【作者】 崔瑞；

【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， 2012， 博士

【摘要】 老年人和老年动物常表现出运动平衡障碍、运动不协调和力量减弱，这些运动相关行为的变化是衰老的标志之一。在正常衰老过程中，参与运动调控的黑质-纹状体多巴胺能神经元发生退行性改变，表现为反映多巴胺（dopamine，DA）能神经元功能活动的两大标志物酪氨酸羟化酶(tyrosinehydroxylase, TH)和多巴胺转运体(dopamine transportet, DAT)的表达减弱。黑质-纹状体DA能神经元功能活动的减弱显著影响老年机体的运动相关行为。研究显示，雄性甾体激素（anabolic androgenic steroids,AAS）能够影响机体的行为，改变中枢DA能神经元的功能状态。AAS增加老年雄性小鼠的抗焦虑行为和认知行为、改变成年大鼠探索行为等多个旷场行为指标、增强雄性大鼠脑内DA的代谢，补充睾酮逆转去势引起的雄性大鼠纹状体TH活性的降低。胶质细胞源性神经生长因子可以提高老年大鼠黑质和纹状体的TH活性、增强老年大鼠的运动能力，暗示外源性干预因素可以作用于老年大鼠黑质-纹状体DA能神经元并影响老年大鼠的运动行为。研究发现老年雄性大鼠血清睾酮水平的显著降低，补充睾酮可提高老年大鼠内侧视前区的DA能神经元的活动，维持其正常的性活动。补充雄激素能否改变老年雄性大鼠黑质-纹状体DA能神经元的功能活动，从而改善其运动相关行为的障碍目前尚不清楚。临床资料显示睾酮参与帕金森病(Parkinson’s disease,PD)的神经病理生理改变，伴有睾酮缺乏的帕金森病患者在睾酮替代治疗后，静止性震颤、精细运动调控和非运动症状均得到显著改善，而且运动症状的改善程度与血清睾酮水平的升高有关。我们推测对老年雄性大鼠给予睾酮处理可能会提高黑质-纹状体DA能神经元的功能状态，从而改善老年雄性大鼠的运动相关行为。因此，本研究利用旷场实验、倾斜面实验、水平绳实验和粘附物去除实验，分析丙酸睾酮(testosterone propionate, TP)处理对老年大鼠运动相关行为的影响；通过免疫细胞化学、免疫印迹以及RT-PCR技术观察TP处理后老年雄性大鼠黑质-纹状体TH和DAT的表达变化；通过细胞培养分析睾酮（testosterone, T）处理后SH-SY5Y细胞TH和DAT的表达，推测雄激素改善老年大鼠黑质-纹状体DA能神经元功能状态的可能机制，期望所获结果为以雄激素替代辅助治疗老年运动相关行为障碍提供一定的实验依据。第一部分：丙酸睾酮改善老年雄性大鼠的运动相关行为目的：探讨TP处理对老年雄性大鼠运动相关行为的影响。方法：通过旷场实验、倾斜面实验、水平绳实验和粘附物去除实验观察TP处理后老年雄性大鼠运动相关行为的变化，放射免疫分析法检测TP处理后老年雄性大鼠血清睾酮含量的变化。结果：1.通过旷场实验发现，24Mon组老年大鼠的静止闻嗅次数，Walking、Climbing、Rearing、Sniffing次数，垂直运动和水平运动次数，总径长，理毛次数和理毛持续时间比6Mon组分别降低了39%（P<0.01）、52%（P<0.01）、39%（P<0.01）、69%（P<0.01）、51%（P<0.01）、50%（P<0.01）、56%（P<0.01）、55%（P<0.01）、59%（P<0.01）和55%（P<0.01），理毛潜伏期比6Mon组延长了392%（P<0.01）；长期TP处理后，老年大鼠的静止闻嗅次数，Climbing、Rearing、Sniffing次数，垂直运动次数比24Mon组分别升高了27%（P<0.05）、37%（P<0.05）、97%（P<0.01)、44%（P<0.05）和52%（P<0.05），差异显著；其中Climbing次数恢复至6Mon组水平。2.通过倾斜面实验发现，24Mon组老年大鼠的倾斜面下滑角度比6Mon组降低了22%（P<0.01），50°夹角滑下次数增加了550%（P<0.01）；长期TP处理后，老年大鼠的倾斜面下滑角度显著增加（P<0.05），50°夹角滑下次数显著减少（P<0.01）。3.通过水平绳实验发现，24Mon组老年大鼠的悬挂时间比6Mon组大鼠缩短了62%（P<0.01）；长期TP处理后，老年大鼠的悬挂时间比24Mon组明显延长（P<0.01）。4.通过粘附物去除实验发现，24Mon组老年大鼠的左鼻和右鼻粘附物去除时间比6Mon组大鼠分别延长了382%（P<0.01）和577%（P<0.01），左前爪和右前爪粘附物去除时间比6Mon组分别延长了1410%（P<0.01）和1156%（P<0.01）；长期TP处理后，老年大鼠的左鼻（P<0.05）、右鼻（P<0.05）和左前爪（P<0.01）、右前爪（P<0.01）粘附物去除时间均显著降低，但仍与6Mon组水平有显著差异。5.24Mon组大鼠的血清睾酮含量比6Mon组降低了61%（P<0.05），TP处理使老年大鼠血清睾酮水平显著升高（P<0.01）。结论：1.24月龄老年雄性大鼠的旷场运动行为显著低于6月龄大鼠；长期TP处理可以改善老年大鼠的旷场运动行为。2.24月龄老年雄性大鼠平衡反应能力、肌张力和运动协调能力比6月龄大鼠显著降低；长期TP处理可以改善这些受损的运动技能。3. TP处理可以显著升高老年雄性大鼠血清睾酮水平。第二部分：丙酸睾酮增强老年雄性大鼠黑质-纹状体DA能神经元TH和DAT的表达目的：探讨丙酸睾酮处理对老年雄性大鼠黑质-纹状体DA能神经元TH和DAT表达的影响。方法：通过免疫细胞化学和免疫印记检测TH和DAT在老年雄性大鼠黑质和尾壳核的表达。结果：1.免疫细胞化学实验发现：24Mon组老年大鼠SN的TH表达比6Mon组降低了25%（P<0.01），CPu的TH表达比6Mon组降低了28%（P<0.01），长期TP处理后SN和CPu的TH表达分别升高了14%（P<0.01）和26%（P<0.01），差异显著。24Mon组老年大鼠SN和CPu的DAT表达比6Mon组分别降低了22%（P<0.01）和32%（P<0.01），TP处理使SN（P<0.01）和CPu（P<0.05）的DAT表达显著升高，其中24Mon-TP组大鼠SN的DAT表达恢复到了6Mon组水平。2.免疫印记实验发现：24Mon组老年大鼠SN和CPu的TH表达分别比6Mon组降低了28%（P<0.01）和36%（P<0.01），长期TP处理后SN和CPu的TH相对表达分别升高了22%（P<0.01）和68%（P<0.01），CPu的TH表达恢复至6Mon组水平。24Mon组老年大鼠SN的Glyco-DAT的相对表达比6Mon降低了54%（P<0.01），Non-Glyco-DAT降低了26%（P<0.05）；CPu的Glyco-DAT相对表达比6Mon组降低了71%（P<0.01），Non-Glyco-DAT降低了39%（P<0.01）；TP处理后，老年大鼠SN和CPu的Glyco-DAT、CPu的Non-Glyco-DAT的相对表达分别升高了74%（P<0.01）、89%（P<0.01）和35%（P<0.05），SN的Non-Glyco-DAT的相对表达没有显著变化。结论：1.24月龄老年雄性大鼠SN和CPu多巴胺能神经元TH的表达明显降低；长期给予TP能够显著增加老年雄性大鼠SN和CPu的TH表达。