本文選題：囊泡單胺轉(zhuǎn)運(yùn)體2 + 丙酸睪酮　； 參考：《河北醫(yī)科大學(xué)》2012年碩士論文

【摘要】：囊泡單胺轉(zhuǎn)運(yùn)體2(vesicular monoamine transporter type2, VMAT2)也稱為囊泡單胺轉(zhuǎn)運(yùn)蛋白，是位于單胺能神經(jīng)元突觸囊泡膜上的一種跨膜糖蛋白，其表達(dá)有三種形式，即75KDa的糖基化VMAT2（Glyco-VMAT2）、55KDa的部分糖基化VMAT2（Partially glyco-VMAT2）以及45KDa的原型VMAT2（Native-VMAT2）。Glyco-VMAT2是成熟形式，其作用是將胞漿內(nèi)由相應(yīng)前體物質(zhì)合成的單胺神經(jīng)遞質(zhì)轉(zhuǎn)運(yùn)至突觸囊泡內(nèi)，對(duì)胞漿內(nèi)游離的單胺神經(jīng)遞質(zhì)含量進(jìn)行調(diào)節(jié)，控制胞漿內(nèi)游離的單胺神經(jīng)遞質(zhì)水平。VMAT2表達(dá)的減少一方面使單胺能神經(jīng)元突觸囊泡內(nèi)可利用的單胺神經(jīng)遞質(zhì)降低；另一方面由于胞漿內(nèi)游離的單胺神經(jīng)遞質(zhì)含量增加，其被氧化后產(chǎn)生較多的氧自由基，，進(jìn)而影響線粒體功能而導(dǎo)致單胺能神經(jīng)元的退行性變。 單胺神經(jīng)遞質(zhì)主要包括多巴胺（dopamine, DA）、5-羥色胺（serotonin,5-HT）以及去甲腎上腺素（norepinephrine, NE）等，這些神經(jīng)遞質(zhì)均含有一個(gè)氨基基團(tuán)，可被單胺氧化酶氧化，產(chǎn)生自由基。研究表明腦內(nèi)單胺能神經(jīng)元參與機(jī)體多種神經(jīng)精神活動(dòng)的調(diào)節(jié)，其功能的障礙可導(dǎo)致不同形式的神經(jīng)精神性疾病。臨床神經(jīng)配基顯像資料表明，某些神經(jīng)精神性疾病患者腦內(nèi)多同時(shí)存在兩種以上單胺能神經(jīng)體系的損傷，如帕金森病（Parkinson's disease, PD）。研究發(fā)現(xiàn)PD患者除表現(xiàn)因黑質(zhì)DA能神經(jīng)元損傷所致的運(yùn)動(dòng)障礙外，還存在5-HT能神經(jīng)元損傷所致的非運(yùn)動(dòng)癥狀，如抑郁等。研究證實(shí)PD患者腦內(nèi)普遍存在VMAT2的表達(dá)顯著減少，推測(cè)相應(yīng)神經(jīng)元胞漿內(nèi)游離的DA/5-HT含量增多，自由基增加，這可能是導(dǎo)致黑質(zhì)DA能神經(jīng)元以及腦干5-HT能神經(jīng)元退形變的原因之一。提高VMAT2的表達(dá)則很可能延緩甚至保護(hù)這些神經(jīng)元免受進(jìn)一步的損傷，特別是當(dāng)相關(guān)神經(jīng)元處于代償性合成過多單胺神經(jīng)遞質(zhì)時(shí)期。 除生殖功能外，雄激素還參與腦內(nèi)神經(jīng)結(jié)構(gòu)與功能活動(dòng)的調(diào)節(jié)。在機(jī)體衰老的過程中，體內(nèi)雄激素-睪酮逐漸減少，而有些神經(jīng)精神性疾病如PD又多發(fā)生于老年男性，其發(fā)病是否與雄激素有關(guān)聯(lián)目前尚不清楚。但臨床資料表明，PD患者雄激素替代后，其癥狀得到部分緩解，推測(cè)補(bǔ)充雄激素可能提高了腦內(nèi)單胺能神經(jīng)元VMAT2的表達(dá)。因此本課題以成年雄性大鼠去勢(shì)處理為實(shí)驗(yàn)因素，建立動(dòng)物模型，觀察去勢(shì)大鼠給予丙酸睪酮（testosterone propionate, TP）處理后對(duì)VMAT2表達(dá)的影響，期望所獲結(jié)果為相關(guān)神經(jīng)精神性疾病的發(fā)病及治療提供一定的實(shí)驗(yàn)依據(jù)。 目的：觀察TP處理對(duì)大鼠VMAT2表達(dá)的影響，探討鼻腔及皮下兩種TP給藥方式對(duì)VMAT2表達(dá)的影響效果，期望所獲結(jié)果為相關(guān)神經(jīng)精神性疾病的發(fā)病及治療提供一定的實(shí)驗(yàn)依據(jù)。 方法：將成年雄性Wistar大鼠隨機(jī)分為鼻腔給藥組和皮下給藥組。