丙酸睾酮改变大鼠发育早期的旷场行为及中脑DA能神经元在旷场行为改变中的参与
发布时间：2018-07-22 10:36
【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%（P0.01），，睾丸的平均重量降低了24%（P0.01）；Flu组精囊的重量降低了16%（P0.05）；Flu+TP组精囊的重量增加了201%（P0.01），睾丸的平均重量降低了60%（P0.01）。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%（P0.01）和47%（P0.01）。与Flu组相比，Flu+TP组精囊的重量增加了256%（P0.01），睾丸的平均重量降低了62%（P0.01）。各组垂体的重量无明显变化（P0.05）。在7周龄时，与对照组相比，TP组睾丸的平均重量降低了25%（P0.01），精囊腺没有差异；Flu组睾丸的平均重量无明显变化（P0.05）；Flu+TP组睾丸的平均重量降低了62%（P0.01）。与TP组相比，Flu+TP组睾丸的平均重量降低了49%（P0.01）。与Flu组相比，Flu+TP组睾丸的平均重量降低了58%（P0.01）。各组精囊和垂体的重量无明显变化（P0.05）。在6月龄时，各组精囊、垂体和睾丸重量无明显变化（P0.05）。 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%（P0.01）；Flu+TP组血清T的浓度增加了4229%（P0.01）。与Flu组相比，Flu+TP组血清T的浓度增加了3996%（P0.01）。各组血清LH和FSH水平无明显变化（P0.05）。在7周龄时，与对照组相比，TP组血清T的浓度降低了53%（P0.01）；Flu+TP组血清T的浓度降低了88%（P0.01）。与TP组相比，Flu+TP组血清T的浓度降低了74%（P0.01）。与Flu组相比，Flu+TP组血清T的浓度低了88%（P0.01）。各组血清LH和FSH水平无明显变化（P0.05）。在6月龄时，各组血清T、LH和FSH水平无明显变化（P0.05） 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。
[Abstract]:Tourette Syndrome (TS) is a common clinical child behavior disorder syndrome. It is a comprehensive syndrome characterized by involuntary movements of the facial, extremities and trunk muscles accompanied by abnormal speech of the larynx, which is characterized by abnormality of the larynx, that is, the indecent language of the larynx. Most of them are born in boys, the incidence of male and female is 3-9:1, and in some parts of China, even 10.6: is up to 10.6: 1.TS patients often have at least one kind of behavioral or emotional disorder, and about 1/3 patients are associated with obsessive-compulsive disorder. The study found that TS has a more obvious course of disease, characterized by childhood onset, exacerbation of puberty, progressive improvement in adult motor symptoms, and even disappearance. Studies have reported that urine of children with severe TS symptoms contains a higher level of testosterone. It can be accompanied by androplastic symptoms, and anti androgen treatment significantly alleviates the symptoms of TS adult patients. These suggest that the disease may be associated with the disorder of androgen levels in the child.
Recent clinical data suggest that the dysfunction of the dopamine (DA) energy system is closely related to TS. The signal enhancement of the new striatal dopamine transporter (DAT) signal in children with TS (6-12 years old) is enhanced by ligand neural imaging, and the activity of abnormal dopa decarboxylase in the caudate nucleus of TS adolescents (12-17 years old) is enhanced. Clinical data show that anAfrican Life can enhance the release of dopamine in the shell and ventral striatum of TS patients, and the dopamine receptor antagonist has the effect of inhibiting the symptoms of TS in children. These results suggest that the pathogenesis of TS may have a close relationship with the dopaminergic nervous system in the brain.
Testosterone is produced mainly by the stromal cells of the testicle and is fat soluble and can be used in the central nervous system through the blood brain barrier. A large number of studies have shown that androgens can affect the functional activity of dopaminergic neurons. The embryonic stage is given to androgens, which can increase the function of the dopamine neurons in the prefrontal cortex of adult rats. High level of androgen can significantly increase the attack behavior of rats and increase the expression of dopamine D2 receptor in the hypothalamus, and the long-term administration of androgen in adult rats promotes the expression of DAT in the striatum.
The DA neurons in the brain are mainly concentrated in the mesencephalic dense part of the substantia nigra (SNc) and the ventral tegmental area (VTA). The dopamine nigrostriatal loop and the VTA cortical loop are involved in physical movement and cognition respectively. The thinking ability regulates the abnormal behavior of.TS in the development of.TS, and then changes the dopamine energy in the brain. The functional activity of the nervous system is not yet known.
