丙酸睾酮增加大鼠脑内VMAT2的表达

发布时间：2018-04-18 01:36

本文选题：囊泡单胺转运体2 + 丙酸睾酮　；参考：《河北医科大学》2012年硕士论文

【摘要】：囊泡单胺转运体2(vesicular monoamine transporter type2, VMAT2)也称为囊泡单胺转运蛋白，是位于单胺能神经元突触囊泡膜上的一种跨膜糖蛋白，其表达有三种形式，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，其作用是将胞浆内由相应前体物质合成的单胺神经递质转运至突触囊泡内，对胞浆内游离的单胺神经递质含量进行调节，控制胞浆内游离的单胺神经递质水平。VMAT2表达的减少一方面使单胺能神经元突触囊泡内可利用的单胺神经递质降低；另一方面由于胞浆内游离的单胺神经递质含量增加，其被氧化后产生较多的氧自由基，，进而影响线粒体功能而导致单胺能神经元的退行性变。单胺神经递质主要包括多巴胺（dopamine, DA）、5-羟色胺（serotonin,5-HT）以及去甲肾上腺素（norepinephrine, NE）等，这些神经递质均含有一个氨基基团，可被单胺氧化酶氧化，产生自由基。研究表明脑内单胺能神经元参与机体多种神经精神活动的调节，其功能的障碍可导致不同形式的神经精神性疾病。临床神经配基显像资料表明，某些神经精神性疾病患者脑内多同时存在两种以上单胺能神经体系的损伤，如帕金森病（Parkinson's disease, PD）。研究发现PD患者除表现因黑质DA能神经元损伤所致的运动障碍外，还存在5-HT能神经元损伤所致的非运动症状，如抑郁等。研究证实PD患者脑内普遍存在VMAT2的表达显著减少，推测相应神经元胞浆内游离的DA/5-HT含量增多，自由基增加，这可能是导致黑质DA能神经元以及脑干5-HT能神经元退形变的原因之一。提高VMAT2的表达则很可能延缓甚至保护这些神经元免受进一步的损伤，特别是当相关神经元处于代偿性合成过多单胺神经递质时期。除生殖功能外，雄激素还参与脑内神经结构与功能活动的调节。在机体衰老的过程中，体内雄激素-睾酮逐渐减少，而有些神经精神性疾病如PD又多发生于老年男性，其发病是否与雄激素有关联目前尚不清楚。但临床资料表明，PD患者雄激素替代后，其症状得到部分缓解，推测补充雄激素可能提高了脑内单胺能神经元VMAT2的表达。因此本课题以成年雄性大鼠去势处理为实验因素，建立动物模型，观察去势大鼠给予丙酸睾酮（testosterone propionate, TP）处理后对VMAT2表达的影响，期望所获结果为相关神经精神性疾病的发病及治疗提供一定的实验依据。目的：观察TP处理对大鼠VMAT2表达的影响，探讨鼻腔及皮下两种TP给药方式对VMAT2表达的影响效果，期望所获结果为相关神经精神性疾病的发病及治疗提供一定的实验依据。方法：将成年雄性Wistar大鼠随机分为鼻腔给药组和皮下给药组。（1）鼻腔给药组：30只大鼠被随机分为3组，即假手术处理组（Sham）、去势鼻腔安慰剂组（GDX）、去势鼻腔给药组（GDX-TP），每组10只（各5只分别用于免疫组织化学和免疫印迹）。（2）皮下给药组：30只大鼠被随机分为3组，即假手术处理组（Sham-sc）、去势皮下安慰剂组（GDX-sc）、去势皮下给药组（GDX-sc.TP），每组10只（各5只分别用于免疫组织化学和免疫印迹）。鼻腔给药组通过鼻腔给予睾酮制剂-TP，皮下给药组通过背部皮下注射TP，给药剂量为2.0mg/kg.d，于5:00~6:00PM期间给药，每天1次。对照组分别按上述方式给予芝麻油作为安慰剂，连续21天。大鼠以4%多聚甲醛经心灌注固定或直接断头取脑，相应脑组织分别以免疫组织化学和免疫印迹检测大鼠脑内VMAT2的表达变化。结果： 1免疫组织化学染色的一般观察在所观察的脑区中，黑质（substantia nigra, SN）、腹侧被盖区（ventraltegmental area,VTA）的VMAT2阳性标记结构主要以神经元胞体为主，仅有少量神经元突起被标记；尾壳核（caudate-putamen, CPu）及伏核（accumbens nucleus,Acb）的VMAT2阳性标记结构呈点状密集分布于核团的基质区、两核团未见神经元胞体的阳性标记。去势大鼠SN、VTA的VMAT2阳性神经元胞体的免疫反应强度、以及CPu、Acb的VMAT2阳性免疫反应强度均比相应对照组减弱；去势大鼠鼻腔或皮下给予TP后相应脑区VMAT2的免疫反应强度增加。 