base mutations, including one nonsense mutation at the hinge region of the receptor. These molecular lesions occurred in the steroid-binding domain (SBD) and all but one affected the first nucleotide of their respective codons. A nonsense mutation in exon 4, which converts a glutamine into a premature termination signal (Q657stop), a missense mutation changing arginine instead of glycine (G743R) and a conservative substitution of leucine with valine at amino acid 830 (L830V) were detected in patients with CAIS. Three other missense mutations located in exons 4 (L701I), 5 (A765S), and 6 (Q802R) were present in individuals bearing a partial form of AIS. These data allow us to reaffirm the view that nonsense mutations in the AR results almost invariably in a CAIS phenotype and underly the importance of the SBD for the AR functional activity.     

Mechanisms of inhibition of synaptic transmission in the sympathetic ganglia of rats with alloxan diabetes

In experiments on isolated cranial sympathetic ganglia of rats with alloxan diabetes the preganglionic nerve was stimulated and combined presynaptic action potentials (APs) and EPSPs of neurons of the ganglion were recorded. In rats with moderately severe alloxan diabetes progressive depression of rhythmic APs of the ganglion correlated completely with inhibition of the excitatory power of the presynaptic endings, i.e., with a decrease in the liberation of mediator and exhaustion of its operative fraction. In rats with the severe form of diabetes postsynaptic inhibition of neurons of the ganglion also was observed. The dynamic characteristics of conversions of the mediator, assessed on the basis of examination of posttetanic potentiation patterns, showed a very small change in the output of mediator but a substantial (by 38%) depression of replenishment of the mediator reserves per second compared with the control.Laboratory of Physiology of the Autonomic Nervous System, I. P. Pavlov Institute of Physiology, Academy of Medical Sciences of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 9, pp. 272–274, September, 1978.     

完全型雄激素不敏感综合征雄激素受体基因突变分析

目的 对完全型雄激素不敏感综合征一家系雄激素受体(androgen receptor,AR)基因进行突变检测;并对发现突变的基因进行分析.方法 应用PCR扩增、DNA序列测定等技术分析所有AR基因外显子及其邻近DNA序列片段;应用核苷酸内切酶诊断方法观察其是否存在于正常人群;应用跨物种比对方法探讨突变所在位置的保守性.结果 3例患者AR基因第4外显子均发生E681D(GAG→GAT)错义突变,患者母亲为此突变杂合子携带者;患者父亲未见异常;正常人群未发现AR基因E681D突变;681位谷氨酸在不同物种间高度保守.结论 AR基因E681D(GAG→GAT)突变可能是导致完全型雄激素不敏感综合征新的突变方式.     

雄激素受体基因新突变引起的完全型雄激素不敏感综合征

目的 对1例完全型雄激素不敏感综合征(complete androgen insensitivity syndrom,CAIS)的雄激素受体(androgen receptor,AR)基因进行分析,寻找致病突变.方法 提取外周血全基因组DNA,扩增位于X染色体上的AR基因所有8个外显子及邻近外显子与内含子之间的剪切位点DNA序列,对扩增片段直接进行DNA序列测定,与基因库中的序列进行比对.结果 该病例AR基因第1外显子的441位密码子发生无义突变(GAA→TAA),由编码谷氨酸(Glu)变为终止密码,导致雄激素受体蛋白翻译至441位时终止,产生没有功能的氨基酸多肽残段.结论 Glu441stop(GAA→TAA)是一种导致CAIS的AR基因新的突变方式,之前在世界范围内均未见报道,丰富了AR基因突变谱,增加对CAIS发病机制的了解.

Abstract：

Objective To identify the mutation of human androgen receptor gene(AR) in a patient with complete androgen insensitivity syndrome (CAIS). Methods DNA sequences of 8 exons and their exon/intron boundaries of the AR gene in the patient were amplified by PCR and directly sequenced. Results DNA sequencing revealed a nonsense mutation in exon 1, resulting in a change of codon 441 GAA (glutamic acid) to a stop codon (TAA). Conclusion A novel mutation Glu441stop (GAA to TAA) of the androgen receptor gene leading to complete androgen insensitivity syndrome was identified in this study in a Chinese patient. It may help us further understanding the pathogenesis of CAIS.     

Absence of hepatotoxicity after long-term, low-dose flutamide in hyperandrogenic girls and young women

BACKGROUND: Flutamide is a pure non-steroidal anti-androgen that may be hepatotoxic, when given in high-dose (750 mg/d). Low- to ultralow-doses (250-62.5 mg/day) have been recently explored in patients with Polycystic Ovary Syndrome (PCOS), and these lower doses were found to confer benefit on multiple PCOS markers. There is a need for evidence on the potential hepatotoxicity of low- and ultralow-dose flutamide therapy. METHODS: We assessed circulating levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as markers of hepatotoxicity in a total of 190 hyperandrogenic girls and young women receiving low- or ultralow-dose flutamide because of established (n = 150) or incipient (n = 40) PCOS without obesity. Assessments were performed before start of flutamide, after 3 months, and subsequently at least twice yearly. RESULTS: AST and ALT results were normal at baseline, and they remained so on flutamide treatment, including between 3 months and last assessment, which was after a mean time of 19 months on low- or ultralow-dose flutamide (range 3-54 months). None of the AST or ALT levels at any time during flutamide treatment was > or = 45 U/L. CONCLUSION: We found no evidence for hepatotoxicity in 190 hyperandrogenic girls or young women receiving low- or ultralow-dose flutamide for up to 54 months. These results may represent a first step in a long process whereby the status of low- and ultralow-dose flutamide may gradually evolve from 'absence of evidence on toxicity' towards 'evidence of absence of hepatic toxicity'. Ultralow-dose flutamide may become a key component within future therapies for hyperandrogenic states in girls and young women.     