Therefore, in this study, the experimental animal model was established by early subcutaneous injection of testosterone propionate (testosterone propionate, group TP), androgen receptor inhibitor flutoamine (flutamide, Flu group) and flutamide + testosterone (group Flu+TP). The experimental animal model was established by open field experiment, liquid chromatography tandem mass spectrometry (liquid chromatography tandem mass spectrometry). The immunoblotting and RT-PCR technique were used to observe the behavior of 3 weeks old (young), 7 weeks old (puberty) and 6 month old (adult) experimental rats and the changes in the expression of DA energy neurons in the middle brain. The effect of androgen on the behavior and its mechanism during the early development were discussed, and the role of the changes of the DA energy neurons in the middle brain in this process was analyzed. The results are expected to provide some experimental evidence for exploring the pathogenesis of TS. Part I: testosterone propionate changes the open field behavior of male rats at the early stage of development.
Objective: To investigate the effect of early postnatal treatment of TP on open field behavior in male rats.
Methods: the changes in behavior related parameters of rats at 3 weeks, 7 weeks and 6 month old open fields were observed by the open field experiment. The weight of the rats and their gonadal organs and the pituitary gland were weighed, and the changes of serum testosterone, follicular stimulating hormone and luteinizing hormone were measured by radioimmunoassay at the three time points of rats.
Result:
1 through the open field experiment, it was found that at the age of 3 weeks and 7 weeks, compared with the control group, there was a significant increase in all kinds of stillness smelling, exercise behavior, exploring behavior and behavior parameters in the TP group, while the number of behavior parameters in the Flu+TP group was not obviously changed, the number of crawling only and the number of hair cut at 7 weeks increased. Compared with the group TP, the group Flu+TP was compared with the group of TP. The static sniffing, exercise behavior, exploratory behavior and hair behavior indexes significantly reduced the early treatment of.TP and the early intervention of Flu on 3 weeks, 7 weeks and the.6 months of the 6 month old rats, and there was no significant change in the behavior of the rats in each group.
2TP did not show significant difference in body weight between 3 week old rats, 7 weeks old and 6 month old groups.
Changes in weight of pituitary gland and testis in early stage of 3TP treatment in rats
At 3 weeks of age, the weight of the seminal vesicle in the TP group increased by 1028% (P0.01), the average weight of the testicles decreased by 24% (P0.01), the weight of the seminal vesicles in the Flu group decreased by 16% (P0.05), the weight of the seminal vesicles in the group Flu+TP was 201% (P0.01), the average weight of the testis decreased by 60% (P0.01). The weight of the seminal vesicle and the testicles in the Flu+TP group, compared with the group TP, and the testis. The weight of the average weight decreased by 73% (P0.01) and 47% (P0.01). Compared with the Flu group, the weight of the seminal vesicle in the Flu+TP group increased by 256% (P0.01) and the average weight of the testis decreased by 62% (P0.01). The weight of the pituitary gland in each group was not significantly changed (P0.05). At the age of 7 weeks, the average weight of the testis in the TP group decreased by 25% (P0.01), and the seminal vesicle glands were not different at the age of 7. The average weight of testis in group Flu was not significantly changed (P0.05); the average weight of testicle in group Flu+TP decreased by 62% (P0.01). Compared with group TP, the average weight of testis in group Flu+TP decreased by 49% (P0.01). Compared with group Flu, the average weight of testis in group Flu+TP decreased by 58% (P0.01). The weight of seminal vesicles and hypophysis in each group was not significantly changed (P0.05). In 6 month old There was no significant change in seminal vesicle, pituitary gland and testis weight in each group (P0.05).
Changes of serum T, LH and FSH levels in rats treated with 4TP
At the age of 3 weeks, the serum concentration of T in the TP group increased by 4400% (P0.01), and the concentration of serum T increased by 4229% (P0.01) in the Flu+TP group. The concentration of serum T increased by 3996% (P0.01) compared with the Flu group. The serum LH and FSH levels were not significantly changed. At the age of 7 weeks, the concentration of serum was decreased compared with the control group. 53% (P0.01); the concentration of serum T in group Flu+TP decreased by 88% (P0.01). Compared with group TP, the concentration of serum T decreased by 74% (P0.01). Compared with group Flu, the concentration of T in serum of Flu+TP group was 88% lower than that in group Flu.