2经鼻腔给予TP对VMAT2表达的影响 1）SN、CPu的VMAT2表达对免疫组织化学测量结果的统计分析发现，GDX组SN的VMAT2阳性神经元胞体免疫反应强度的AOD值比Sham组降低13%（P0.05）；CPu的背内侧（dorsomedial, DM）、腹内侧（ventromedial, VM）、腹外侧（ventrolateral, VL）和背外侧（dorsolateral, DL）四个亚区的VMAT2免疫反应强度AOD值比Sham组分别降低16%（P0.01）、11%（P0.01）、11%（P0.01）和17%（P0.01）。去势大鼠鼻腔给予TP后上述脑区VMAT2的表达恢复到Sham组水平。免疫印记结果显示，雄性大鼠去势后SN和CPu的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2与GAPDH免疫印迹条带相对吸光度的比值均明显降低， GDX组SN的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2比Sham组分别降低了33%（P0.05）、27%（P0.01）和51%（P0.05）；CPu分别降低54%（P0.01）、31%（P0.05）和26%（P0.05）。去势大鼠鼻腔给予TP后上述脑区VMAT2的表达恢复到Sham组水平。 2）VTA和Acb的VMAT2表达 GDX组VTA神经元的VMAT2免疫反应强度的AOD值比Sham组降低15%（P0.01）；Acb的Core和Shell两个亚区的AOD值分别降低12%（P0.01）和11%（P0.01）。去势大鼠鼻腔给予TP后VTA和Acb的VMAT2的表达恢复至Sham组水平。免疫印记结果显示，雄性大鼠去势后VTA的Partially glyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2与GAPDH免疫印迹条带相对吸光度比值均明显降低，GDX组VTA的Partiallyglyco-VMAT2和Native-VMAT2与GAPDH免疫印迹条带相对吸光度的比值比Sham组分别降低41%（P0.05）和46%（P0.01）；Acb分别降低19%（P0.05）、50%（P0.01）和47%（P0.01）。去势大鼠鼻腔给予TP后VTA和Acb的VMAT2表达恢复到Sham组水平；Acb的Glyco-VMAT2的表达甚至超过了Sham组水平的190%（P0.05）。 3皮下给予TP对VMAT2表达的影响 1）SN、CPu的VMAT2表达雄性大鼠去势后显著降低了SN和CPu的四个亚区VMAT2免疫反应强度。去势大鼠皮下给予TP后增加了SN和CPu四个亚区VMAT2的表达，但仍低于Sham-sc组水平。免疫印记结果显示，雄性大鼠去势后SN和CPu的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2与GAPDH免疫印迹条带相对吸光度的比值均明显降低，去势大鼠皮下给予TP后虽增加了SN和CPu三种VMAT2的表达，但尚未恢复到Sham-sc组水平。 2）VTA、Acb的VMAT2表达雄性大鼠去势后显著降低了VTA和Acb两个亚区VMAT2免疫反应强度。去势大鼠皮下给予TP后增加VTA和Acb的VMAT2表达，但仍低于Sham-sc组水平。免疫印记结果显示，雄性大鼠去势后VTA的Partiallyglyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partiallyglyco-VMAT2和Native-VMAT2与GAPDH免疫印迹条带相对吸光度的比值均明显降低。去势大鼠皮下给予TP后只增加了VTA的Partiallyglyco-VMAT2和Native-VMAT2以及Acb的Partially glyco-VMAT2的表达，GDX-sc.TP组VTA的Partially glyco-VMAT2和Native-VMAT2以及Acb的Glyco-VMAT2、Partially glyco-VMAT2和Native-VMAT2的表达仍低于Sham-sc组水平。 4鼻腔及皮下给予TP对VMAT2表达影响的比较通过免疫组织化学和免疫印迹方法共观察了SN、CPu、VTA和Acb四个脑区VMAT2的表达变化。其中免疫组织化学分析了8个位置，免疫印迹分析了3个免疫印迹条带。雄性大鼠去势显著降低上述脑区VMAT2的表达；鼻腔给予TP能够恢复去势大鼠VMAT2的表达；皮下给予TP仅增加去势大鼠上述脑区VMAT2的表达，但未恢复到假手术水平。结论： 1.去势降低雄性大鼠SN、CPu、VTA、Acb的VMAT2表达。 2.丙酸睾酮增加去势大鼠SN、CPu、VTA、Acb的VMAT2表达。 3.鼻腔给予丙酸睾酮对VMAT2表达的改善优于皮下给药途径。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R338

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