（1）鼻腔給藥組：30只大鼠被隨機(jī)分為3組，即假手術(shù)處理組（Sham）、去勢(shì)鼻腔安慰劑組（GDX）、去勢(shì)鼻腔給藥組（GDX-TP），每組10只（各5只分別用于免疫組織化學(xué)和免疫印跡）。（2）皮下給藥組：30只大鼠被隨機(jī)分為3組，即假手術(shù)處理組（Sham-sc）、去勢(shì)皮下安慰劑組（GDX-sc）、去勢(shì)皮下給藥組（GDX-sc.TP），每組10只（各5只分別用于免疫組織化學(xué)和免疫印跡）。鼻腔給藥組通過鼻腔給予睪酮制劑-TP，皮下給藥組通過背部皮下注射TP，給藥劑量為2.0mg/kg.d，于5:00~6:00PM期間給藥，每天1次。對(duì)照組分別按上述方式給予芝麻油作為安慰劑，連續(xù)21天。 大鼠以4%多聚甲醛經(jīng)心灌注固定或直接斷頭取腦，相應(yīng)腦組織分別以免疫組織化學(xué)和免疫印跡檢測(cè)大鼠腦內(nèi)VMAT2的表達(dá)變化。 結(jié)果： 1免疫組織化學(xué)染色的一般觀察 在所觀察的腦區(qū)中，黑質(zhì)（substantia nigra, SN）、腹側(cè)被蓋區(qū)（ventraltegmental area,VTA）的VMAT2陽性標(biāo)記結(jié)構(gòu)主要以神經(jīng)元胞體為主，僅有少量神經(jīng)元突起被標(biāo)記；尾殼核（caudate-putamen, CPu）及伏核（accumbens nucleus,Acb）的VMAT2陽性標(biāo)記結(jié)構(gòu)呈點(diǎn)狀密集分布于核團(tuán)的基質(zhì)區(qū)、兩核團(tuán)未見神經(jīng)元胞體的陽性標(biāo)記。 去勢(shì)大鼠SN、VTA的VMAT2陽性神經(jīng)元胞體的免疫反應(yīng)強(qiáng)度、以及CPu、Acb的VMAT2陽性免疫反應(yīng)強(qiáng)度均比相應(yīng)對(duì)照組減弱；去勢(shì)大鼠鼻腔或皮下給予TP后相應(yīng)腦區(qū)VMAT2的免疫反應(yīng)強(qiáng)度增加。 2經(jīng)鼻腔給予TP對(duì)VMAT2表達(dá)的影響 1）SN、CPu的VMAT2表達(dá) 對(duì)免疫組織化學(xué)測(cè)量結(jié)果的統(tǒng)計(jì)分析發(fā)現(xiàn)，GDX組SN的VMAT2陽性神經(jīng)元胞體免疫反應(yīng)強(qiáng)度的AOD值比Sham組降低13%（P0.05）；CPu的背內(nèi)側(cè)（dorsomedial, DM）、腹內(nèi)側(cè)（ventromedial, VM）、腹外側(cè)（ventrolateral, VL）和背外側(cè)（dorsolateral, DL）四個(gè)亞區(qū)的VMAT2免疫反應(yīng)強(qiáng)度AOD值比Sham組分別降低16%（P0.01）、11%（P0.01）、11%（P0.01）和17%（P0.01）。去勢(shì)大鼠鼻腔給予TP后上述腦區(qū)VMAT2的表達(dá)恢復(fù)到Sham組水平。免疫印記結(jié)果顯示，雄性大鼠去勢(shì)后SN和CPu的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2與GAPDH免疫印跡條帶相對(duì)吸光度的比值均明顯降低， GDX組SN的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2比Sham組分別降低了33%（P0.05）、27%（P0.01）和51%（P0.05）；CPu分別降低54%（P0.01）、31%（P0.05）和26%（P0.05）。去勢(shì)大鼠鼻腔給予TP后上述腦區(qū)VMAT2的表達(dá)恢復(fù)到Sham組水平。 2）VTA和Acb的VMAT2表達(dá) GDX組VTA神經(jīng)元的VMAT2免疫反應(yīng)強(qiáng)度的AOD值比Sham組降低15%（P0.01）；Acb的Core和Shell兩個(gè)亞區(qū)的AOD值分別降低12%（P0.01）和11%（P0.01）。去勢(shì)大鼠鼻腔給予TP后VTA和Acb的VMAT2的表達(dá)恢復(fù)至Sham組水平。