發(fā)布時間：2018-07-22 10:36

【摘要】：抽動-穢語綜合癥（Tourette Syndrome，TS）是臨床較為常見的兒童行為障礙綜合征，是以面部、四肢以及軀干部肌肉不自主抽動伴有喉部異常發(fā)音即猥穢語言為特征的綜合癥候群，多發(fā)生于男童，男女發(fā)病比例為3-9:1，我國部分地區(qū)甚至高達10.6:1。TS患者常伴有至少一種行為或情緒障礙，大約1/3的病人伴有強迫癥。研究發(fā)現(xiàn)TS具有較明顯的病程特征，表現(xiàn)為兒童期發(fā)病，青春期病癥加重，成年后運動癥狀逐漸好轉(zhuǎn)，甚至消失。有研究報道，TS癥狀嚴(yán)重的患兒尿液含有較高水平的睪酮；可伴有雄激素增多癥的表現(xiàn)；抗雄激素處理則顯著緩解TS成年患者的癥狀。這些表明其發(fā)病可能和患兒體內(nèi)雄激素水平紊亂有關(guān)。 近年來的臨床資料暗示多巴胺（DA）能神經(jīng)體系的功能障礙與TS密切相關(guān)。利用配基神經(jīng)顯像技術(shù)檢測到TS患兒（6-12歲）新紋狀體多巴胺轉(zhuǎn)運體（DAT）信號增強，TS青少年患者（12-17歲）尾狀核有異常的多巴脫羧酶活性增強。臨床資料顯示，安非他命可以增強TS患者殼及腹側(cè)紋狀體多巴胺的釋放，多巴胺受體拮抗劑則具有抑制TS患兒抽動癥狀的功效。上述研究結(jié)果表明TS發(fā)病可能和腦內(nèi)多巴胺能神經(jīng)體系存在密切的關(guān)系。 睪酮在男性主要由睪丸間質(zhì)細胞產(chǎn)生，具有脂溶性特點，可透過血腦屏障，作用于中樞神經(jīng)系統(tǒng)。大量研究顯示雄激素可以影響多巴胺能神經(jīng)元的功能活動。胚胎期給予雄激素，可以導(dǎo)致成年期大鼠前額葉皮質(zhì)多巴胺神經(jīng)元功能活動增強；青春期給予雄激素可以明顯引起大鼠的攻擊行為增多以及下丘腦多巴胺D2受體表達增加；長期給予成年大鼠雄激素促進了紋狀體DAT的表達。 腦內(nèi)DA能神經(jīng)元主要集中在中腦黑質(zhì)致密部（SNc）及腹側(cè)被蓋區(qū)（VTA）等處。多巴胺黑質(zhì)紋狀體環(huán)路及VTA皮質(zhì)環(huán)路分別參與軀體運動及認(rèn)知、思維能力的調(diào)控。TS患兒發(fā)育過程中的異常行為是否與雄激素水平紊亂、進而改變了腦內(nèi)多巴胺能神經(jīng)體系的功能活動有關(guān)，目前尚不清楚。 因此，本研究以雄性Wistar乳鼠為實驗對象，分別通過早期皮下注射丙酸睪酮(testosterone propionate, TP組）、雄激素受體抑制劑氟他胺（flutamide，F(xiàn)lu組）以及氟他胺+丙酸睪酮（(Flu+TP組），建立大鼠實驗動物模型。利用曠場實驗、液相色譜-串聯(lián)質(zhì)譜、免疫印跡和RT-PCR技術(shù)觀察3周齡（幼年）、7周齡（青春期）和6月齡（成年）實驗大鼠的曠場行為以及中腦DA能神經(jīng)元相關(guān)指標(biāo)的表達變化，探討雄激素在早期發(fā)育過程中對行為的影響及其機制，分析中腦DA能神經(jīng)元改變在這一過程中所起的作用，期望所獲結(jié)果能為探尋TS的發(fā)病提供一定的實驗依據(jù)。第一部分：丙酸睪酮改變雄性大鼠發(fā)育早期的曠場行為 目的：探討生后TP早期處理對雄性大鼠曠場行為的影響。 方法：利用曠場實驗觀察大鼠模型在3周齡、7周齡及6月齡曠場行為相關(guān)參數(shù)的變化；稱量大鼠及其性腺器官和腦垂體的重量；以放射免疫法檢測上述三個時間點大鼠模型血清睪酮、卵泡刺激素、黃體生成素含量的變化。 結(jié)果： 1通過曠場實驗發(fā)現(xiàn)，在3周齡和7周齡時，與對照組相比，TP組各種靜止聞嗅、運動行為、探索行為及理毛行為參數(shù)明顯增加；而Flu+TP組曠場實驗多項行為參數(shù)未表現(xiàn)明顯改變，僅爬行的數(shù)量降低以及7周齡理毛次數(shù)增加。