乌司他丁对脂多糖诱导的小胶质细胞活化的抑制作用及其机制的初步研究

发布时间：2018-07-04 18:38

本文选题：神经退行性病变 + 小胶质细胞　；参考：《第四军医大学》2012年硕士论文

【摘要】：【研究背景】神经退行性病变是一类年龄相关的以神经元功能和结构丧失甚至死亡为病理特征的疾病，典型疾病如阿兹海默症（Alzheimer’s disease,AD），帕金森症（Parkinson’s disease, PD）等，临床症状根据损伤的不同类型表现出不同的行为、精神、认知、运动性障碍。在病理学上，炎症与氧化应激对神经退行性病变有十分重要的作用。小胶质细胞作为神经系统的固有免疫单位，和神经退行性病变有非常重要的联系。静息的小胶质细胞对于神经系统有支持、监视、修复等功能；小胶质细胞对脑内环境变化十分敏感，一些刺激能使小胶质细胞活化，小胶质细胞活化可以产生很多炎症趋化因子和自由基，这些物质大部分具有细胞毒性，蓄积过高可以使神经元退化、变性、死亡，进一步会导致神经退行性病变。多种信号通路都参与了小胶质细胞的活化过程，如JAK-STAT、MAPK等，且不同的通路之间互相联系，互相影响，信号通路调控对于调节小胶质细胞状态具有重要意义。因此找到抑制小胶质细胞的活化的方法和途径在预防和治疗神经退行性病变中有重要的意义。以小胶质细胞作为神经退行性病变的治疗靶点已经成为一种可行的方式，，目前已证实多种药物均能调控小胶质细胞的活化，且具有一定的临床治疗或预防作用。乌司他丁（Ulinastain，UTI）是一种蛋白酶抑制剂，在脑及其他多种器官系统中均被证实具有明确的抗炎抗氧化作用，它对于小胶质细胞活化的过程是否有调控作用还未被证实，本文将基于此问题展开一些讨论。【目的】通过利用脂多糖（lipopolysaccharide，LPS）建立体内外小胶质细胞活化的模型，观察UTI治疗对于小胶质细胞活化的作用和对学习记忆功能的影响。并进一步探讨其中的信号通路机制。【方法】 1、利用LPS注射大鼠建立小胶质细胞活化的体内模型。 2、利用LPS刺激BV2细胞建立小胶质细胞的体外模型。 3、 Morris水迷宫（Morris Water Maze，MWM）观察大鼠学习记忆功能的变化。 4、免疫组织化学观察小胶质细胞形态学的变化。 5、 ELISA检测TNF-α和IL-1β的释放。 6、 Western Blot检测相关信号通路的变化。【结果】 1、 LPS诱导小胶质细胞细胞活化模型的建立体内实验部分，LPS腹腔注射后，大鼠的Morris水迷宫成绩下降，免疫组织化学可观察到脑内海马区大量活化的小胶质细胞，脑组织ELISA检测提示TNF-α和IL-1β的含量增高。体外实验部分，LPS刺激可以使BV2细胞TNF-α和IL-1β的释放增高。 2、 UTI对LPS诱导的小胶质细胞活化的抑制效应体内试验部分，100kU/kg的UTI对于LPS注射引起的Morris水迷宫成绩下降有改善作用，免疫组织化学结果显示小胶质细胞的活化减少，ELISA结果提示TNF-α和IL-1β含量下降。体外实验部分，UTI处理的BV2细胞在LPS刺激下TNF-α和IL-1β的释放减少。 3、 UTI抑制LPS诱导的小胶质细胞活化的信号转导机制体内外实验均发现，在LPS的刺激可诱导海马组织和BV2细胞的MAPKs和JAK-STAT通路活性上调，在UTI处理后他们的活性下降。通过抑制部分MAPKs通路的表达可以抑制LPS诱导的STAT3活性的升高。【结论】 1、 LPS可以使小胶质细胞活化，炎症因子释放增多，并影响学习记忆功能。 2、 UTI对LPS引起的小胶质细胞活化有抑制作用，从而降低炎症因子的释放，改善学习记忆功能。 3、 UTI对LPS引起的小胶质细胞活化的抑制作用可能是通过抑制MAPKs和JAK-STAT通路实现的。在这个过程中，MAPKs和JAK-STAT通路存在联系，MAPKs参与调控STAT3的磷酸化。综上所述，UTI对于神经系统有一定的保护作用，可望作为一种新的药物用于神经退行性病变的治疗和预防。
[Abstract]:[research background]
Neurodegenerative disease is a class of age-related diseases characterized by neuronal function and loss of structure or even death. Typical diseases such as Alzheimer 's disease (AD), Parkinson's (Parkinson' s disease, PD) and so on. Clinical symptoms show different behavior, mental, cognitive, and exercise according to the different types of damage. In pathology, inflammation and oxidative stress play an important role in neurodegenerative diseases.
Microglia, as an intrinsic immune unit of the nervous system, has a very important relationship with neurodegenerative diseases. Resting microglia can support, monitor, repair and other functions of the nervous system. Microglia are sensitive to changes in the brain environment. Some stimulations can activate microglia and microglia can be activated. Many inflammatory chemokines and free radicals are produced. Most of these substances are cytotoxic. Excessive accumulation of these substances can cause degeneration, degeneration, death, and further cause neurodegenerative diseases. Many signal pathways are involved in the activation process of microglia, such as JAK-STAT, MAPK, and the interconnections between different pathways. It is of great significance to regulate the state of microglia. Therefore, it is important to find ways and ways to prevent the activation of microglia in the prevention and treatment of neurodegenerative diseases. It is a feasible way to treat microglia as a target for treatment of neurodegenerative diseases. It has been confirmed that a variety of drugs can regulate the activation of microglia and have a certain clinical or preventive effect. Ulinastain (UTI) is a protease inhibitor, which has been proved to have a clear anti-inflammatory and antioxidant effect in the brain and many other organ systems. It has a regulation on the process of microglia activation. The effect has not been confirmed. This article will discuss some problems based on this problem.