免疫印記結(jié)果顯示，雄性大鼠去勢(shì)后VTA的Partially glyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2與GAPDH免疫印跡條帶相對(duì)吸光度比值均明顯降低，GDX組VTA的Partiallyglyco-VMAT2和Native-VMAT2與GAPDH免疫印跡條帶相對(duì)吸光度的比值比Sham組分別降低41%（P0.05）和46%（P0.01）；Acb分別降低19%（P0.05）、50%（P0.01）和47%（P0.01）。去勢(shì)大鼠鼻腔給予TP后VTA和Acb的VMAT2表達(dá)恢復(fù)到Sham組水平；Acb的Glyco-VMAT2的表達(dá)甚至超過了Sham組水平的190%（P0.05）。 3皮下給予TP對(duì)VMAT2表達(dá)的影響 1）SN、CPu的VMAT2表達(dá) 雄性大鼠去勢(shì)后顯著降低了SN和CPu的四個(gè)亞區(qū)VMAT2免疫反應(yīng)強(qiáng)度。去勢(shì)大鼠皮下給予TP后增加了SN和CPu四個(gè)亞區(qū)VMAT2的表達(dá)，但仍低于Sham-sc組水平。免疫印記結(jié)果顯示，雄性大鼠去勢(shì)后SN和CPu的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2與GAPDH免疫印跡條帶相對(duì)吸光度的比值均明顯降低，去勢(shì)大鼠皮下給予TP后雖增加了SN和CPu三種VMAT2的表達(dá)，但尚未恢復(fù)到Sham-sc組水平。 2）VTA、Acb的VMAT2表達(dá) 雄性大鼠去勢(shì)后顯著降低了VTA和Acb兩個(gè)亞區(qū)VMAT2免疫反應(yīng)強(qiáng)度。去勢(shì)大鼠皮下給予TP后增加VTA和Acb的VMAT2表達(dá)，但仍低于Sham-sc組水平。免疫印記結(jié)果顯示，雄性大鼠去勢(shì)后VTA的Partiallyglyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partiallyglyco-VMAT2和Native-VMAT2與GAPDH免疫印跡條帶相對(duì)吸光度的比值均明顯降低。去勢(shì)大鼠皮下給予TP后只增加了VTA的Partiallyglyco-VMAT2和Native-VMAT2以及Acb的Partially glyco-VMAT2的表達(dá)，GDX-sc.TP組VTA的Partially glyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2的表達(dá)仍低于Sham-sc組水平。 4鼻腔及皮下給予TP對(duì)VMAT2表達(dá)影響的比較 通過免疫組織化學(xué)和免疫印跡方法共觀察了SN、CPu、VTA和Acb四個(gè)腦區(qū)VMAT2的表達(dá)變化。其中免疫組織化學(xué)分析了8個(gè)位置，免疫印跡分析了3個(gè)免疫印跡條帶。雄性大鼠去勢(shì)顯著降低上述腦區(qū)VMAT2的表達(dá)；鼻腔給予TP能夠恢復(fù)去勢(shì)大鼠VMAT2的表達(dá)；皮下給予TP僅增加去勢(shì)大鼠上述腦區(qū)VMAT2的表達(dá)，但未恢復(fù)到假手術(shù)水平。 結(jié)論： 1.去勢(shì)降低雄性大鼠SN、CPu、VTA、Acb的VMAT2表達(dá)。 2.丙酸睪酮增加去勢(shì)大鼠SN、CPu、VTA、Acb的VMAT2表達(dá)。 3.鼻腔給予丙酸睪酮對(duì)VMAT2表達(dá)的改善優(yōu)于皮下給藥途徑。
[Abstract]:Vesicular monoamine transporter 2 (vesicular monoamine transporter type2, VMAT2) also known as the vesicular monoamine transporter is located in monoaminergic synaptic vesicles is a transmembrane glycoprotein membrane, its expression has three forms, namely 75KDa glycosylation of VMAT2 (Glyco-VMAT2), part of the glycosylation of VMAT2 55KDa (Partially glyco-VMAT2) and 45KDa VMAT2 (Native-VMAT2).Glyco-VMAT2 prototype is a mature form, its role is to the