2.24月龄老年雄性大鼠SN和CPu糖基化与非糖基化DAT的表达均显著降低；长期TP处理可显著升高SN糖基化DAT、CPu的糖基化和非糖基化DAT的表达，而对SN非糖基化DAT表达没有显著影响，说明TP可能影响DAT的糖基化过程。第三部分：丙酸睾酮增强老年雄性大鼠黑质-纹状体DA能神经元THmRNA和DAT mRNA的表达目的：探讨丙酸睾酮在转录水平对老年雄性大鼠黑质-纹状体DA能神经元的影响。方法：通过RNA提取、RT-PCR检测丙酸睾酮处理后老年雄性大鼠黑质TH mRNA和DAT mRNA的表达。结果：与6Mon组大鼠相比，24Mon组大鼠SN的TH mRNA（P<0.01）和DAT mRNA（P<0.01）的表达都显著下降；长期TP处理使老年雄性大鼠黑质TH mRNA和DAT mRNA表达显著升高，恢复至6Mon组水平。结论：长期TP处理升高老年雄性大鼠黑质TH mRNA和DAT mRNA的表达，表明TP可以转录水平影响黑质-纹状体DA能神经元TH和DAT的表达。第四部分：睾酮对分化的SH-SY5Y细胞TH和DAT表达的影响目的：探讨睾酮对分化的SH-SY5Y细胞DA能神经元功能状态的影响。方法：RA/TPA诱导SH-SY5Y细胞分化6天，通过免疫印迹、RT-PCR检测睾酮处理对RA/TPA分化的SH-SY5Y细胞TH和DAT表达的影响。结果：1. RA/TPA分化6天后，SH-SY5Y细胞形态发生显著变化，突起明显增多、变长，细胞NeuN表达增加。2.台盼蓝活细胞计数和MTT检测均显示10nM和100nM T处理24小时和48小时对细胞活性没有显著影响。3.免疫印迹结果显示，与Con-24h组细胞相比，10nM-24h组RA/TPA分化的SH-SY5Y细胞TH的表达升高了123%（P<0.01），100nM-24h组分化细胞TH的表达降低了58%（P<0.01）;与Con-48h组细胞相比，10nM-48h组RA/TPA分化的SH-SY5Y细胞TH的表达降低了31%（P<0.01），100nM-48h组分化细胞TH的表达降低了89%（P<0.01）。与Con-24h组细胞相比，T处理使10nM-24h组RA/TPA分化的SH-SY5Y细胞DAT的表达升高了90%（P<0.01），100nM-24h组分化细胞DAT的表达升高了91%（P<0.01）；与Con-48h组细胞相比，T处理后10nM-48h组RA/TPA分化的SH-SY5Y细胞DAT的表达升高了47%（P<0.05），100nM-48h组分化细胞DAT的表达升高了84%（P<0.01）。4. RT-PCR结果显示，与Con-24h组细胞相比，T处理后10nM-24h组细胞TH mRNA的表达升高了41%（P<0.05），100nM-24h组细胞THmRNA的表达无显著变化；Con-48h、10nM-48h和100nM-48h三组细胞间TH mRNA的表达无显著差异。结论：1. RA/TPA可以诱导SH-SY5Y细胞向成熟神经元分化。2.10nM和100nM的T处理分化的SH-SY5Y细胞24小时和48小时对细胞活力没有显著影响。3.10nM T处理24小时可以显著增加分化的SH-SY5Y细胞的THmRNA和蛋白表达，而其他剂量和处理时间的T则降低TH的表达，表明T增加DA能神经元TH表达的作用是低剂量短时效性的。4.10nM和100nM T处理24和48小时均使分化的SH-SY5Y细胞DAT表达升高，表明T增强了DA能神经元功能状态。更多还原

【Abstract】 The motor behavior and the motor performance involving balance,coordination, and strength decline significantly in aged man and aged animals,which is the hallmark of aging. The nigrostriatal dopaminergic (NSDA)system, which is involved in motor control, also accordingly undergoes aprogressive decline during normal aging, such as a decrease in tyrosinehydroxylase (TH) and its mRNA, as well as in dopamine transporter (DAT)and its mRNA, which implicate the impairment of dopaminergic system inaged animals. The impaired NSDA system may contribute to the significantdecline in motor behavior and motor performance in aged animals.The influence of anabolic androgenic steroids (AAS) on centraldopaminergic activity and behaviors of organisms has been reported in animalstudies. Supplement of testosterone increases anti-anxiety behavior andenhances cognitive performance in aged intact male mice. Some parameters ofthe open field activity in rats are improved by testosterone. Relatively highdoses of AAS increases dopaminergic metabolism in male rat brain. Theadministration of testosterone to castrated rats completely prevents thecastration-induced reduction of striatal TH activity.Injections with glial cell line-derived neurotrophic factor can increaselocomotor activity of aged rats through enhancing TH activity in the substantianigra (SN) and striatum of aged rats. It implies exogenous interventions act onthe NSDA system and affect locomotor activity of aged rats. Aged rats havelower level of testosterone in serum. Long term testosterone replacement mayfavorably alter the decline in the process of sexual activity with aging and therestoration by testosterone replacement of