[Objective]
By using lipopolysaccharide (LPS) to build a stereoscopic model of microglia activation, the effect of UTI therapy on the activation of microglia and the effects on the learning and memory function were observed and the signal pathway mechanism was further explored.
[method]
1, the rat model of microglia activation was established by injecting LPS into rats.
2, we use LPS to stimulate BV2 cells to establish microglia models in vitro.
3, Morris Water Maze (MWM) was used to observe the changes of learning and memory function in rats. Morris.
4, the morphological changes of microglia were observed by immunohistochemistry.
5, ELISA was used to detect the release of TNF- alpha and IL-1 beta.
6, Western Blot detected the changes in the related signaling pathways.
[results]
1, LPS induced microglia cell activation model was established.
In the experimental part of the body, after LPS intraperitoneal injection, the results of Morris water maze decreased in rats. Immunohistochemical staining could observe a large number of activated microglia in the hippocampus of the brain. The ELISA detection of brain tissue suggested that the content of TNF- A and IL-1 beta was increased. In vitro, LPS stimulation could increase the release of TNF- A and IL-1 beta in BV2 cells.
2, the inhibitory effect of UTI on microglia activation induced by LPS.
In the body test part, UTI of 100kU/kg could improve the decrease of Morris water maze caused by LPS injection. The results of immunohistochemistry showed that the activation of microglia decreased, and the result of ELISA suggested that the content of TNF- alpha and IL-1 beta decreased. In vitro experimental part, BV2 cells treated by UTI were reduced by LPS stimulated TNF- and IL-1 beta.
3, the signal transduction mechanism of UTI inhibiting LPS induced microglial activation.
Both in vitro and in vivo experiments showed that the activation of MAPKs and JAK-STAT pathways in hippocampal and BV2 cells was up-regulated by LPS stimulation, and their activity decreased after UTI treatment. By inhibiting the expression of partial MAPKs pathway, the activity of STAT3 induced by LPS could be inhibited.
[Conclusion]
1, LPS can activate microglia, increase the release of inflammatory factors, and affect learning and memory function.
2, UTI inhibits the activation of microglia induced by LPS, thereby reducing the release of inflammatory factors and improving learning and memory function.
3, the inhibitory effect of UTI on the activation of microglia caused by LPS may be achieved by inhibiting the MAPKs and JAK-STAT pathways. In this process, there is a link between the MAPKs and JAK-STAT pathways, and MAPKs is involved in the regulation of STAT3 phosphorylation.
To sum up, UTI has a certain protective effect on the nervous system, and is expected to be used as a new drug for the treatment and prevention of neurodegenerative diseases.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R741;G804.2

【共引文献】