【作者】 岂春晓；

【导师】 石葛明；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2014， 博士

【摘要】 抽动-秽语综合症（Tourette Syndrome，TS）是临床较为常见的儿童行为障碍综合征，是以面部、四肢以及躯干部肌肉不自主抽动伴有喉部异常发音即猥秽语言为特征的综合症候群，多发生于男童，男女发病比例为3-9:1，我国部分地区甚至高达10.6:1。TS患者常伴有至少一种行为或情绪障碍，大约1/3的病人伴有强迫症。研究发现TS具有较明显的病程特征，表现为儿童期发病，青春期病症加重，成年后运动症状逐渐好转，甚至消失。有研究报道，TS症状严重的患儿尿液含有较高水平的睾酮；可伴有雄激素增多症的表现；抗雄激素处理则显著缓解TS成年患者的症状。这些表明其发病可能和患儿体内雄激素水平紊乱有关。近年来的临床资料暗示多巴胺（DA）能神经体系的功能障碍与TS密切相关。利用配基神经显像技术检测到TS患儿（6-12岁）新纹状体多巴胺转运体（DAT）信号增强，TS青少年患者（12-17岁）尾状核有异常的多巴脱羧酶活性增强。临床资料显示，安非他命可以增强TS患者壳及腹侧纹状体多巴胺的释放，多巴胺受体拮抗剂则具有抑制TS患儿抽动症状的功效。上述研究结果表明TS发病可能和脑内多巴胺能神经体系存在密切的关系。睾酮在男性主要由睾丸间质细胞产生，具有脂溶性特点，可透过血脑屏障，作用于中枢神经系统。大量研究显示雄激素可以影响多巴胺能神经元的功能活动。胚胎期给予雄激素，可以导致成年期大鼠前额叶皮质多巴胺神经元功能活动增强；青春期给予雄激素可以明显引起大鼠的攻击行为增多以及下丘脑多巴胺D2受体表达增加；长期给予成年大鼠雄激素促进了纹状体DAT的表达。脑内DA能神经元主要集中在中脑黑质致密部（SNc）及腹侧被盖区（VTA）等处。多巴胺黑质纹状体环路及VTA皮质环路分别参与躯体运动及认知、思维能力的调控。TS患儿发育过程中的异常行为是否与雄激素水平紊乱、进而改变了脑内多巴胺能神经体系的功能活动有关，目前尚不清楚。因此，本研究以雄性Wistar乳鼠为实验对象，分别通过早期皮下注射丙酸睾酮(testosterone propionate, TP组）、雄激素受体抑制剂氟他胺（flutamide，Flu组）以及氟他胺+丙酸睾酮（(Flu+TP组），建立大鼠实验动物模型。利用旷场实验、液相色谱-串联质谱、免疫印迹和RT-PCR技术观察3周龄（幼年）、7周龄（青春期）和6月龄（成年）实验大鼠的旷场行为以及中脑DA能神经元相关指标的表达变化，探讨雄激素在早期发育过程中对行为的影响及其机制，分析中脑DA能神经元改变在这一过程中所起的作用，期望所获结果能为探寻TS的发病提供一定的实验依据。第一部分：丙酸睾酮改变雄性大鼠发育早期的旷场行为目的：探讨生后TP早期处理对雄性大鼠旷场行为的影响。方法：利用旷场实验观察大鼠模型在3周龄、7周龄及6月龄旷场行为相关参数的变化；称量大鼠及其性腺器官和脑垂体的重量；以放射免疫法检测上述三个时间点大鼠模型血清睾酮、卵泡刺激素、黄体生成素含量的变化。结果：1通过旷场实验发现，在3周龄和7周龄时，与对照组相比，TP组各种静止闻嗅、运动行为、探索行为及理毛行为参数明显增加；而Flu+TP组旷场实验多项行为参数未表现明显改变，仅爬行的数量降低以及7周龄理毛次数增加。与TP组相比，Flu+TP组的静止闻嗅、运动行为、探索行为及理毛行为指标明显减低。TP早期处理和Flu早期干预对3周龄、7周龄以及6月龄大鼠的趋触行为无明显改变。6月龄时，各组大鼠行为均无明显改变。2TP早期处理大鼠3周龄、7周龄及6月龄各组大鼠之间体重没有明显差异。3TP早期处理大鼠精囊、垂体和睾丸重量的改变在3周龄时，与对照组相比，TP组精囊的重量增加了1028%（P<0.01），睾丸的平均重量降低了24%（P<0.01）；Flu组精囊的重量降低了16%（P<0.05）；Flu+TP组精囊的重量增加了201%（P<0.01），睾丸的平均重量降低了60%（P<0.01）。与TP组相比，Flu+TP组精囊的重量和睾丸的平均重量分别降低了73%（P<0.01）和47%（P<0.01）。与Flu组相比，Flu+TP组精囊的重量增加了256%（P<0.01），睾丸的平均重量降低了62%（P<0.01）。各组垂体的重量无明显变化（P>0.05）。在7周龄时，与对照组相比，TP组睾丸的平均重量降低了25%（P<0.01），精囊腺没有差异；Flu组睾丸的平均重量无明显变化（P>0.05）；Flu+TP组睾丸的平均重量降低了62%（P<0.01）。与TP组相比，Flu+TP组睾丸的平均重量降低了49%（P<0.01）。与Flu组相比，Flu+TP组睾丸的平均重量降低了58%（P<0.01）。各组精囊和垂体的重量无明显变化（P>0.05）。在6月龄时，各组精囊、垂体和睾丸重量无明显变化（P>0.05）。4TP早期处理大鼠血清T、LH和FSH水平的改变在3周龄时，与对照组相比，TP组血清T的浓度增加了4400%（P<0.01）；Flu+TP组血清T的浓度增加了4229%（P<0.01）。与Flu组相比，Flu+TP组血清T的浓度增加了3996%（P<0.01）。各组血清LH和FSH水平无明显变化（P>0.05）。在7周龄时，与对照组相比，TP组血清T的浓度降低了53%（P<0.01）；Flu+TP组血清T的浓度降低了88%（P<0.01）。与TP组相比，Flu+TP组血清T的浓度降低了74%（P<0.01）。与Flu组相比，Flu+TP组血清T的浓度低了88%（P<0.01）。各组血清LH和FSH水平无明显变化（P>0.05）。在6月龄时，各组血清T、LH和FSH水平无明显变化（P>0.05）结论：1皮下注射TP可使乳鼠保持在高水平的血睾状态。2乳鼠TP早期处理导致幼年和青春期的旷场行为实验多项行为参数显著增加，暗示发育早期高水平的雄激素可能改变了脑内有关神经信号的传递。第二部分：丙酸睾酮促进雄性大鼠发育早期中脑DA能神经元DA、DOPAC及HVA的表达目的：观察生后TP早期处理对雄性大鼠黑质-尾壳核及腹侧被盖区-伏核DA能神经元神经递质DA及其代谢产物的影响，探讨TP改变发育早期旷场行为与DA信号传递变化可能存在的联系。方法：利用液相色谱-串联质谱（LC-MS/MS）法检测3周龄、7周龄及6月龄实验大鼠中脑DA能神经元投射靶区尾壳核、伏核的DA、DOPAC和HVA的表达变化。结果：与对照组相比，3周龄TP组尾壳核DA、DOPAC、 HVA以及伏核DA、DOPAC的表达显著增加。DOPAC+HVA/DA、DOPAC/DA和HVA/DA无明显变化；Flu组尾壳核和伏核DA的表达明显减低，尾壳核DOPAC+HVA/DA和DOPAC/DA的比值增加，伏核HVA/DA的比值增加；Flu+TP组尾壳核DA的表达降低，DOPAC+HVA/DA和DOPAC/DA的比值明显增加，伏核无显著改变。与TP组相比，Flu+TP组两个核团DA、DOPAC的浓度明显降低，伏核HVA降低不明显。与对照组相比，7周龄TP组尾壳核和伏核DA、DOPAC和HVA的表达明显增加，DOPAC+HVA/DA、DOPAC/DA和HVA/DA的比值无明显变化；Flu组尾壳核HVA的表达增加了25%，伏核所有指标没有变化；Flu+TP组尾壳核各项指标均无明显变化。与TP组相比，Flu+TP组尾壳核各项指标均无明显变化，伏核DA的浓度降低了26%。6月龄各组大鼠神经递质及代谢产物各项指标比值均无明显变化。结论：乳鼠TP早期处理促进其幼年和青春期中脑DA能神经元DA、DOPAC及HVA的表达，给予Flu则抑制了外源性TP的这种作用。