Conclusion:
1 subcutaneous injection of TP can keep suckling mice in a high level of blood testis.
The early treatment of TP in 2 milk rats resulted in a significant increase in behavioral parameters in young and puberty behavior experiments, suggesting that high levels of androgen in early development may change the transmission of neural signals in the brain.
The second part: testosterone propionate promotes the expression of DA, DOPAC and HVA in the mesencephalic DA neurons of male rats at the early stage of development.
Objective: To observe the effect of TP early treatment on the neurotransmitter DA and its metabolites in the substantia nigra caudate nucleus and ventral tegmental area of the male rats, and to explore the possible relationship between the changes of the early development of TP and the change of DA signal transmission in the early development of TP.
Methods: liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to detect the expression of DA, DOPAC and HVA in the nucleus accumbens of the DA energy neurons in the middle brain of the middle brain of 3 weeks, 7 weeks and 6 month old experimental rats.
Compared with the control group, the expression of DA, DOPAC, HVA and DA in the nucleus accumbens of the 3 week old TP group increased significantly by.DOPAC+HVA/DA, DOPAC/DA and HVA/DA had no significant changes, and the DA expression in the tail putamen and nucleus accumbens in the Flu group decreased obviously, the ratio of the DOPAC+HVA/DA to the caudate putamen increased and the ratio of the nucleus accumbens increased; the expression of the tail putamen was expressed. The ratio of DOPAC+HVA/DA to DOPAC/DA increased obviously, and the nucleus accumbens had no significant change. Compared with the TP group, the concentration of DA, DOPAC in the two group of Flu+TP group decreased obviously, and the HVA in the nucleus accumbens decreased not obviously.
Compared with the control group, the expression of DA, DOPAC and HVA in the caudate putamen and nucleus accumbens of the 7 week old TP group increased obviously, and the ratio of DOPAC+HVA/DA, DOPAC/DA and HVA/DA had no obvious change, the HVA expression of the caudate putamen in Flu group increased by 25%, all the indexes of the nucleus accumbens were not changed, and the indexes of the tail putamen in the Flu+TP group were not obviously changed. Compared with TP group, the tail putamen nucleus of Flu+TP group There was no significant change in the indexes, and the concentration of DA in the nucleus accumbens decreased by 26%.
6 month old there was no significant change in the ratios of neurotransmitters and metabolites among the rats in each group.
Conclusion: the early treatment of TP in neonatal rats promoted the expression of DA, DOPAC and HVA in the DA neurons of puberty and adolescence, while giving Flu inhibited the effect of exogenous TP.
The third part: the early treatment of testosterone propionate promoted the expression of DA, TH, DAT, MAOA, MAOB and COMT in the mesencephalon of male rats at the early stage of development.
Objective: To observe the effect of early treatment of TP on the expression of functional activity of DA neurons in the mesencephalon, and to explore the relationship between the expression of TP and the expression of DA, DOPAC and HVA in DA neurons.
Methods: the expression of TH, DAT, mRNA and mRNA of TH, DAT, MAOA, MAOB, COMT in male rats after testosterone propionate treatment was detected by immunoblotting and real-time quantitative RT-PCR.
Results: the changes of TH and DAT in three developmental stages of substantia nigra (SN), caudate putamen (CPu), ventral tegmental area (VTA) and nucleus accumbens (Acb) were observed in the early stage of TP treatment.
At 3 weeks and 7 weeks of age, compared with the control group, the expression of substantia nigra, caudate putamen, ventral tegmental area and nucleus accumbens TH and DAT increased significantly in the TP group. The expression of TH mRNA and DAT mRNA in the substantia nigra and ventral tegmental area increased. Compared with the TP group, the expression of TH and DAT in the substantia nigra, caudate nucleus, ventral tegmentum, ventral tegmentum, and ventral tegmentum area were significantly reduced. The expression of TH mRNA and DAT mRNA decreased. At 6 month old, there was no significant change in the expression of substantia nigra, caudate putamen, ventral tegmental area, nucleus accumbens TH and DAT in each group.
