原儿茶酸抑制Aβ诱导的PC12细胞的毒性作用及机制研究

发布时间：2018-01-25 16:24

本文关键词： 阿尔兹海默病 β-淀粉样蛋白原儿茶酸 PC12细胞自噬　出处：《广州中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景：阿尔茨海默症(Alzheimer's Disease, AD),是痴呆最常见的原因,在临床上称为原发性老年痴呆症,是一种常见的起病隐匿的进行性发展的中枢神经系统退行性疾病,患者可因继发感染和并发症而死亡。AD发病机制复杂,病因不明,随着人类社会进入老龄化的步伐加快,我国65岁以上老年人口数目逐年增加,有研究显示,我国痴呆患者人数已超过800万,到2040年,将超过2600万。痴呆致残率高,会给家庭和社会带来沉重的负担。因此,预防和治疗阿尔茨海默病(AD)具有积极的社会性意义,中医中药在AD防治中具有独特的地位和广阔的前景。需明确阿尔兹海默症的现状,深入阐明发病机制,发现新的药物靶点,寻找治疗阿尔兹海默症的有效药物。原儿茶酸(Protocatechuic acid, PCA)是存在于许多蔬菜和水果中的一种天然酚酸类化合物,并且是很多中药的活性成分,例如益智仁、丹参、芙蓉等。而益智仁是我国著名的“四大南药”之一,药理学研究报告发现其具有抗癌,保护心血管等作用,对神经细胞具有抗衰老、抗氧化、降低兴奋性毒性等作用。研究发现,益智仁的主要成分原儿茶酸具有良好的神经保护作用。关于原儿茶酸的药理作用已有大量报道,原儿茶酸具有抗炎、抗氧化、抑制肿瘤的作用；原儿茶酸对缺血、缺氧神经元细胞具有一定的保护作用；原儿茶酸可显著抑制MPP+、H202或鱼藤酮诱导的PC12细胞调亡和氧化应激,具有良好的抗氧化和神经保护作用目的：本实验研究以Aβ1-42寡聚体诱导PC12细胞Aβ沉积的AD细胞模型,研究原儿茶酸对Aβ1-42寡聚体诱导PC12细胞毒性的保护作用及机制,为更好明确原儿茶酸神经保护作用及作用机制,进一步开发益智仁及其有效单体原儿茶酸而提供实验依据。方法：首先采用免疫荧光法鉴定Aβ1-42纤维寡聚体,将Aβ1-42作用于PC12细胞,并于不同时间点(0、3、6、9、12、24 h)观察PC12细胞的形态,运用MTT比色法检测细胞存活率以确定造模条件。并且通过MTT比色法检测原儿茶酸对PC12细胞的毒性作用。然后采用预报护的给药方式,检测不同浓度的原儿茶酸对PC12细胞的干预作用。通过使用Western-blot的方法来检测与自噬相关标记蛋白Beclinl的表达水平,用以验证原儿茶酸对PC12细胞Aβ沉积的AD细胞模型的保护作用机制。结果：1.在显微镜下可观察到当12h和24 h时,A β1-42淀粉样白色团块聚集基本稳定,无明显差异,MTT比色法检测PC12细胞的损伤率均达40%,由此可以确定12 h和24 h均可做为AD的造模条件。2.当原儿茶酸在200 μmol/L浓度时作用24 h的条件下,细胞活力有所上升。同时,Western-blot结果显示,原儿茶酸增加了Beclinl的表达水平。结论：原儿茶酸可明显降低Aβ1-42对PC12细胞毒性,其作用机制可能与增加自噬水平有关,对于其抗AD作用有更深层次的研究价值。
[Abstract]:Background: Alzheimer's disease (Alzheimer's disease) is the most common cause of dementia, known as primary Alzheimer's disease (AD). It is a common and concealed progressive degenerative disease of the central nervous system. Patients can die from secondary infection and complications. The pathogenesis of AD is complex and the etiology is unknown. With the accelerated pace of aging in human society, the number of elderly population over 65 years old in China has increased year by year. Some studies show that the number of dementia patients in China has exceeded 8 million, until 2040. Dementia will be more than 26 million. The high rate of dementia will bring a heavy burden to families and society. Therefore, the prevention and treatment of Alzheimer's disease (ADD) has a positive social significance. Traditional Chinese medicine has a unique position and broad prospects in AD prevention and treatment. It is necessary to clarify the current situation of Alzheimer's disease, to clarify the pathogenesis of Alzheimer's disease, and to find new drug targets. Protocatechuic acid.Protocatechuic acid.Protocatechuic acid.Protocatechuic acidis is a natural phenolic acid compound found in many vegetables and fruits. And is a lot of Chinese medicine active ingredients, such as Yi Zhiren, Salvia miltiorrhiza, Furong and so on. And Yi Zhiren is one of the famous "four South Medicines" in China, pharmacological research report found that it has anti-cancer, cardiovascular protection and so on. It has the effects of anti-aging, anti-oxidation, reducing excitotoxicity and so on. Protocatechuic acid, the main component of Yizhiren, has a good neuroprotective effect. The pharmacological effect of protocatechuic acid has been widely reported. Protocatechuic acid has anti-inflammatory, antioxidant and tumor inhibition effects. Protocatechuic acid has protective effect on ischemic and hypoxic neuron cells. Protocatechuic acid significantly inhibited the apoptosis and oxidative stress of PC12 cells induced by MPP H202 or rotenone. Objective: to study the AD cell model with A 尾 1-42 oligomer induced A 尾 deposition in PC12 cells. To study the protective effect and mechanism of protocatechuic acid on the cytotoxicity of PC12 cells induced by A 尾 1-42 oligomer, in order to better understand the neuroprotective effect and mechanism of protocatechuic acid. Methods: firstly, A 尾 1-42 fiber oligomer was identified by immunofluorescence method, A 尾 1-42 was treated with A 尾 1-42 in PC12 cells. The morphology of PC12 cells was observed at different time points. MTT colorimetric assay was used to detect the survival rate of cells in order to determine the model conditions, and the toxicity of protocatechuic acid to PC12 cells was detected by MTT colorimetry. The effects of protocatechuic acid at different concentrations on PC12 cells were detected. The expression level of autophagy-associated marker protein (Beclinl) was detected by using Western-blot. To verify the protective mechanism of protocatechuic acid on AD cell model of A 尾 deposition in PC12 cells. Results: 1. Under microscope, 12h and 24h were observed. The aggregation of A 尾 1-42 amyloid white lumps was basically stable, and the damage rate of PC12 cells detected by MTT colorimetry was 40%. Therefore, 12 h and 24 h can be used as the model conditions of AD. 2. When protocatechuic acid was exposed to 200 渭 mol/L for 24 h. At the same time, the results of Western-blot showed that the cell viability was increased. Conclusion: protocatechuic acid can significantly reduce the cytotoxicity of A 尾 1-42 to PC12 cells, and its mechanism may be related to the increase of autophagy. It has further research value for its anti-AD effect.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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