cytoplasm by the corresponding precursors of monoamine neurotransmitter transporters to synaptic vesicle synthesis of vesicles, on monoamine neurotransmitter content of cytosolic free to regulate, control of monoamine neurotransmitter levels.VMAT2 intracellular free hand reduced the expression of monoamine neurotransmitters in synaptic vesicles can be reduced by increased; on the other hand the monoamine neurotransmitter content of cytosolic free, which is produced after oxidation More oxygen free radicals, thus affecting the function of mitochondria, lead to the degenerative changes of monoamine neurons.
Monoamine neurotransmitters including dopamine (dopamine, DA), 5- (serotonin, 5-HT) and serotonin norepinephrine (norepinephrine, NE), these neurotransmitters contain an amino group, which can be oxidized by monoamine oxidase to produce free radicals. Studies show that monoaminergic neurons are involved in a variety of regulation mental activity, its dysfunction could lead to neuropsychiatric diseases in different forms. The clinical neural imaging data show that the ligand, some neuropsychiatric disorders in patients with brain at the same time, there are more than two kinds of monoaminergic nerve system damage, such as Parkinson's disease (Parkinson's, disease, PD). The study found that patients with PD addition due to movement disorders of nigral DA neuron injury caused by the outside, there are 5-HT neuron injury caused by non motor symptoms, such as depression. Studies have confirmed that expression of VMAT2 is ubiquitous in the brain of PD patients. Reduce the content of DA/5-HT, presumably corresponding neurons of cytosolic free radicals increased, increased, this may be the cause of the substantia nigra DA neurons and 5-HT neurons of brainstem back deformation. Increase the expression of VMAT2 is likely to delay or even protect the neurons from further damage, especially when the neurons in compensatory excessive synthesis of monoamine neurotransmitters.