與TP組相比，F(xiàn)lu+TP組的靜止聞嗅、運動行為、探索行為及理毛行為指標(biāo)明顯減低。TP早期處理和Flu早期干預(yù)對3周齡、7周齡以及6月齡大鼠的趨觸行為無明顯改變。6月齡時，各組大鼠行為均無明顯改變。 2TP早期處理大鼠3周齡、7周齡及6月齡各組大鼠之間體重沒有明顯差異。 3TP早期處理大鼠精囊、垂體和睪丸重量的改變 在3周齡時，與對照組相比，TP組精囊的重量增加了1028%（P0.01），，睪丸的平均重量降低了24%（P0.01）；Flu組精囊的重量降低了16%（P0.05）；Flu+TP組精囊的重量增加了201%（P0.01），睪丸的平均重量降低了60%（P0.01）。與TP組相比，F(xiàn)lu+TP組精囊的重量和睪丸的平均重量分別降低了73%（P0.01）和47%（P0.01）。與Flu組相比，F(xiàn)lu+TP組精囊的重量增加了256%（P0.01），睪丸的平均重量降低了62%（P0.01）。各組垂體的重量無明顯變化（P0.05）。在7周齡時，與對照組相比，TP組睪丸的平均重量降低了25%（P0.01），精囊腺沒有差異；Flu組睪丸的平均重量無明顯變化（P0.05）；Flu+TP組睪丸的平均重量降低了62%（P0.01）。與TP組相比，F(xiàn)lu+TP組睪丸的平均重量降低了49%（P0.01）。與Flu組相比，F(xiàn)lu+TP組睪丸的平均重量降低了58%（P0.01）。各組精囊和垂體的重量無明顯變化（P0.05）。在6月齡時，各組精囊、垂體和睪丸重量無明顯變化（P0.05）。 4TP早期處理大鼠血清T、LH和FSH水平的改變 在3周齡時，與對照組相比，TP組血清T的濃度增加了4400%（P0.01）；Flu+TP組血清T的濃度增加了4229%（P0.01）。與Flu組相比，F(xiàn)lu+TP組血清T的濃度增加了3996%（P0.01）。各組血清LH和FSH水平無明顯變化（P0.05）。在7周齡時，與對照組相比，TP組血清T的濃度降低了53%（P0.01）；Flu+TP組血清T的濃度降低了88%（P0.01）。與TP組相比，F(xiàn)lu+TP組血清T的濃度降低了74%（P0.01）。與Flu組相比，F(xiàn)lu+TP組血清T的濃度低了88%（P0.01）。各組血清LH和FSH水平無明顯變化（P0.05）。在6月齡時，各組血清T、LH和FSH水平無明顯變化（P0.05） 結(jié)論： 1皮下注射TP可使乳鼠保持在高水平的血睪狀態(tài)。 2乳鼠TP早期處理導(dǎo)致幼年和青春期的曠場行為實驗多項行為參數(shù)顯著增加，暗示發(fā)育早期高水平的雄激素可能改變了腦內(nèi)有關(guān)神經(jīng)信號的傳遞。 第二部分：丙酸睪酮促進雄性大鼠發(fā)育早期中腦DA能神經(jīng)元DA、DOPAC及HVA的表達 目的：觀察生后TP早期處理對雄性大鼠黑質(zhì)-尾殼核及腹側(cè)被蓋區(qū)-伏核DA能神經(jīng)元神經(jīng)遞質(zhì)DA及其代謝產(chǎn)物的影響，探討TP改變發(fā)育早期曠場行為與DA信號傳遞變化可能存在的聯(lián)系。 方法：利用液相色譜-串聯(lián)質(zhì)譜（LC-MS/MS）法檢測3周齡、7周齡及6月齡實驗大鼠中腦DA能神經(jīng)元投射靶區(qū)尾殼核、伏核的DA、DOPAC和HVA的表達變化。結(jié)果： 與對照組相比，3周齡TP組尾殼核DA、DOPAC、 HVA以及伏核DA、DOPAC的表達顯著增加。DOPAC+HVA/DA、DOPAC/DA和HVA/DA無明顯變化；Flu組尾殼核和伏核DA的表達明顯減低，尾殼核DOPAC+HVA/DA和DOPAC/DA的比值增加，伏核HVA/DA的比值增加；Flu+TP組尾殼核DA的表達降低，DOPAC+HVA/DA和DOPAC/DA的比值明顯增加，伏核無顯著改變。