dopaminergic activity in the medialpreoptic area may be involved in the maintenance of sexual function in aged rats. To date, it is not known whether exogenous testosterone intervention toaged male rats ameliorates the impaired motor behavior and motorperformance by altering NSDA system. Testosterone involved in theneuropathophysiology of Parkinson’s disease. The resting tremor, fine motorcontrol and nonmotor symptom are significantly improved in a parkinsonianpatient with testosterone deficiency following testosterone treatment and theimprovement in motor symptoms is correlated with serum testosteronesupplement levels. We presume that androgen supplement might amelioratethe declined motor behavior and motor performance of aged rats by alteringNSDA system.Therefore, in the present study, the motor-related behaviors of aged malerats after testosterone propionate (TP) treatment were observed by open fieldtest, tilting-plane test, horizontal-wire test as well as adhesive removal test andthe effect of chronic administration of TP on aged rats was investigated byanalysing the altered expression of TH and DAT in NSDA system. In order tospeculate the mechanism of ameliorate effects of androgen on NSDA system,the effect of T on the expression of TH and DAT in differentiated SH-SY5Ycell was tested. It is hoped that supplement of androgen could be used inadjutant therapy for age-dependent motor deficits.Part1: The amelioratory effect of TP on motor-related behaviors in agedmale ratsObjective: To study the effects of chronic administration of TP onmotor-related behaviors in aged male rats.Methods: The open field test, tilting-plane test, horizontal-wire test andadhesive removal test were used to analyse the effects of TP on motor-relatedbehaviors in aged male rats. Radioimmunoassay (RIA) was used to detecttestosterone concentration in serum after administration of TPResults:1. Open field test: The amount of immobile-sniffing events, walking，climbing，rearing，sniffing, vertical activity, horizontal activity, total path length, number of grooming events and the duration of grooming wasdecreased in24Mon rats by39%(P<0.01),52%(P<0.01),39%(P<0.01),69%(P<0.01),51%(P<0.01),50%(P<0.01),56%(P<0.01),55%(P<0.01),59%(P<0.01) and55%(P<0.01) respectively, and the latency of groomingwas increased by392%(P<0.01) compared to6Mon rats. After TP treatment,the amount of immobile-sniffing events, climbing，rearing，sniffing andvertical activity was significantly increased in24Mon-TP rats by27%(P<0.05)、37%(P<0.05)、97%(P<0.01)、44%(P<0.05) and52%(P<0.05)respectively compared to24Mon rats. The amount of climbing in24Mon-TPrats failed to differ significantly from that in6Mon rats.2. Tilting-plane test: The angle of sliding off decreased by22%(P<0.01)and the number of sliding off at50°angle increased by550%(P<0.01) in24Mon rats compared to6Mon rats. After TP treatment, the angle of slidingoff was significantly increased (P<0.05) and the number of sliding off at50°angle was significantly decreased (P<0.01).3. Horizontal-wire test: The duration time of hanging was shorter by62%(P<0.01) in24Mon rats compared to6Mon rats. Administration of