Expression changes of MAOA, MAOB and COMT mRNA
At the age of 3 weeks and at the age of 7, the expression of MAOA mRNA, MAOB mRNA and COMT mRNA in the TP group increased significantly compared with the control group. Compared with the TP group, the expressions of MAOAmRNA, MAOBmRNA and COMT volumes in the substantia nigra and ventral tegmental area of the Flu+TP group were significantly lower than those in the TP group. At 6 month old, each group of substantia nigra and ventral tegmentum areas There was no obvious change in the expression of AOBmRNA and COMT mRNA.
Conclusion: TP early treatment changes the expression level of TH, DAT, mRNA, MAOA, MAOB, COMT mRNA in young and puberty mesencephalic DA neurons, showing the enhancement effect of TP, and Flu can inhibit the effect of exogenous TP, suggesting the change of signal transmission of neural nerve in the middle brain and the regulation of neurotransmitter metabolism related enzymes. It is related to the expression of the DA transporter.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.94
本文编号：2137163
[Abstract]:Tourette Syndrome (TS) is a common clinical child behavior disorder syndrome. It is a comprehensive syndrome characterized by involuntary movements of the facial, extremities and trunk muscles accompanied by abnormal speech of the larynx, which is characterized by abnormality of the larynx, that is, the indecent language of the larynx. Most of them are born in boys, the incidence of male and female is 3-9:1, and in some parts of China, even 10.6: is up to 10.6: 1.TS patients often have at least one kind of behavioral or emotional disorder, and about 1/3 patients are associated with obsessive-compulsive disorder. The study found that TS has a more obvious course of disease, characterized by childhood onset, exacerbation of puberty, progressive improvement in adult motor symptoms, and even disappearance. Studies have reported that urine of children with severe TS symptoms contains a higher level of testosterone. It can be accompanied by androplastic symptoms, and anti androgen treatment significantly alleviates the symptoms of TS adult patients. These suggest that the disease may be associated with the disorder of androgen levels in the child.
Recent clinical data suggest that the dysfunction of the dopamine (DA) energy system is closely related to TS. The signal enhancement of the new striatal dopamine transporter (DAT) signal in children with TS (6-12 years old) is enhanced by ligand neural imaging, and the activity of abnormal dopa decarboxylase in the caudate nucleus of TS adolescents (12-17 years old) is enhanced. Clinical data show that anAfrican Life can enhance the release of dopamine in the shell and ventral striatum of TS patients, and the dopamine receptor antagonist has the effect of inhibiting the symptoms of TS in children. These results suggest that the pathogenesis of TS may have a close relationship with the dopaminergic nervous system in the brain.
Testosterone is produced mainly by the stromal cells of the testicle and is fat soluble and can be used in the central nervous system through the blood brain barrier. A large number of studies have shown that androgens can affect the functional activity of dopaminergic neurons. The embryonic stage is given to androgens, which can increase the function of the dopamine neurons in the prefrontal cortex of adult rats. High level of androgen can significantly increase the attack behavior of rats and increase the expression of dopamine D2 receptor in the hypothalamus, and the long-term administration of androgen in adult rats promotes the expression of DAT in the striatum.
The DA neurons in the brain are mainly concentrated in the mesencephalic dense part of the substantia nigra (SNc) and the ventral tegmental area (VTA). The dopamine nigrostriatal loop and the VTA cortical loop are involved in physical movement and cognition respectively. The thinking ability regulates the abnormal behavior of.TS in the development of.TS, and then changes the dopamine energy in the brain. The functional activity of the nervous system is not yet known.
Therefore, in this study, the experimental animal model was established by early subcutaneous injection of testosterone propionate (testosterone propionate, group TP), androgen receptor inhibitor flutoamine (flutamide, Flu group) and flutamide + testosterone (group Flu+TP). The experimental animal model was established by open field experiment, liquid chromatography tandem mass spectrometry (liquid chromatography tandem mass spectrometry). The immunoblotting and RT-PCR technique were used to observe the behavior of 3 weeks old (young), 7 weeks old (puberty) and 6 month old (adult) experimental rats and the changes in the expression of DA energy neurons in the middle brain. The effect of androgen on the behavior and its mechanism during the early development were discussed, and the role of the changes of the DA energy neurons in the middle brain in this process was analyzed. The results are expected to provide some experimental evidence for exploring the pathogenesis of TS. Part I: testosterone propionate changes the open field behavior of male rats at the early stage of development.
