【作者】 周莉；

【导师】 肖志成； 周新富；

【作者基本信息】 昆明医科大学 ， 外科学， 2013， 博士

【摘要】 研究背景：酒精中毒和抑郁症均是神经退行性疾病,其共同特征均是在特定脑区出现大量神经元凋亡,并伴随神经再生减少。并且,酒精中毒和抑郁症表现出很高的共病率。临床证据表明,抑郁症状常出现在慢性酒精中毒的戒断过程中,并且对戒断后复饮有很大的影响。虽然没有一种单一的神经生物学机制可以解释这2种对大脑均有巨大破坏性疾病的发生,但不断有证据提示该2种疾病均与神经再生减少有关。脑源性神经营养因子(BDNF)能够影响中枢神经系统内神经元的发育和存活。许多研究提示酒精中毒和抑郁症均是成年期海马的神经再生发生了改变引起的。由此,我们提出这样一个设想：神经元再生的改变是否就是这2种神经精神疾病——酒精中毒和抑郁症共同的神经生物学机制。因此,本实验的主要内容包括：BDNF和proBDNF及其受体分别在抑郁症和酒精中毒中表达的变化,并探讨在酒精戒断过程中出现抑郁症状时这些因子的变化及2种疾病的相互作用。方法：1. proBDNF及其受体在人类抑郁症及酒精中毒中表达的研究：抽取2010年至2011年在云南省精神病医院住院的满足ICD-10对抑郁症(40例)及慢性酒中毒(30例)诊断标准的病人血样以及与其相匹配的正常人血样男女各50例。从中分离血清,并提取RNA和蛋白质,通过western blotting、 RT-qPCR和ELISA等技术,观察proBDNF、 BDNF、 p75NTR、 sotilin及TrkB这5个因子与这2种疾病的关系。同时,在酒精中毒实验中,我们在酒精戒断后采用PANSS量表对酒精中毒患者的阳性和阴性症状做出评价,利用其中的抑郁因子得分比较酒精戒断后的抑郁症状,探讨当酒精戒断伴随抑郁症状出现时proBDNF及其受体的变化。2. proBDNF及其受体在慢性酒中毒小鼠中表达的研究：以50例小鼠为研究对象,建立慢性酒中毒小鼠模型,分为正常对照组、慢性酒中毒组、酒精戒断1天组(简称戒断1天组),酒精戒断3天组(简称戒断3天组)和慢性酒中毒侧脑室注射anti-proBDNF组(简称侧脑室注射anti-proBDNF组)。观察BDNF. proBDNF因子及其受体和慢性酒中毒的关系。并且观察连续酒精戒断3天内小鼠的抑郁行为,探讨当酒精戒断过程中出现抑郁症状时proBDNF及其受体的变化。结果：1.重症抑郁患者及慢性酒精中毒患者与正常对照组比较,proBDNF, sortilin和p75NTR的蛋白水平较高,而TrkB的蛋白质水平降低。proBDNF,p75NTR,sortilin和TrkB表达的血.清蛋白水平与淋巴细胞蛋白水平的趋势是一致的。2.在重症抑郁患者中,BDNF,TrkB和p75NTR在mRNA水平的改变与其在蛋白水平的改变一致。重症抑郁症患者中,血清中mBDNF浓度明显低于正常对照组的水平(P<0.05)。且在重症抑郁症患者中,血清中proBNDF的表达水平与血清mBDNF的水平呈反比。3. proBDNF、 p75NTR及sortilin在淋巴细胞蛋白及血清中的表达与重症抑郁程度(HRSD-21项评分)呈正相关：而mBDNF及TrkB与HRDS-21项的得分呈负相关。4.在人类血液样本中,慢性酒中毒患者酒精戒断前无论是淋巴细胞蛋白还是血清中的proBDNF, sortilin和p75NTR的表达水平均高于正常对照组,而nBDNF和TrkB的水平较低。在进行了一周的戒断治疗后,proBDNF,sortilin和p75NTR的表达水平较戒断前明显降低了,而mBDNF和TrkB的水平则明显升高。通过ELISA检测时发现血清中mBDNF的水平在酒精戒断前较正常对照组低,而戒断一周后其水平就明显增加了。比较戒断前proBDNF和mBDNF的表达比例时发现,在慢性酒中毒患者中proBDNF/mBDNF的平衡被打破了。5.人类血液样本中,mBDNF和TrkB的表达水平与慢性酒中毒患者平均每天酒精消耗量呈负相关,proBDNF,p75NTR和sortilin的表达水平与慢性酒中毒患者平均每天酒精消耗量呈正相关。6.酒精戒断1周后,酒精中毒患者的抑郁症状较戒断前及正常对照组明显,但未达到统计学意义。7.通过行为学检查发现,经过56天的慢性酒精溶液灌胃后实验组小鼠的体重、毛色、精神状态、水平动机活动、探究行为及认知功能都有明显改变,可以说明慢性酒精中毒小鼠造模成功。8.戒断的行为学实验发现,随着戒断时间的延长,戒断组小鼠水平动机活动、探究行为及认知功能都逐渐恢复,有的已经接近或超过正常对照组。但戒断1天时,强迫游泳实验中戒断组小鼠的绝对不动时间最长,与慢性酒中毒组和正常对照组均有统计学差异。随着戒断时间的延长,戒断组小鼠的绝对不动时间有所减少,但仍比正常对照组小鼠长。9.侧脑室注射anti-proBDNF实验中,侧脑室注射anti-proBDNF组小鼠的认知功能较慢性酒中毒组明显改善,但仍未达到正常水平。10.慢性酒中毒组小鼠中proBDNF、 sortilin和p75NTR的脑蛋白水平高于戒断组及正常对照组,而mBDNF和TrkB水平较低。且随着戒断时间的延长,戒断组小鼠proBDNF、 sortilin、 p75NTR和TrkB水平都逐渐恢复,有的已经接近或超过正常对照组。侧脑室注射anti-proBDNF实验中,侧脑室注射anti-proBDNF组小鼠脑蛋白proBDNF、 sortilin、 p75NTR和TrkB的表达水平都有所恢复,但仍未达到正常水平。11.实时荧光定量PCR的结果发现,鼠脑BDNF、 sortilin、 p75NTR和TrkB的mRNA表达水平与其蛋白表达水平一致。即戒断后小鼠BDNF和TrkB的mRNA表达较慢性酒中毒组有所升高,但未恢复到正常水平；sortilin和p75NTR的mRNA表达较慢性酒中毒组有所降低,但未恢复到正常水平。结论：1.我们的初步研究结果显示,在重症抑郁患者和慢性酒精中毒患者中proBDNF及其受体p75NTR和sortilin的表达上调,而TrkB和BDNF的水平下调了。也就是,在proBDNF/p75NTR/sortilin和mBDNF/TrkB信号途径之间的平衡被破坏了。尽管我们的研究存在许多局限性,但其研究结果表明,在重症抑郁和慢性酒中毒中proBDNF/p75NTR/sortilin信号途径发挥着潜在的作用。也表明,抑郁症和酒精中毒的发病存在共同的分子机制,这可能也是2种疾病共病的分子基础。但这仅仅只是一个研究的开始,关于BDNF在抑郁症和慢性酒中毒中的作用还需要进一步的深入研究,以便更多地认识抑郁症和酒中毒这2种神经精神疾病,为其治疗提供新的方向及靶点。2.无论在人类慢性酒中毒样本还是小鼠慢性酒精中毒模型中我们均发现proBDNF/p75NTR/soritlin信号通路的表达增强了,由此引发了慢性酒中毒小鼠的海马及前额叶皮层等脑区的功能障碍。而通过对他们进行干预(戒断或侧脑室注射anti-proBDNF)后,减弱了proBDNF/p75NTR/soritlin信号通路对神经元发生的抑制作用,且mBDNF和TrkB的表达水平却能够明显回升,促进了大脑中神经元的再生,也改善慢性酒中毒引起的认知功能及行为能力障碍。3.无论在人类慢性酒中毒样本还是小鼠慢性酒精中毒模型中,在酒精戒断后均出现了抑郁样症状或行为。这可能是由于在慢性酒精中毒的发展过程中,proBDNF/mBDNF的平衡失调,超过了机体的可调节范围,使得脑内神经发生被明显抑制。但随着酒精戒断的时间逐渐延长,机体的代偿功能逐渐恢复,神经元再生也开始增多,抑郁症状才逐步减轻。更多还原

