發(fā)布時間：2018-01-25 16:24

  本文關(guān)鍵詞： 阿爾茲海默病 β-淀粉樣蛋白 原兒茶酸 PC12細(xì)胞 自噬　出處：《廣州中醫(yī)藥大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：背景：阿爾茨海默癥(Alzheimer's Disease, AD),是癡呆最常見的原因,在臨床上稱為原發(fā)性老年癡呆癥,是一種常見的起病隱匿的進(jìn)行性發(fā)展的中樞神經(jīng)系統(tǒng)退行性疾病,患者可因繼發(fā)感染和并發(fā)癥而死亡。AD發(fā)病機(jī)制復(fù)雜,病因不明,隨著人類社會進(jìn)入老齡化的步伐加快,我國65歲以上老年人口數(shù)目逐年增加,有研究顯示,我國癡呆患者人數(shù)已超過800萬,到2040年,將超過2600萬。癡呆致殘率高,會給家庭和社會帶來沉重的負(fù)擔(dān)。因此,預(yù)防和治療阿爾茨海默病(AD)具有積極的社會性意義,中醫(yī)中藥在AD防治中具有獨(dú)特的地位和廣闊的前景。需明確阿爾茲海默癥的現(xiàn)狀,深入闡明發(fā)病機(jī)制,發(fā)現(xiàn)新的藥物靶點(diǎn),尋找治療阿爾茲海默癥的有效藥物。原兒茶酸(Protocatechuic acid, PCA)是存在于許多蔬菜和水果中的一種天然酚酸類化合物,并且是很多中藥的活性成分,例如益智仁、丹參、芙蓉等。而益智仁是我國著名的“四大南藥”之一,藥理學(xué)研究報告發(fā)現(xiàn)其具有抗癌,保護(hù)心血管等作用,對神經(jīng)細(xì)胞具有抗衰老、抗氧化、降低興奮性毒性等作用。研究發(fā)現(xiàn),益智仁的主要成分原兒茶酸具有良好的神經(jīng)保護(hù)作用。關(guān)于原兒茶酸的藥理作用已有大量報道,原兒茶酸具有抗炎、抗氧化、抑制腫瘤的作用；原兒茶酸對缺血、缺氧神經(jīng)元細(xì)胞具有一定的保護(hù)作用；原兒茶酸可顯著抑制MPP+、H202或魚藤酮誘導(dǎo)的PC12細(xì)胞調(diào)亡和氧化應(yīng)激,具有良好的抗氧化和神經(jīng)保護(hù)作用目的：本實(shí)驗(yàn)研究以Aβ1-42寡聚體誘導(dǎo)PC12細(xì)胞Aβ沉積的AD細(xì)胞模型,研究原兒茶酸對Aβ1-42寡聚體誘導(dǎo)PC12細(xì)胞毒性的保護(hù)作用及機(jī)制,為更好明確原兒茶酸神經(jīng)保護(hù)作用及作用機(jī)制,進(jìn)一步開發(fā)益智仁及其有效單體原兒茶酸而提供實(shí)驗(yàn)依據(jù)。方法：首先采用免疫熒光法鑒定Aβ1-42纖維寡聚體,將Aβ1-42作用于PC12細(xì)胞,并于不同時間點(diǎn)(0、3、6、9、12、24 h)觀察PC12細(xì)胞的形態(tài),運(yùn)用MTT比色法檢測細(xì)胞存活率以確定造模條件。并且通過MTT比色法檢測原兒茶酸對PC12細(xì)胞的毒性作用。然后采用預(yù)報護(hù)的給藥方式,檢測不同濃度的原兒茶酸對PC12細(xì)胞的干預(yù)作用。通過使用Western-blot的方法來檢測與自噬相關(guān)標(biāo)記蛋白Beclinl的表達(dá)水平,用以驗(yàn)證原兒茶酸對PC12細(xì)胞Aβ沉積的AD細(xì)胞模型的保護(hù)作用機(jī)制。結(jié)果：1.在顯微鏡下可觀察到當(dāng)12h和24 h時,A β1-42淀粉樣白色團(tuán)塊聚集基本穩(wěn)定,無明顯差異,MTT比色法檢測PC12細(xì)胞的損傷率均達(dá)40%,由此可以確定12 h和24 h均可做為AD的造模條件。2.當(dāng)原兒茶酸在200 μmol/L濃度時作用24 h的條件下,細(xì)胞活力有所上升。同時,Western-blot結(jié)果顯示,原兒茶酸增加了Beclinl的表達(dá)水平。結(jié)論：原兒茶酸可明顯降低Aβ1-42對PC12細(xì)胞毒性,其作用機(jī)制可能與增加自噬水平有關(guān),對于其抗AD作用有更深層次的研究價值。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R749.16

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