In addition to the reproductive function, regulation of androgen is also involved in brain structure and function activities. In the process of aging in vivo androgen testosterone decreased gradually, while some neuropsychiatric disorders such as PD and occurs in older men, the incidence and the associated androgen is not clear. But clinical data suggest that instead, PD patients with androgen, the symptoms of partial remission, that androgen supplementation may improve the monoaminergic neurons in the expression of VMAT2. Therefore the issue of adult male castrated rats as experimental factors, the establishment of the animal model, observe the ovariectomized rats given testosterone propionate (testosterone propionate, TP) on the expression of influence after VMAT2 treatment, expected results and provide experimental basis for the pathogenesis and treatment of associated neuropsychiatric disorders.
Objective: To observe the effect of TP treatment on the expression of VMAT2 in rats, and to explore the effect of two kinds of TP administration modes on the expression of VMAT2 in nasal cavity and subcutaneous tissue. The expected results will provide some experimental evidence for the pathogenesis and treatment of related neuropsychiatric diseases.
Methods: adult male Wistar rats were randomly divided into nasal administration group and subcutaneous administration group. (1) nasal administration group: 30 rats were randomly divided into 3 groups: sham operation group (Sham), ovariectomized group (GDX), placebo nasal castration intranasal administration group (GDX-TP), 10 rats in each group (5 rats in each group were used for immunohistochemistry and Western blotting). (2) subcutaneous administration group: 30 rats were randomly divided into 3 groups: sham operation group, ovariectomized (Sham-sc) subcutaneous placebo group (GDX-sc), ovariectomized group administered subcutaneously (GDX-sc.TP). 10 rats in each group (5 rats in each group were used for immunohistochemistry and Western blotting). Nasal administration group by nasal administration of testosterone preparations -TP, subcutaneous administration group by subcutaneous injection of TP, the dosage of 2.0mg/kg.d, 5:00~6:00PM during the administration, 1 times a day. The control group were given according to the above way as sesame oil placebo, for 21 consecutive days.
The rats were killed by 4% paraformaldehyde and fixed directly or directly, and the corresponding brain tissues were detected by immunohistochemistry and Western blot. The expression of VMAT2 in the brain of rats was detected.
Result:
1 general observation of immunohistochemical staining
In the brain was observed in the substantia nigra (substantia, nigra, SN), the ventral tegmental area (ventraltegmental, area, VTA) VMAT2 labeled structure is mainly composed of neurons, only a small number of neurites were labeled; caudate putamen (caudate-putamen, CPu) and nucleus accumbens (accumbens nucleus, Acb VMAT2) labeled structures are densely distributed in the matrix region punctate nucleus and nucleus were not labeled two neurons.
The immunoreactive intensity of VMAT2 positive neurons in SN and VTA of ovariectomized rats, as well as the VMAT2 positive immunoreaction intensity of CPu and Acb were all lower than those of the corresponding control group. The immunoreaction intensity of VMAT2 in the corresponding brain area increased after castration or after subcutaneous administration of TP in castrated rats.
2 the effect of TP on the expression of VMAT2 in the nasal cavity
1) VMAT2 expression of SN, CPu
Statistics on the measurement results of immunohistochemical analysis showed that VMAT2 positive neurons were immunoreactive intensity in GDX group SN AOD decreased 13% than in the Sham group (P0.05); dorsomedial CPu (dorsomedial, DM), ventromedial (ventromedial, VM), ventrolateral (ventrolateral, VL) and dorsolateral (dorsolateral, DL, VMAT2) immune response to AOD four sub regions decreased 16% respectively compared with Sham group (P0.01), 11% (P0.01), 11% (P0.01) and 17% (P0.01). The nasal cavity of ovariectomized rats after administration of TP VMAT2 expression in the brain is restored to the level in Sham group. Western blotting results showed that Glyco-VMAT2 SN and CPu in castrated male rats, Partially glyco-VMAT2 and Native-VMAT2 GAPDH and immunoblot band ratio of relative absorbance decreased significantly in group GDX, SN Glyco-VMAT2, Partially glyco-VMAT2 and Native-VMAT2 than in the Sham group were decreased by 33% (P0.05), 27% (P0.01) and 51% (P0.05) CPu decreased by 54% (P0.01), 31% (P0.05) and 26% (P0.05), respectively. The expression of VMAT2 in the brain region of the castrated rat's nasal cavity was restored to the level of the Sham group after TP.