第三部分：丙酸睾酮早期处理促进雄性大鼠发育早期中脑DA能神经元TH、DAT、MAOA、MAOB及COMT的表达目的：观察乳鼠TP早期处理对中脑DA能神经元功能活动强弱相关指标表达的影响，探讨其表达变化与TP促进DA能神经元表达DA、DOPAC及HVA的相互关系。方法：通过免疫印迹方法以及实时定量RT-PCR方法来检测丙酸睾酮处理后雄性大鼠黑质TH，DAT及其mRNA以及TH、DAT、MAOA、MAOB、COMT的mRNA的表达。结果： TP早期处理对三个发育阶段黑质（SN）、尾壳核（CPu）、腹侧被盖区（VTA）和伏核（Acb）脑区TH和DAT的改变在3周龄和7周龄时，与对照组相比，TP组大鼠处理黑质,尾壳核，腹侧被盖区及伏核TH和DAT表达均明显增加。黑质、腹侧被盖区TH mRNA和DAT mRNA表达升高。与TP组相比，Flu+TP组黑质，尾壳核，腹侧被盖区，伏核TH和DAT的表达明显减少。黑质、腹侧被盖区TH mRNA和DAT mRNA表达降低。在6月龄时，各组黑质,尾壳核，腹侧被盖区，伏核TH和DAT的表达无明显变化。MAOA、MAOB及COMT mRNA的表达改变在3周龄和在7周龄时，与对照组相比，TP组处理黑质和腹侧被盖区MAOA mRNA、MAOB mRNA以及COMT mRNA的表达均明显增加。与TP组相比，Flu+TP组黑质和腹侧被盖区MAOAmRNA、MAOBmRNA以及COMT mRNA的表达均明显降低。在6月龄时，各组黑质和腹侧被盖区MAOAmRNA、MAOBmRNA以及COMT mRNA的表达无明显变化。结论：乳鼠TP早期处理改变其幼年和青春期中脑DA能神经元TH、DAT及其mRNA以及MAOA、MAOB、COMT mRNA的表达水平，表现为TP的增强效应；Flu能够抑制外源性TP的这种效应，暗示中脑DA神经元神经的信号传递的改变与TP调控神经递质DA代谢相关酶及DA转运体的表达有关。更多还原

【Abstract】 Tourette Syndrome(TS) is a common children’s behavior disordersyndrome in clinic. It is a comprehensive syndrome characterized byinvoluntary muscles twitch on the face, limbs and trunk with abnormallaryngeal pronunciation, thus dirty language. This disease occurred mosty inboys, and incidence ratio of men and women is3-9:1, while in parts of ourcountry it is even as high as10.6:1. Patients with this disease are accompaniedby at least one behavior or mood disorders, about a third of the patients withOCD. Many studies demonstrated that TS has obvious course characteristics,characterized by childhood-onset, illness is aggravating in adolescence andmotor symptoms gradually improve as an adult, even disappear. It has beenreported that urine of TS children with severe symptoms contains high level oftestosterone. Patients can be with hyperandrogenism performance.Antiandrogen treatment can significantly alleviate TS symptoms in adultpatients. These results showed the incidence may be related to androgen levelsdisorder in children.Recent clinical data suggested that dysfunction of dopamine (DA)nervous system is closely related to the TS. By Genin neural imagingtechnology, TS children (6to12years of age) can be detected with newstriatal dopamine transporter (DAT) signal enhancement, and TS adolescentpatients (12to17years of age) can be detected with abnormal DOPAdecarboxylase activity enhancement in caudate nucleus. Clinical data showedthat amphetamine can enhance striatum dopamine release in putamen andveutro of TS patients, and dopamine receptor antagonist has inhibited twitchsymptoms of TS children. These results indicated that TS may have closerelationship with brain dopamine nervous system. Most of testosterone in men is produced by leydig cells. Testosterone isan important form of androgen in the body, and has the characteristics offat-soluble. It can be used as a male steroid hormones (AAS), and can throughthe blood brain barrier and act on central nervous system. Researches showedthat abuse of AAS will lead to hyperactivity, lack of impulse control andemotional instability. In addition, the animal experiments also found thattestosterone can lead to the increase of motor behavior and excessivegrooming of normal adult male rats. The above experiments proved that AAShas affected the behavior changes in male rats.DA