【作者】 王玉同；

【导师】 窦科峰；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 对于一些常见威胁生命的疾病发病率的增加,应激被认为是一种主要的危险因素。近年来,人们逐渐认识到应激反应不但可以造成一些神经系统依赖的精神疾病,损害空间学习和记忆能力,而且可以造成如心、肝等非神经系统的结构改变,影响心脏和肝脏的功能。到目前为止,关于肝脏功能的损害是继发于脑功能损害或是与脑功能损害同时发生还知之甚少,同时,应激反应对于生命的重要性显而易见,但关于应激病理生理学的细胞分子机制,尤其是那些非神经系统器官而言,尚知之甚少。对肝脏来说,应激对于其功能影响很大而且病情发展相对较快,但是我们对于其病理生理学的分子机制的了解甚至不如心血管系统。仅仅在最近有一些证据阐述了应激反应如何对肝脏疾病产生影响,但到目前为止,对于应激反应如何影响正常肝脏的功能以及隐含的分子机制仍不明了。研究目的:本实验旨在阐明应激反应对于脑功能和肝脏功能的影响之间的关系以及应激对于肝脏功能影响的分子机制,从而更好地为我们针对性地处理和治疗应激所造成的各器官功能损害提供理论依据。研究方法:在应激对于脑功能和肝脏功能影响关系研究中,我们在14天的慢性束缚应激实验(6h/d)模型过程中应用经典的抗抑郁药物帕罗西丁(20mg/kg)和被证明可以保护肝脏功能的中药四逆散(100mg/kg)观察其对脑功能及肝脏功能的保护作用。我们监测不同处理条件下体重的变化情况,同时分别监测血清谷丙转氨酶(GPT)和碱性磷酸酶(ALP)以及Morris水迷宫来分别评价肝脏功能和脑空间学习和记忆功能。在应激对肝脏功能影响的分子机制研究中,我们将BDNF,KDR和Bax作为有可能参与束缚应激肝脏反应的候选分子来研究。为了检验急性和重复/慢性束缚应激的不同作用,我们通过半定量反转录PCR(RT-PCR)检测了受到急性或者15天重复束缚应激的大鼠肝脏内BDNF、KDR和Bax分子的mRNA水平。实验结果:通过体重监测我们发现慢性束缚应激可以明显使大鼠生长速度放缓(P<0.05),而帕罗西丁和四逆散预处理可以逆转慢性束缚应激对体重的影响(P>0.05);慢性束缚应激可以造成大鼠血清谷氨酸氨基转移酶(GPT)和碱性磷酸酶(ALP)水平明显升高(P<0.05)以及大鼠空间学习和记忆能力明显下降(P<0.05);14天帕罗西丁预处理可以提高大鼠空间学习和记忆能力(P<0.05),而对血清谷氨酸氨基转移酶(GPT)和碱性磷酸酶(ALP)的酶活性没有影响(P>0.05);另一方面,14天四逆散预处理对慢性束缚应激大鼠空间学习和记忆能力没有影响(P>0.05),而可以逆转慢性束缚应激造成的血清GPT和ALP酶活性的下降(P<0.05)。在实验二中我们发现:急性束缚应激刺激后,KDR mRNA的水平明显(P < 0.01)增加,慢性/重复束缚应激后,肝脏内BDNF mRNA水平明显(P < 0.01)下降,而,Bax无论在急性还是在慢性/重复束缚应激条件下都保持在正常水平(P>0.05)。实验结论:束缚应激造成的肝脏功能损害与脑功能损害是伴随发生的,慢性束缚应激通过不同的机制损害肝脏和脑功能。BDNF和KDR分别参与了肝脏急性和慢性束缚应激的反应,出乎我们意料的是,对于肝脏急性和慢性束缚应激的反应,Bax似乎不起作用。更多还原

【Abstract】 Stress is one of the major risk factors which accounts for the increased incidence of a number of common life-threatening disorders. It is now getting highlight in that stress not only produces some nervous system depended mental problems, but also results in some substantial structure change beyond the nervous system like the heart, the liver etc,causes the functional impirement of these organs. But little is known on the question whether impairment to non-nervous organs was in parallel with or consequent to that of brain. Meanwhile, although the importance of stress in life appears to be significant, the cellular and molecular mechanisms involved in the pathophysiology of stress remains largely unknown especially for those non-neuronal organs. The case for liver is even worse than that for the cardiovascular system which received strong focus and developed in a relatively fast speed. Only recently has scientific evidence been obtained to demonstrate how stress exacerbates liver diseases. There still lack study on how stress affects the function of normal liver and what are the underlying mechanisms. Objective:This study aims to illuminate the relationship between the stress induced liver function impairment and that of brain and to detect the molecular change of liver under a most commonly used stress model, immobilization.Method:In the current study, the typical antidepressant paroxetine (20 mg/kg) or Chinese prescription Sinisan (100 mg/kg) which is proved to protect the liver were used during the 14 repeated daily RSs (RS, 6 h/d). The body weight was measured under different treatments. The enzyme activities of serum glutamic pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as Morris Water Maze (MWM) were used to evaluate the liver function or spatial learning and memory, respectively. In the other study,we employed BDNF, KDR and Bax as the candidate molecules which are suggested to be involved in the liver response to immobilization stress. To test the different effect of acute and repeated immobilization, we evaluated the mRNA level for BDNF, KDR and Bax in liver tissue after the rats received one acute immobilization or 15 repeated daily immobilization stresses.Result:The 14 RSs caused significant decrease of growing speed as revealed with the body weight (P < 0.05, vs vehicle), while pretreatment with paroxetine or Sinisan kept the stressed rats grow at the normal speed (P > 0.05, vs vehicle). The 14 RSs caused significant increase of sera enzyme acitivities for GPT and ALP (P < 0.05, vs vehicle), as well as significant decrease of spatial learning and memory abilities (P < 0.05, vs vehicle). Fourteen daily paroxetine treatments increased the abilities for spatial learning and memory but had no effect on the elevated enzyme activities. On the other hand, fourteen daily Sinisan treatments did not affect RS impaired spatial learning and memory abilities while reversed the RS induced increase of enzyme activities. In the other study, acute immobilization stress significantly (P < 0.01, vs control) increased the mRNA level for KDR (114.8% of control) but not for BDNF mRNA. While, fifteen repeated immobilization stresses significantly (P < 0.01, vs control) decreased the mRNA level for BDNF (82.22% of control) but not for KDR. Surprisingly, neither acute nor repeated immobilization changed the expression level of Bax.Conclusion:Restraint stress induced impairment of liver function is in parallel with the impairment to brain. Repeated RSs can impair the brain or liver function via different mechanisms. KDR or BDNF is involved in the liver response to acute or repeated immobilization, respectively. Locally synthesized Bax is not strongly involved in the liver response to acute or repeated immobilization stress.更多还原