【Abstract】 Background:Alcoholism and depression are all neurodegenerative diseases, which have common features of a large number neuronal apoptosis in specific brain regions, accompanied by reduced nerve regeneration. Also, alcoholism and depression showed a high rate of comorbidity. Clinical evidence also indicates that depression that emerges during abstinence from chronic alcohol use has a greater negative impact on relapse. Although no single neurobiological mechanism can account for the behavioral pathologies associated with these devastating disorders, converging evidence suggests that aspects of both alcoholism and depression are linked to reductions in neurogenesis.Brain-derived neurotrophic factor (BDNF) can affect the neuronal development and survival in the central nervous system. Many studies have suggested alcoholism and depression are caused by changes of adult hippocampal neurogenesis. Thus, we propose such an idea:changes of neurogenesis are the common neurobiological mechanisms of these two neuropsychiatric disorders-alcoholism and depression. Therefore, this study mainly covers: the expressions of BDNF and proBDNF and their receptors in depression and alcoholism, and to explore the depression symptoms occurred during the process of the alcohol withdrawal; the last are the interactions of the2diseases.Methods:1. The association studies of proBDNF and its receptors in human depression and alcoholism:40female depression patients and30male alcoholism patients were recruited from Yunnan Mental Hospital, which were all met the1CD-10diagnosis criteria of depression and alcoholism in the year from2010to2011. The control groups were the matched men and women50cases respectly. The serum, RNA and protein were extracted from the blood samples, and the proBDNF, BDNF, p75NTR, sotilin and TrkB factors were assayed by western blotting, RT-qPCR and ELISA techniques and the relationships between the factors and the two kinds of diseases were observation. Meanwhile the PANSS were used to evaluate the depression scores in the alcoholism patients before and after alcohol withdrawal and discuss the expression changes of proBDNF and its receptor.2. The researches of the expressions of proBDNF and its receptors in the chronic alcoholism mice model:50mice were used to establish a chronic alcoholism mouse model. And all the mice were divided into normal control group, group of chronic alcoholism, alcohol withdrawal one day group, alcohol withdrawal3days group and anti-proBDNF group. The chronic alcoholism mouse model was used to observe the changes of expressions of BDNF, proBDNF and their receptors. Then the FST test was used to explore the depression behaviors after alcohol withdrawal.Results:1. ProBDNF, sortilin and p75NTR protein levels were higher in major depression and chronic alcoholism patients than in control groups and TrkB protein level was decreased. The expression trends of proBDNF, p75NTR, sortilin and TrkB levels in the