neurons in the brain are mainly concentrated in the midbrainsubstantial nigra compacta (SNc) and ventral tegmental area (VTA), etc. Thesubstantia nigra-striatum and VTA-cortical dopaminergic neurons regulatemovement, cognition and thinking ability respectively. Whether the change ofandrogen levels are related to the abnormal behavior disorders in the TSchildren development or the function of the brain dopaminergic nerve system,it is not very clear.Therefore, the study chose male Wistar rats as experimental object,respectively subcutaneous injection of testosterone propionate (testosteronepropionate, TP group), androgen receptor inhibitor flutamide (flutamide, Flugroup), and flutamide+testosterone propionate (Flu+TP group) to establishthe experimental animal model. The open field experiment, LC-MS/MS,western blot and RT-PCR technique are used to observe behaviors of theWistar rats and the related parameters of the midbrain DA neurons in3weeks(youth),7weeks of age (adolescence) and6months of age (adult). The aimsof the study are to explore the possible mechanisms in androgen acting ondopamine nervous system of rats in development, and it is expected thatprovide certain experiment basis for exploring the pathogenesis of TS.Part1Effects of TP on male rats open field behaviors in the earlydevelopmental stageObjective To study the effects of chronic administration of TP onmotor-related behaviors of male rats in the three developmental stages. Methods Using open field test to observe rat behavior parameters in3weeks,7weeks and6months of age; Measuring the rat body weight, gonadorgans, pituitary weight. Radioimmunoassay (RIA) was used to detecttestosterone, follicle-stimulating hormone and luteinizing hormoneconcentration in serum after administration of TP.Results1At3weeks and7weeks, compared with the control group, TP groupimmobile-sniffing behavior, exploratory behavior, locomotor behavior andgrooming behavior were increased significantly. However, compared with thecontrol group, Flu+TP group open field behavior parameters showed noobvious change but the amount of Walking reduction at3weeks and7weeksand number of grooming increasing in7weeks. Compared with the TP group,TP group immobile sniffing, locomotor behavior, exploration behavior andgrooming behavior significantly reduced. The thigmotaxic behavior has noobvious change in TP or Flu group at3weeks,7weeks and6months. At6months, each behavior had no obvious change in all groups of rats.2There were no significant differences between groups of rats weigh at3weeks,7weeks and6months.3Effect of early TP administration on seminal vesicle, testis and pituitaryweight.At3weeks, compared with the control group, average vesicle weight inTP group increases by1028%（P<0.01），average testis weight decreases by24%（P<0.05）, average testis weight in Flu group decreases by16%（P<0.05）,average seminal vesicle weight in Flu+TP group has increases by201%(P<0.01), average testis weight decreases by60%(P <0.01).Weight of averageseminal vesicle and testis weight in Flu+TP group compared with TPdecreases by73%(P <0.01) and47%(P <0.01). Average seminal vesicleweight in Flu+TP group Compared with Flu group has increases by256%(P<0.01), average