TPimproved the duration time of hanging compared to24Mon rats (P<0.01).4. Adhesive removal test: The latency to remove adhesive paper on leftnose and right nose increased by382%(P<0.01) and577%(P<0.01)respectively in24Mon rats compared to6Mon rats. The latency to removeadhesive paper on left forepaw and right forepaw increased by1410%(P<0.01) and1156%(P<0.01) in24Mon rats respectively. TP treatmentdecreased the latency to remove adhesive paper on the nose (P<0.05) andforepaw (P<0.01) of aged rat significantly.5. Testosterone in serum was decreased by61%(P<0.05) in24Mon ratscompared to6Mon rats. Administration of TP increased the level of serumtestosterone in24Mon-TP rats significantly (P<0.01).Conclusions:1. The open field behavior was significantly lower in24Mon rats than in6Mon rats. Administration of TP ameliorated the open field behavior of aged male rats.2. The balancing reactions, muscular strength performance and motorcoordination ability were significantly reduced in24Mon rats compared to6Mon rats, which were improved after chronic TP administration.3. Administration of TP increased the level of serum testosterone in agedmale rats.Part2: Administration of TP increased the expression of TH and DAT innigrostriatal dopaminergic neurons of aged male ratsObjective: To study the influence of chronic administration of TP on theexpression of TH and DAT in nigrostriatal dopaminergic neurons of aged rats.Methods: Immunohistochemistry and immunoblotting was used to detectthe expression of TH and DAT in nigrostriatal dopaminergic neurons.Results:1. Immunocytochemistry: The expression of TH in24Mon rats wasdecreased by25%(P<0.01) in SN and28%(P<0.01) in CPu. Chronicadministration of TP increased the expression of TH in SN and CPu of agedrats by14%(P<0.01) and26%(P<0.01) respectively. The expression of DATwas decreased by22%(P<0.01) in SN and32%(P<0.01) in CPu respectivelyin24Mon rats compared to6Mon rats. Chronic administration of TP improvedthe expression of DAT in SN (P<0.01) and CPu (P<0.05). The expression ofDAT in SN of24Mon-TP rats restored to the level of6Mon rats.2. Immunoblotting: The expression of TH was decreased by28%(P<0.01),36%(P<0.01) in SN and CPu in24Mon rats compared to6Mon ratsrespectively. Chronic treatment of TP increased the expression of TH by22%(P<0.01),68%(P<0.01) in SN and CPu respectively. The expression ofGlyco-DAT and Non-Glyco-DAT were decreased by54%(P<0.01) and26%(P<0.05) in SN respectively,71%(P<0.01) and39%(P<0.01) in CPurespectively. The expression of Non-Glyco-DAT in CPu and expression ofGlyco-DAT in SN and CPu of aged rats increased by35%(P<0.05),74% (P<0.01) and89%(P<0.01) respectively after chronic TP treatment. Theexpression of Non-Glyco-DAT in SN was not influenced by TP.Conclusions:1. The expression of TH and DAT in SN and CPu of aged rats wassignificantly reduced, which was improved after chronic administration of TP.2. The expression of Glyco-DAT and Non-Glyco-DAT in SN and CPu ofaged rats was significantly decreased. After chronic TP treatment, theexpression of Non-Glyco-DAT in CPu and expression of Glyco-DAT in SNand CPu of aged rats improved significantly, but