serum were consistent with the changes of lymophocytes protein levels.2. In major depressive patients, the expression levels of BDNF, TrkB and p75NTR at the mRNA level changed consistently with their trends in protein levels. The serum concentrations in the major depressive patients were significantly higher than in the control group’s (P<0.05). In the major depressive patients, the proBDNF levels in the serum had negative relationship with the mBDNF levels in the serum.3. In the major depressive patients, the expressions of proBDNF, p75NTR and sortilin in lymphocytes and serum had positive correlations with the extent of depression (HRSD-21score); while the serum levels of mBDNF and TrkB were negatively correlated with HRDS-21item scores4. In the human blood samples, before alcohol withdrawal, either in lymophocytes or in serum, the expression levels of proBDNF, sortilin and p75NTR were higher in chronic alcoholism group than in the control group, while mBDNF and TrkB levels were lower in patients’group. After a week of abstinence, proBDNF, sortilin and p75NTR expression levels were significantly reduced in chronic alcoholism group when compared with control group and mBDNF and TrkB levels were significantly increased. Through ELISA test, serum mBDNF level was found reduced before the alcohol withdrawal when compared with normal control group, but a week after the withdrawal it significantly increased. proBDNF/mBDNF balance was broken in patients with chronic alcoholism.5. In the human blood samples, mBDNF and TrkB expression levels in patients with chronic alcoholism were negatively correlated with average daily alcohol consumption; proBDNF, p75NTR and sortilin expression levels in chronic alcoholism patients were positively correlated with average daily alcohol consumption.6. In the human samples, the depression symptoms during alcohol withdrawal were more abvious after one week’s alcohol withdrawal than before abstinence and control group, but the significance did not reach statistical significance.7. Through behavioral tests, we found that after56days of chronic alcohol solution intragastric administration, the mice body weight, hair color, mental status, level of motivation activity, exploratory behavior, and cognitive function all significantly changed, it suggested that chronic alcoholism mice model was successfully established.8. Withdrawal behavioral experiments of mice, we found that with the withdrawal time, the level of motivation activities, explore behaviour and cognitive functions were gradually restored after alcohol abstinence, and some results had been close to or more than the normal control group. But after one day’s abstinence, the absolute immobility time was the longest among three days FST in the chronic alcohohlism group. With the withdrawal time, the absolute immobility time decreased in chronic alcoholism group, but still longer than normal control mice.9. In the intracerebroventricular injection of anti-proBDNF experiment, after icv anti-proBDNF, cognitive function in the chronic alcoholism group was significantly