Objective: To investigate the effect of early postnatal treatment of TP on open field behavior in male rats.
Methods: the changes in behavior related parameters of rats at 3 weeks, 7 weeks and 6 month old open fields were observed by the open field experiment. The weight of the rats and their gonadal organs and the pituitary gland were weighed, and the changes of serum testosterone, follicular stimulating hormone and luteinizing hormone were measured by radioimmunoassay at the three time points of rats.
Result:
1 through the open field experiment, it was found that at the age of 3 weeks and 7 weeks, compared with the control group, there was a significant increase in all kinds of stillness smelling, exercise behavior, exploring behavior and behavior parameters in the TP group, while the number of behavior parameters in the Flu+TP group was not obviously changed, the number of crawling only and the number of hair cut at 7 weeks increased. Compared with the group TP, the group Flu+TP was compared with the group of TP. The static sniffing, exercise behavior, exploratory behavior and hair behavior indexes significantly reduced the early treatment of.TP and the early intervention of Flu on 3 weeks, 7 weeks and the.6 months of the 6 month old rats, and there was no significant change in the behavior of the rats in each group.
2TP did not show significant difference in body weight between 3 week old rats, 7 weeks old and 6 month old groups.
Changes in weight of pituitary gland and testis in early stage of 3TP treatment in rats
At 3 weeks of age, the weight of the seminal vesicle in the TP group increased by 1028% (P0.01), the average weight of the testicles decreased by 24% (P0.01), the weight of the seminal vesicles in the Flu group decreased by 16% (P0.05), the weight of the seminal vesicles in the group Flu+TP was 201% (P0.01), the average weight of the testis decreased by 60% (P0.01). The weight of the seminal vesicle and the testicles in the Flu+TP group, compared with the group TP, and the testis. The weight of the average weight decreased by 73% (P0.01) and 47% (P0.01). Compared with the Flu group, the weight of the seminal vesicle in the Flu+TP group increased by 256% (P0.01) and the average weight of the testis decreased by 62% (P0.01). The weight of the pituitary gland in each group was not significantly changed (P0.05). At the age of 7 weeks, the average weight of the testis in the TP group decreased by 25% (P0.01), and the seminal vesicle glands were not different at the age of 7. The average weight of testis in group Flu was not significantly changed (P0.05); the average weight of testicle in group Flu+TP decreased by 62% (P0.01). Compared with group TP, the average weight of testis in group Flu+TP decreased by 49% (P0.01). Compared with group Flu, the average weight of testis in group Flu+TP decreased by 58% (P0.01). The weight of seminal vesicles and hypophysis in each group was not significantly changed (P0.05). In 6 month old There was no significant change in seminal vesicle, pituitary gland and testis weight in each group (P0.05).
Changes of serum T, LH and FSH levels in rats treated with 4TP
At the age of 3 weeks, the serum concentration of T in the TP group increased by 4400% (P0.01), and the concentration of serum T increased by 4229% (P0.01) in the Flu+TP group. The concentration of serum T increased by 3996% (P0.01) compared with the Flu group. The serum LH and FSH levels were not significantly changed. At the age of 7 weeks, the concentration of serum was decreased compared with the control group. 53% (P0.01); the concentration of serum T in group Flu+TP decreased by 88% (P0.01). Compared with group TP, the concentration of serum T decreased by 74% (P0.01). Compared with group Flu, the concentration of T in serum of Flu+TP group was 88% lower than that in group Flu.
Conclusion:
1 subcutaneous injection of TP can keep suckling mice in a high level of blood testis.
The early treatment of TP in 2 milk rats resulted in a significant increase in behavioral parameters in young and puberty behavior experiments, suggesting that high levels of androgen in early development may change the transmission of neural signals in the brain.
The second part: testosterone propionate promotes the expression of DA, DOPAC and HVA in the mesencephalic DA neurons of male rats at the early stage of development.
Objective: To observe the effect of TP early treatment on the neurotransmitter DA and its metabolites in the substantia nigra caudate nucleus and ventral tegmental area of the male rats, and to explore the possible relationship between the changes of the early development of TP and the change of DA signal transmission in the early development of TP.