2) VMAT2 expression of VTA and Acb
The intensity of VMAT2 immunoreactivity in GDX group of VTA neurons of the AOD value is 15% lower than Sham group (P0.01); Acb Core and Shell two sub region AOD value decreased by 12% (P0.01) and 11% (P0.01). The nasal cavity in ovariectomized rats given the expression of VTA and Acb VMAT2 Sham TP after the group returned to the level. Western blotting results showed that male castrated rats VTA Partially and glyco-VMAT2 Native-VMAT2 and Acb Glyco-VMAT2, Partially glyco-VMAT2 and Native-VMAT2 GAPDH and Western blot bands with relative absorbance ratio were significantly lower in group GDX, VTA Partiallyglyco-VMAT2 and Native-VMAT2 GAPDH and immunoblot band ratio relative absorbance than in the Sham group were decreased by 41% (P0.05) and 46% (P0.01); Acb were decreased by 19% (P0.05), 50% (P0.01) and 47% (P0.01). The nasal cavity in ovariectomized rats treated with VTA and Acb VMAT2 expression restored to the Sham group level after TP; expression of Acb Glyco-VMAT2 was More than 190% (P0.05) of the Sham group.
3 the effect of subcutaneous administration of TP on VMAT2 expression
1) VMAT2 expression of SN, CPu
Castrated male rats decreased significantly after the four sub regions, the intensity of VMAT2 immunoreactivity of SN and CPu. The subcutaneous of ovariectomized rats after administration of TP increased the expression of SN and CPu four sub district of VMAT2, but still lower than the level of group Sham-sc. Western blot results showed that Glyco-VMAT2, SN and CPu in castrated male rats Partially glyco-VMAT2 and Native-VMAT2 GAPDH and immunoblot band ratio of absorbance decreased significantly, subcutaneous ovariectomized rats after administration of TP was increased by SN and the expression of CPu three VMAT2, but has not been restored to the level in Sham-sc group.
2) VMAT2 expression of VTA, Acb
Castrated male rats decreased significantly after VTA and Acb two sub regions. The intensity of VMAT2 immunoreactivity increased expression of VTA and Acb VMAT2 were ovariectomized rats after administration of TP, but still lower than the level of group Sham-sc. Western blotting results showed that castrated male rats after VTA Partiallyglyco-VMAT2 and Native-VMAT2 Acb, Glyco-VMAT2, and Partiallyglyco-VMAT2 Native-VMAT2 and GAPDH blotting with the ratio of relative absorbance decreased significantly. The subcutaneous ovariectomized rats after administration of TP increased VTA Partiallyglyco-VMAT2 and Native-VMAT2 Acb and Partially glyco-VMAT2 expression, GDX-sc.TP VTA and Native-VMAT2 glyco-VMAT2 Partially group and Acb Glyco-VMAT2, the expression of Partially glyco-VMAT2 and Native-VMAT2 is still lower than the level in Sham-sc group.
Comparison of the effect of 4 nasal cavity and subcutaneous TP on the expression of VMAT2
By immunohistochemistry and Western blot methods were SN, CPu, VTA and Acb VMAT2 expression in four brain regions. The immunohistochemical analysis of the 8 position, Western blot analysis of 3 Western blot bands. The castrated rats were significantly decreased VMAT2 expression in the brain; nasal administration the expression of TP can restore VMAT2 in ovariectomized rats; subcutaneous Administration of TP only increased VMAT2 expression in the brain of ovariectomized rats, but did not return to the level of sham operation.
Conclusion:
The 1. castration reduced the VMAT2 expression of SN, CPu, VTA, and Acb in male rats.
2. testosterone propionate increases the VMAT2 expression of SN, CPu, VTA, and Acb in ovariectomized rats.
3. the improvement of VMAT2 expression by testosterone propionate is better than that of subcutaneous administration.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R338

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