The clinical and molecular spectrum of androgen insensitivity syndromes

Androgen insensitivity syndromes (AIS) are due to end-organ resistance to androgenic steroids in males leading to defective virilization of the external genitalia. The phenotype encompasses a wide array of genital ambiguity and may range from completely female to undervirilized but unequivocally male with infertility. This disorder is caused by mutations of the androgen receptor and is an X-linked recessive trait. We have studied 47 patients with AIS and have characterized the underlying molecular abnormality in the androgen receptor gene. Twenty patients had complete AIS and twenty-seven had partial AIS. Of the latter, 11 were of predominantly female phenotypic appearance and gender was assigned accordingly, while 16 were raised as males. Within the group of complete AIS, two patients had gross deletions within the gene, one had a small deletion, and one had an insertion. In the other patients with complete AIS, as well as all individuals with partial AIS, single nucleotide substitutions within the coding region were detected, each leading to an amino acid alteration. Seven codons were involved in more than one mutation in different cases. In addition, in one patient with spinal and bulbar muscular atrophy, an elongation of a glutamine-repeat was characterized. We conclude that mutations in the androgen receptor gene may be present throughout the whole coding region. However, our study provides evidence that several mutational hot spots exist. © 1996 Wiley-Liss, Inc.     

Rapid detection of a mutation hot-spot in the human androgen receptor

Mutations of the human androgen receptor gene may disturb sexual development in males, and are inherited as an X-linked recessive trait. The vast majority of the mutations are familial. We have identified a large kindred with complete androgen insensitivity syndrome (CAIS) without detectable androgen-binding in genital skin fibroblasts. A single nucleotide substitution (C-to-T transition) was identified, resulting in an Arg⁸⁵⁵ to Cys in the androgen binding domain. To date, four independent CAIS families have been reported with this specific mutation that coincides with the propensity of cytosines at CpG dinucleotides to methylate. An allele-specific oligo-nucleotide assay was developed that allowed for the rapid and specific identification of this mutation hot-spot in individuals with androgen receptor incensitivity syndromes.     

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated cosegregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acids androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.     

DonnahooⅣ型阴茎弯曲畸形阴茎皮肤及纤维性尿道组织中雄激素受体的表达及临床意义

目的：检测Donnahoo Ⅳ型阴茎弯曲畸形阴茎皮肤及纤维性尿道组织中雄激素受体(androgen receptor,AR)的表达情况,探讨AR异常在疾病发病中的作用及临床意义。方法：以Ⅳ型阴茎弯曲患者25例作为研究对象,手术治疗纠正弯曲畸形,术中收集阴茎背、腹侧皮肤和纤维性尿道组织。以包皮过长患者18例作为正常对照,行包皮环切术收集正常阴茎背、腹侧包皮组织。组织标本经免疫组化LSAB法染色后观测AR表达规律,行统计学分析。结果：AR在正常阴茎包皮和Ⅳ型阴茎弯曲标本中有阳性表达。正常对照阴茎背、腹侧包皮AR阳性表达率为(62.94±5.40)%、(62.87±5.33)%;Ⅳ型阴茎弯曲背、腹侧皮肤和纤维性尿道AR阳性表达率为(58.63±2.66)%、(57.23±2.04)%、(53.71±2.15)%,AR表达较正常对照均显著减少(P<0.05);Ⅳ型阴茎弯曲自身背、腹侧皮肤间相比,AR表达无统计学差异(P>0.05),AR在纤维性尿道的表达较自身皮肤显著减少(P<0.05)。分层分析发现,轻中度弯曲背、腹侧皮肤及纤维性尿道AR阳性表达率为：(59.37±3.11)%、(58.75±3.20)%、(55.48±2.86)%,AR表达较正常对照无统计学差异(P>0.05);重度弯曲背、腹侧皮肤及纤维性尿道AR阳性表达率为：(55.21±3.32)%、(53.69±4.09)%、(46.17±3.65)%,AR表达较正常对照均显著减少(P<0.05);与轻中度弯曲比较,重度弯曲纤维性尿道AR表达显著减少(P<0.05)。结论：Donnahoo Ⅳ型阴茎弯曲畸形阴茎皮肤及纤维性尿道组织中AR表达显著减少,自身背、腹侧皮肤间AR表达并无差异,重度弯曲纤维性尿道AR表达减少更为明显。     

一种导致完全性雄激素不敏感综合征的AR基因移码突变的鉴定

目的 对1个男性假两性畸形完全性雄激素不敏感综合征的家系雄激素受体(androgen receptor,AR)基因进行突变检测,并分析其致病原因.方法 用PCR扩增及DNA测序等技术分析男性假两性畸形先证者候选基因AR的外显子及外显子内含子接头序列,根据检测到的突变位点情况,检测患者及其家系其他成员的相应DNA区段的碱基序列.结果 先证者及其家庭成员共3例患者均为AR基因1910delA的移码突变.其母亲为AR基因突变杂合子,是此疾病的携带者.该突变导致AR基因的N637I(AAU→AUC)、L638*(CTG→TGA)改变,导致AR蛋白283个氨基酸的截短.正常人群未发现该移码突变,该突变尚未见文献报道.结论 基因水平确定了该家系为AR基因突变引起的完全性雄激素不敏感综合征男性假两性畸形家系,同时发现了1种AR基因病理性新突变.