Methods: liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to detect the expression of DA, DOPAC and HVA in the nucleus accumbens of the DA energy neurons in the middle brain of the middle brain of 3 weeks, 7 weeks and 6 month old experimental rats.
Compared with the control group, the expression of DA, DOPAC, HVA and DA in the nucleus accumbens of the 3 week old TP group increased significantly by.DOPAC+HVA/DA, DOPAC/DA and HVA/DA had no significant changes, and the DA expression in the tail putamen and nucleus accumbens in the Flu group decreased obviously, the ratio of the DOPAC+HVA/DA to the caudate putamen increased and the ratio of the nucleus accumbens increased; the expression of the tail putamen was expressed. The ratio of DOPAC+HVA/DA to DOPAC/DA increased obviously, and the nucleus accumbens had no significant change. Compared with the TP group, the concentration of DA, DOPAC in the two group of Flu+TP group decreased obviously, and the HVA in the nucleus accumbens decreased not obviously.
Compared with the control group, the expression of DA, DOPAC and HVA in the caudate putamen and nucleus accumbens of the 7 week old TP group increased obviously, and the ratio of DOPAC+HVA/DA, DOPAC/DA and HVA/DA had no obvious change, the HVA expression of the caudate putamen in Flu group increased by 25%, all the indexes of the nucleus accumbens were not changed, and the indexes of the tail putamen in the Flu+TP group were not obviously changed. Compared with TP group, the tail putamen nucleus of Flu+TP group There was no significant change in the indexes, and the concentration of DA in the nucleus accumbens decreased by 26%.
6 month old there was no significant change in the ratios of neurotransmitters and metabolites among the rats in each group.
Conclusion: the early treatment of TP in neonatal rats promoted the expression of DA, DOPAC and HVA in the DA neurons of puberty and adolescence, while giving Flu inhibited the effect of exogenous TP.
The third part: the early treatment of testosterone propionate promoted the expression of DA, TH, DAT, MAOA, MAOB and COMT in the mesencephalon of male rats at the early stage of development.
Objective: To observe the effect of early treatment of TP on the expression of functional activity of DA neurons in the mesencephalon, and to explore the relationship between the expression of TP and the expression of DA, DOPAC and HVA in DA neurons.
Methods: the expression of TH, DAT, mRNA and mRNA of TH, DAT, MAOA, MAOB, COMT in male rats after testosterone propionate treatment was detected by immunoblotting and real-time quantitative RT-PCR.
Results: the changes of TH and DAT in three developmental stages of substantia nigra (SN), caudate putamen (CPu), ventral tegmental area (VTA) and nucleus accumbens (Acb) were observed in the early stage of TP treatment.
At 3 weeks and 7 weeks of age, compared with the control group, the expression of substantia nigra, caudate putamen, ventral tegmental area and nucleus accumbens TH and DAT increased significantly in the TP group. The expression of TH mRNA and DAT mRNA in the substantia nigra and ventral tegmental area increased. Compared with the TP group, the expression of TH and DAT in the substantia nigra, caudate nucleus, ventral tegmentum, ventral tegmentum, and ventral tegmentum area were significantly reduced. The expression of TH mRNA and DAT mRNA decreased. At 6 month old, there was no significant change in the expression of substantia nigra, caudate putamen, ventral tegmental area, nucleus accumbens TH and DAT in each group.
Expression changes of MAOA, MAOB and COMT mRNA
At the age of 3 weeks and at the age of 7, the expression of MAOA mRNA, MAOB mRNA and COMT mRNA in the TP group increased significantly compared with the control group. Compared with the TP group, the expressions of MAOAmRNA, MAOBmRNA and COMT volumes in the substantia nigra and ventral tegmental area of the Flu+TP group were significantly lower than those in the TP group. At 6 month old, each group of substantia nigra and ventral tegmentum areas There was no obvious change in the expression of AOBmRNA and COMT mRNA.
Conclusion: TP early treatment changes the expression level of TH, DAT, mRNA, MAOA, MAOB, COMT mRNA in young and puberty mesencephalic DA neurons, showing the enhancement effect of TP, and Flu can inhibit the effect of exogenous TP, suggesting the change of signal transmission of neural nerve in the middle brain and the regulation of neurotransmitter metabolism related enzymes. It is related to the expression of the DA transporter.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.94
【引证文献】
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本文编号：2137163
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