improved, but had not yet reached the normal levels.10. The proBDNF, sortilin and p75NTR protein levels in mice brain were the highest in the chronic alcoholism group than in the withdrawal group and the normal control group, while mBDNF and TrkB level were the lowest. And with the withdrawal time, the proBDNF, sortilin, p75NTR and TrkB levels were gradually restored, and some had been close to or more than the normal control group. After intracerebroventricular injection of anti-proBDNF, proBDNF, sortilin, p75NTR and TrkB expression levels were all recovered, but had not yet reached normal levels.11. The real-time quantitative PCR results showed that BDNF, sortilin, p75NTR and TrkB mRNA levels in mice brain were consistent with their protein expression levels. That was after the withdrawal, BDNF and TrkB mRNA levels were increased, but did not return to the normal levels; sortilin and p75NTR mRNA levels were decreased, but did not return to normal levels.Conlusions:1. Our preliminary findings showed that in major depressive patients and chronic alcoholism patients, proBDNF and its receptors p75NTR and sortilin were upregulated, and TrkB and BDNF levels lowered. That was that the balance between proBDNF/p75NTR/sortilin and mBDNF/TrkB signaling pathways was destroyed. Although there are many limitations of our study, but the results suggested that in major depressive patients and chronic alcoholism patients, the proBDNF/p75NTR/sortilin signaling pathway played a potential role in the neuropsychiatries disorders. It also suggested that there was common molecular mechanism in the pathology of depression and alcoholism, which might also be the two molecular basis of the comorbidity of these two diseases. But this was just a start of the study, the role of BDNF in depression and chronic alcoholism still require further in-depth study in order to know more about the two diseases and provide a new treatment direction.2. Whether chronic alcohol intoxication in humans or mice model, we had found that proBDNF/p75NTR/soritlin signaling pathway expression increased, which led to dysfunctios of hippocampus and prefrontal cortex and other brain regions in chronic alcoholism mice. Through the intervention (withdrawal or intracerebroventricular injection of anti-proBDNF), the weakened proBDNF/p75NTR/soritlin signaling pathway had less inhibition of neurogenesis and mBDNF and TrkB expression level was able to rebound significantly so that to promoted regeneration of neurons in the brain, but also to improve cognitive and behavioral skills caused by chronic alcohol poisoning.3. Whether in human chronic alcoholism blood samples or chronic alcoholism mice model, the depression symptoms occurred during alcohol abstinence. The reason might be the balance of proBDNF/mBDNF was broken during chronic alcohol intoxication, which beyond the ability of the body and led to the inhibition of neurogenesis in the brain. However, with the abstinence time, the compensation functions of the body recovered and the neuroregeneration began, so the depression symptoms alleviated.更多还原