testis weight has decreases by62%(P <0.01). Pituitary weightof each group has no obvious change (P>0.05)At7weeks, compared with the control group，average testis weight decreases25%（P<0.01）in TP group，average testis weight in Flu group hasno obvious difference, average testis weight in Flu+TP group has decreasedby62%(P <0.01).Average weight of testis in Flu+T P group compared withTP reduce49%(P <0.01).Average weight of testis in Flu+TP groupcompared with Flu group decreases58%(P <0.01). Average pituitary weightof each group has no obvious change (P>0.05)At6months, each above organ has no obvious change in all groups（P>0.05）.4Effect of Early TP administration on serum T, LH and FSH in threedevelopmental stagesAt3weeks, compared with the control group， concentration of serumTin TP group increases4400%（P<0.01）, serum T in Flu+TP group increases4229%（P<0.01）. Serum T in Flu+TP group compared with Flu increases3996%（P<0.01）. serum T, LH and FSH in each group has no obvious change（P>0.05）.At7weeks, compared with the control group，concentration of serum Tin TP group decreases53%（P<0.01）, serum T in Flu+TP group decreases88%（P<0.01）. Serum T in Flu+TP group compared with TP decreases74%（P<0.01）.Serum T in Flu+TP group compared with Flu decreases88%（P<0.01）. serum T, LH and FSH in each group has no obvious change（P>0.05）.At6months, concentration of serum T, LH and FSH has no obviouschange in all groups（P>0.05）Conclusions:1Subcutaneous administration of TP lead rats to remain at a high level ofblood testosterone.2Early administration of TP on pup rats leads to open field behaviorparameters increasing significantly in childhood and puberty period,suggesting that high levels of androgens in the early development time maychange the brain neural signal transmission. Part2Testosterone propionate promotes the expression of DA,DOPAC and HVA in the midbrain dopaminergic neurons in the earlydevelopment stageObjective To observe the effect of TP on DA neurotransmitters and theirmetabolic products in the substantia nigr-caudate putamen and the ventraltegmental area-the nucleus accumbens neurons, discussed the relationshipbetween early behavior changes by administration of TP and dopaminergicnerve signal transmissionMethods Using LC-MS/MS to observe the expressions of DA, DOPACand HVA on rat corpus striatum and nucleus accumbens in3weeks,7weeksand6months.ResultsThe changes of DA and metabolite in corpus striatum, nucleusaccumbens.At3weeks, compared with control group, the expression of DA,DOPAC and HVA in corpus striatum are increased, the expression of DA,DOPAC in nucleus accumbens in TP group are increased（P<0.01）, there is nosignificant change on DOPAC+HVA/DA, DOPAC/DA and HVA/DA. Theexpression of DA in corpus striatum and nucleus accumbens in Flu group aredecreased, the specific value of DOPAC+HVA/DA and DOPAC/DA incorpus striatum are increased（P<0.01）, the specific value of corpus striatumHVA/DA is increased by40%. The expression of DA in corpus striatum inFlu+TP group is reduced by43%, the specific value of DOPAC+HVA/DAand DOPAC/DA are increased（P<0.01）, there is no significant change oncorpus striatum. Compared with TP group, the expression of DA and DOPACin