小鼠脑缺血再灌注损伤相关的脂类对神经元的作用及机理

The effects and mechanisms of lipids on neurons in mouse cerebral ischemia-reperfusion model

司腾霄

11749223

硕士

生物工程

侯圣陶

2019-05-31

2019-06-20

哈尔滨工业大学

深圳

研究目的目前脑卒中已成为高致死率和致残率的疾病之一，因此对于人类的生活及生活质量造成严重影响，但目前最有效的治疗方式还是前期的溶栓。本实验使用动物中风模型MCAO模型，利用高灵敏度的UPLC-MS质谱平台，研究小鼠脑缺血/再灌注期间损伤的大脑皮层内脂质的变化，因此本实验通过脂组学方法来寻找在小鼠缺血再灌注脑损伤功能恢复过程中显著性变化的脂质分子并研究其对神经元作用活性的影响及作用的分子机制等。研究方法1. 建立小鼠MCAO中风模型，然后用超高效液相色谱对小鼠大脑皮层进行相对性定量和绝对性定量分析，筛选出具有显著性差异的脂类分子。2. 取胚胎期15-16天的胎鼠，取其剥离血管膜以后的大脑皮层，用做原代神经元的培养，并用免疫荧光染色(MAP-2)来鉴定是否为神经元。3. 将先前培养的原代神经元，接种在96孔板上，在细胞培养箱中培养5d。当用前期筛选到的脂类处理原代神经元后，再加入CCK 8溶液，测每孔的OD值，检测脂类对神经元活力的影响。4. 前期结果表明一定浓度的脂类会显著性降低神经元的活力，导致神经元死亡。接下来分别购买了几种抑制细胞死亡的抑制剂和脂类共同孵育神经元，同样用CCK 8测细胞活力，探究细胞死亡的方式。5. 将先前培养的原代神经元，接种在含玻璃底的35mm 的培养皿中，在细胞培养箱中培养5d。然后加入不同浓度的脂类，检测细胞内钙离子信号变化；当提前加入MK-801孵育，再加脂类去检测此时钙离子信号变化，探究脂类是否能引起神经元兴奋性。研究结果1. 建立小鼠MCAO中风模型后，然后用超高效液相色谱对小鼠大脑皮层进行相对性定量和绝对性定量分析，共筛选到578种脂类，然后分析各组间差异脂质，共筛选到84种显著性差异的脂质分子。2. 在中风中一些显著性变化的脂类分子可以显著性降低原代神经元的活力（脂类浓度为25µM-50µM时），导致神经元死亡。3. 对脂类诱导神经元死亡方式的探究，发现有些细胞凋亡抑制剂可以显著性增加神经元的活力，MK-801也能阻断一些脂类引起的神经元兴奋性毒性。研究结论小鼠脑缺血再灌注过程中，一些显著性变化的脂类在中风恢复过程中起重要的调节作用。其中一些脂类例如磷脂酰胆碱(Phosphatidylcholine)、溶血卵磷脂(Lysophosphatidylcholine)、神经酰胺(Ceramides )、磷脂酰乙醇胺(Phosphatidylethanolamine)、溶血磷脂酰乙醇胺(Lysophosphatidylethanolamine)等在中风恢复过程中可诱导小胶质细胞激活，引起炎症反应和神经元的兴奋性毒性，导致神经元死亡。

Research objectiveAt present, stroke has become one of the diseases with high mortality and disability, which has a serious impact on the quality of life and quality of life of human beings. However, the most effective treatment method is the previous thrombolysis. In this experiment, the MCAO model of the animal stroke model was used to study the changes of lipids in the cerebral cortex damaged by cerebral ischemia/reperfusion in mice using the highly sensitive UPLC-MS mass spectrometry platform. Therefore, this experiment used lipidomics to find Lipid molecules with significant changes in the function of brain ischemia-reperfusion injury in mice were studied and their effects on neuronal activity and molecular mechanisms of action were investigated.Research methods1. Establish a mouse MCAO stroke model, and then use the ultra-high performance liquid chromatography to quantitatively and quantitatively analyze the mouse cerebral cortex, and screen out the lipid molecules with significant differences.2. Take the embryonic period 15-16 days of fetal rats, take the cerebral cortex after the vascular membrane is removed, use it as the primary neuron culture, and use immunofluorescence staining (MAP-2) to identify whether it is a neuron.3. The previously cultured primary neurons were seeded on 96-well plates and cultured for 5 days in a cell culture incubator. When the primary neurons were treated with the lipids screened in the previous stage, CCK 8 solution was added, and the OD value of each well was measured to measure the effect of lipids on the vitality of the neurons.4. Previous results indicate that a certain concentration of lipids significantly reduces neuronal viability, leading to neuronal death. Next, several inhibitors of cell death and lipids were purchased to incubate neurons together, and cell viability was also measured by CCK 8 to explore the way of cell death.5. The previously cultured primary neurons were seeded in a 35 mm culture dish containing a glass bottom and cultured for 5 days in a cell culture incubator. Then add different concentrations of lipids to detect intracellular calcium ion signal changes; when added to MK-801 in advance, add lipids to detect changes in calcium ion signal at this time, to explore whether lipids can cause neuronal excitability.Research results1. Establish a mouse MCAO stroke model, then use the ultra-high performance liquid chromatography to quantitatively and quantitatively analyze the mouse cerebral cortex. A total of 578 lipids were screened, and then the differential lipids between the groups were analyzed. A total of 84 significant differences in lipid molecules were screened.2. Some significant changes in lipid molecules in stroke can significantly reduce the viability of primary neurons (lipid concentrations from 25 μM to 50 μM), leading to neuronal death.3. To explore the ways of lipid-induced neuronal death, it was found that some inhibitors of apoptosis can significantly increase the viability of neurons, and MK-801 can also block the excitatory toxicity of neurons caused by some lipids.ConclusionDuring the cerebral ischemia-reperfusion in mice, some significant changes in lipids play an important regulatory role in the recovery of stroke. Some of the lipids such as Phosphatidylcholine, Lysophosphatidylcholine, Ceramides, Phosphatidylethanolamine, Lysophosphatidylethanolamine, etc. can induce small glue during stroke recovery. Activation of cytoplasmic cells causes inflammatory responses and excitatory toxicity of neurons, leading to neuronal death.

脑卒中 缺血再灌注 脂质组学 神经元 细胞凋亡 兴奋性毒性

troke ischemia-reperfusion lipidomics neurons excitotoxicity apoptosis

中文

联合培养

南方科技大学

司腾霄. 小鼠脑缺血再灌注损伤相关的脂类对神经元的作用及机理[D]. 深圳. 哈尔滨工业大学,2019.