與TP組相比，F(xiàn)lu+TP組兩個核團DA、DOPAC的濃度明顯降低，伏核HVA降低不明顯。 與對照組相比，7周齡TP組尾殼核和伏核DA、DOPAC和HVA的表達明顯增加，DOPAC+HVA/DA、DOPAC/DA和HVA/DA的比值無明顯變化；Flu組尾殼核HVA的表達增加了25%，伏核所有指標(biāo)沒有變化；Flu+TP組尾殼核各項指標(biāo)均無明顯變化。與TP組相比，F(xiàn)lu+TP組尾殼核各項指標(biāo)均無明顯變化，伏核DA的濃度降低了26%。 6月齡各組大鼠神經(jīng)遞質(zhì)及代謝產(chǎn)物各項指標(biāo)比值均無明顯變化。 結(jié)論：乳鼠TP早期處理促進其幼年和青春期中腦DA能神經(jīng)元DA、DOPAC及HVA的表達，給予Flu則抑制了外源性TP的這種作用。 第三部分：丙酸睪酮早期處理促進雄性大鼠發(fā)育早期中腦DA能神經(jīng)元TH、DAT、MAOA、MAOB及COMT的表達 目的：觀察乳鼠TP早期處理對中腦DA能神經(jīng)元功能活動強弱相關(guān)指標(biāo)表達的影響，探討其表達變化與TP促進DA能神經(jīng)元表達DA、DOPAC及HVA的相互關(guān)系。 方法：通過免疫印跡方法以及實時定量RT-PCR方法來檢測丙酸睪酮處理后雄性大鼠黑質(zhì)TH，DAT及其mRNA以及TH、DAT、MAOA、MAOB、COMT的mRNA的表達。 結(jié)果： TP早期處理對三個發(fā)育階段黑質(zhì)（SN）、尾殼核（CPu）、腹側(cè)被蓋區(qū)（VTA）和伏核（Acb）腦區(qū)TH和DAT的改變 在3周齡和7周齡時，與對照組相比，TP組大鼠處理黑質(zhì),尾殼核，腹側(cè)被蓋區(qū)及伏核TH和DAT表達均明顯增加。黑質(zhì)、腹側(cè)被蓋區(qū)TH mRNA和DAT mRNA表達升高。與TP組相比，F(xiàn)lu+TP組黑質(zhì)，尾殼核，腹側(cè)被蓋區(qū)，伏核TH和DAT的表達明顯減少。黑質(zhì)、腹側(cè)被蓋區(qū)TH mRNA和DAT mRNA表達降低。在6月齡時，各組黑質(zhì),尾殼核，腹側(cè)被蓋區(qū)，伏核TH和DAT的表達無明顯變化。 MAOA、MAOB及COMT mRNA的表達改變 在3周齡和在7周齡時，與對照組相比，TP組處理黑質(zhì)和腹側(cè)被蓋區(qū)MAOA mRNA、MAOB mRNA以及COMT mRNA的表達均明顯增加。與TP組相比，F(xiàn)lu+TP組黑質(zhì)和腹側(cè)被蓋區(qū)MAOAmRNA、MAOBmRNA以及COMT mRNA的表達均明顯降低。在6月齡時，各組黑質(zhì)和腹側(cè)被蓋區(qū)MAOAmRNA、MAOBmRNA以及COMT mRNA的表達無明顯變化。 結(jié)論：乳鼠TP早期處理改變其幼年和青春期中腦DA能神經(jīng)元TH、DAT及其mRNA以及MAOA、MAOB、COMT mRNA的表達水平，表現(xiàn)為TP的增強效應(yīng)；Flu能夠抑制外源性TP的這種效應(yīng)，暗示中腦DA神經(jīng)元神經(jīng)的信號傳遞的改變與TP調(diào)控神經(jīng)遞質(zhì)DA代謝相關(guān)酶及DA轉(zhuǎn)運體的表達有關(guān)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R749.94

【引證文獻】

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1 李許;EPO與“腎精”相似性的體內(nèi)驗證及“補腎益精”中藥健腦作用機制研究[D];西南大學(xué);2016年

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