Flu+TP group is obviously decline. There is no obvious decreased onnucleus accumbens HVA.At7weeks, compared with control group, the expression of DA,DOPAC and HVA in corpus striatum and nucleus accumbens in TP group areobviously increased（P<0.01）, there is no significant change on the specificvalue of DOPAC+HVA/DA,DOPAC/DA and HVA/DA. The expression ofcorpus striatum HVA in Flu group are increased by25%, there is no change on all targets of nucleus accumbens; there is no obvious change on all targetsof corpus striatum in Flu+TP group. Compared with TP group. There is noobvious change on corpus striatum in Flu+TP group, the expression ofnucleus accumbens DA is reduced by26%.At6months, there is no obvious change on DA neurotransmitter and itsmetabolite of male rats in each group.Conclusion The early TP treatment on neonatal rats can promote themidbrain DA, DOPAC and HVA expression in childhood and adolescenceperiods, while administration of flutamide inhibited the effect of exogenousTP.Part3Testosterone propionate early treatment increase theexpression of TH, DAT, MAOA and MAOB and COMT in midbraindopaminergic neurons of the male ratsObjective To observe the effect of TP administration on pup rats on therelevant activities indicators of the midbrain dopaminergic neurons. Then weexplore in depth the relationship between these effects and how TP canpromote DA, DOPAC and HVA expressionMethods Detecting the expressions of nigra TH, DAT protein, TH, DATof male rats, MAO, MAO and COMT mRNA after treating by testosteronepropionate by western blot and realtime RT-PCR.Results The effect of TP’s earlier treatment on TH and DAT in SN,CPu, VTA, Acb.1At3weeks and7weeks, compared with control group, the expressionsof TH， DAT in substantial nigra, ventral tegmental area and nucleusaccumbens in TP group are increased. Compared with TP group, theexpressions of TH, DAT in substantial nigra, ventral tegmental area andnucleus accumbens in Flu+TP group are obviously reduced.At6months, there is no obviously change on the expressions ofsubstantial nigra, ventral tegmental area and nucleus accumbens TH, DAT ineach group.2The effect of TP’s earlier treatment on TH, DAT, MAOA, MAOB, COMT gene in SN, VTA.At3weeks and7weeks, compared with control group, the expressionsof TH mRNA, DAT mRNA, MAOA mRNA, MAOB mRNA and COMTmRNA in substantial nigra, ventral tegmental area are obviously increased inTP group. Compared with TP group, the expressions of TH mRNA,DATmRNA, MAOA mRNA, MAOB mRNA and COMT mRNA insubstantial nigra, ventral tegmental area are obviously reduced in Flu+TPgroup.At6months, there is no obviously change on the expressions ofsubstantial nigra, ventral tegmental area TH mRNA, DATmRNA, MAOAmRNA, MAOB mRNA and COMT mRNA in each group.Conclusion Early TP administration on pup rats leads to change on TH,DAT and its mRNA and MAOA and MAOB, COMT mRNA expression levelin childhood and adolescence midbrain DA neurons, which performance theenhancement effect of TP. Flu inhibited the effect of exogenous TP, whichsuggested that the midbrain neural signal transmission of DA changesassociated with TP regulation of neurotransmitter DA metabolic enzymes andthe expression of DA transporter.更多还原