the expression ofNon-Glyco-DAT in SN was not influenced. It implies that TP may act on theglycosylation of DAT.Part3: The chronic treatment of TP increased the expression of THmRNA and DAT mRNA in nigrostriatal dopaminergic neurons of agedmale ratsObjective: To study the influence of TP on the expression of TH andDAT in nigrostriatal dopaminergic neurons of aged rats at the transcriptionallevel.Methods: Total RNA extraction and RT-PCR was used to detect theexpression of TH mRNA and DAT mRNA in SN.Results: The expression of TH mRNA (P<0.01) and DAT mRNA(P<0.01) in SN of24Mon rats was significantly reduced compared to6Monrats. The expression of TH mRNA (P<0.01) and DAT mRNA (P<0.01) in24Mon-TP rats increased significantly and failed to differ significantly fromthat in6Mon rats.Conclusions: Chronic treatment of TP restored the decreased expressionof TH mRNA and DAT mRNA in aged rats to the level of6Mon rats. Itsuggests TP could influence the expression of TH and DAT at transcriptionallevel. Part4: Influence of T on the expression of TH and DAT in differentiatedSH-SY5Y cellsObjective: To study the influence of T on the dopaminergic activity indifferentiated SH-SY5Y cells.Methods: SH-SY5Y cells treated with RA/TPA for6days.Immunoblotting and RT-PCR was used to detect the expression of TH andDAT in differentiated SH-SY5Y cells.Results:1. After treated with RA/TPA for6days, the shape of SH-SY5Y cellschanged obviously with long neuritic processes and the expresssion of NeuNwas elevated.2. After treated with10nM or100nM T for24h or48h, the viability ofdifferentiated SH-SY5Y cells did not change by the trypan blue exclusionassay and MTT assay.3. Immunoblotting: The expression of TH was increased by123%(P<0.01) in10nM-24h cells compared to Con-24h cells while decreased by58%(P<0.01) in100nM-24h cells. Compared to Con-48h cells, theexpression of TH was decreased by31%(P<0.01) and89%(P<0.01) in10nM-48h and100nM-48h cells respectively. The expression of DAT wasincreased by90%(P<0.01) and91%(P<0.01) in10nM-24h and100nM-24hcells respectively compared to Con-24h cells. Compared to Con-48h cells, theexpression of DAT was increased by47%(P<0.05) and84%(P<0.01) in10nM-48h and100nM-48h respectively.4. RT-PCR：Compared to Con-24h cells, the expression of TH mRNAwas increased by41%(P<0.05) in10nM-24h cells, but the expression of THmRNA in100nM-24h cells didn’t show any difference with Con-24h cells.Group differences among Con-48h,10nM-48h and100nM-48h cells in theexpression of TH mRNA were not found.Conclusions:1. RA/TPA induced SH-SY5Y cell to develop phenotype of maturedopaminergic neuron. 2. Treatment with10nM or100nM T for24h or48h didn’t influence theviability of differentiated SH-SY5Y cells.3. Treatment with10nM T for24h increased the expression of THmRNA and protein in differentiated SH-SY5Y cells while other dosages andduration of T decreased the expression of TH mRNA and protein. It impliesthat T only improves the experssion of TH in low dosage and short duration.4. Treatment with10nM or100nM T for24h or48h increased theexpression of DAT significantly. It suggests T improve the activity ofdopaminergic neuron.更多还原