本文選題：β-淀粉樣蛋白 + β-細(xì)辛醚�。� 參考：《廣東藥學(xué)院》2012年碩士論文

【摘要】：研究背景： 阿爾茲海默癥(Alzheimer's Disease,AD)是一種最為常見(jiàn)的中樞神經(jīng)系統(tǒng)退行性疾病,為癡呆的最主要類型。目前“Aβ假說(shuō)”被廣泛地接受為AD最主要的發(fā)病機(jī)理,即老年斑的主要組成成分——β-淀粉樣蛋白(amyloid-β,AB)是AD發(fā)病進(jìn)程的中心機(jī)制,其在特定腦區(qū)內(nèi)聚集,引發(fā)相應(yīng)的神經(jīng)毒性作用,造成突觸損傷,神經(jīng)元死亡,從而導(dǎo)致了AD患者記憶衰退和認(rèn)知功能障礙等相應(yīng)癥狀的產(chǎn)生。 目前,AD的治療研究策略主要包括三個(gè)方面：一是開(kāi)發(fā)新的低毒性的擬膽堿藥物；二是神經(jīng)營(yíng)養(yǎng)物質(zhì)的應(yīng)用,包括直接腦室灌注神經(jīng)營(yíng)養(yǎng)因子和神經(jīng)營(yíng)養(yǎng)因子基因修飾細(xì)胞的腦內(nèi)移植治療；三是干細(xì)胞的移植治療。但這些療法基本在于減慢神經(jīng)元的退變或加速神經(jīng)元的產(chǎn)生,并沒(méi)有從根本上阻止AD患者的病變,其遠(yuǎn)期治療前景不容樂(lè)觀。而中醫(yī)藥臨床表明,以石菖蒲為主的藥方是治療AD方劑的基本結(jié)構(gòu),位居單味藥使用頻次第1位,療效肯定。但石菖蒲抗AD的藥效部位及抗AD的“Aβ”機(jī)制尚未闡明,影響了石菖蒲抗AD療效的進(jìn)一步提高。 我室前期工作證明,石菖蒲揮發(fā)油部位對(duì)AD模型小鼠學(xué)習(xí)記憶能力有明顯提升作用,作用機(jī)制可能與其拮抗凋亡相關(guān)因子表達(dá)有關(guān),提示揮發(fā)油部位有可能用于AD的防治。為進(jìn)一步探討石菖蒲揮發(fā)油部位抗AD及其發(fā)揮神經(jīng)保護(hù)作用的有效成分,為石菖蒲抗AD及用于神經(jīng)保護(hù)藥物的研發(fā)提供科學(xué)依據(jù),本實(shí)驗(yàn)首先用Aβ1-42寡聚體對(duì)體外培養(yǎng)的原代皮層神經(jīng)元進(jìn)行優(yōu)化篩選,建立了Aβ1-42寡聚體致神經(jīng)元損傷模型。在此模型上探討β-細(xì)辛醚(β-asarone)、丁香酚(Eugenol)以及β-細(xì)辛醚 聯(lián)合丁香酚共孵育對(duì)Aβ1-42致神經(jīng)元損傷模型的保護(hù)作用及機(jī)制,為石菖蒲抗AD及用于神經(jīng)保護(hù)藥物的研發(fā)提供科學(xué)依據(jù)。 研究目的： 1.觀察β-細(xì)辛醚和丁香酚對(duì)正常狀態(tài)下原代皮層神經(jīng)元和PC12細(xì)胞的形態(tài)和細(xì)胞活力的影響,初步了解β-細(xì)辛醚和丁香酚的神經(jīng)保護(hù)作用； 2.觀察p-細(xì)辛醚、丁香酚及p-細(xì)辛醚聯(lián)合丁香酚共孵育對(duì)Aβ1-42寡聚體誘導(dǎo)的原代皮層神經(jīng)元和PC12細(xì)胞損傷模型的保護(hù)作用,并探討其作用機(jī)理。 研究方法： 實(shí)驗(yàn)一 1.首先取胎鼠的皮層組織進(jìn)行原代皮層神經(jīng)元的培養(yǎng),培養(yǎng)第六天進(jìn)行免疫細(xì)胞化學(xué)染色鑒定神經(jīng)元； 2. Aβ1-42寡聚體誘導(dǎo)的皮層神經(jīng)元損傷模型的建立,ELISA檢測(cè)相關(guān)因子的釋放水平；3.觀察β-細(xì)辛醚和丁香酚對(duì)皮層神經(jīng)元的細(xì)胞活力的影響； 4.原代皮層神經(jīng)元培養(yǎng)第六天加入Aβ1-42寡聚體建立細(xì)胞模型,24小時(shí)后再用β-細(xì)辛醚和丁香酚作用于細(xì)胞。三天后提取蛋白進(jìn)行凋亡相關(guān)因子的檢測(cè)； 實(shí)驗(yàn)二1.石菖蒲主要有效成分β-細(xì)辛醚和丁香酚對(duì)正常狀態(tài)下PC12細(xì)胞形態(tài)和細(xì)胞活力的影響； 2.建立PC12細(xì)胞的Aβ1-42寡聚體誘導(dǎo)損傷模型； 3.PC12細(xì)胞培養(yǎng)第六天加入Aβ1-42寡聚體建立細(xì)胞模型,24小時(shí)后再用p-細(xì)辛醚和丁香酚作用于細(xì)胞。三天后提取RNA進(jìn)行凋亡相關(guān)基因的檢測(cè)； 4.通過(guò)Tunel法探討p-細(xì)辛醚和丁香酚對(duì)Aβ1-42寡聚體誘導(dǎo)的PC12細(xì)胞的保護(hù)作用機(jī)理。 研究結(jié)果： 實(shí)驗(yàn)一 1.培養(yǎng)至第六天的原代皮層神經(jīng)元胞體的突起明顯,免疫細(xì)胞化學(xué)染色顯示,神經(jīng)元標(biāo)志物呈陽(yáng)性； 2.通過(guò)ELISA檢測(cè)TNF-α的釋放水平,建立起Aβ1-42寡聚體誘導(dǎo)的皮層神經(jīng)元損傷模型； 3.培養(yǎng)至第三天的皮層神經(jīng)元分別與10-10-10-8Mp-細(xì)辛醚、丁香酚以及p-細(xì)辛醚聯(lián)合丁香酚共孵育24h,與正常培養(yǎng)的神經(jīng)元相比,10-6β-細(xì)辛醚和10-7M丁香酚組細(xì)胞存活率最高； 4.Western blotting技術(shù)檢測(cè)凋亡相關(guān)蛋白,顯示Aβ1-42寡聚體作用于皮層神經(jīng)元6h后,促凋亡蛋白Bax表達(dá)開(kāi)始增加,至24h達(dá)到較高水平；與之相反,抗凋亡蛋白Bcl-2表達(dá)隨Aβ1-42寡聚體作用時(shí)間延長(zhǎng)逐漸降低； 5.10-10-10-8Mp-細(xì)辛醚和丁香酚可明顯拮抗Aβ1-42寡聚體引起的促凋亡蛋白Bax表達(dá)上調(diào)以及抗凋亡蛋白Bcl-2表達(dá)下降。 實(shí)驗(yàn)二1.培養(yǎng)至第三天的PC12細(xì)胞與10-10-10-8Mβ-細(xì)辛醚和丁香酚分別共孵育24h,與正常培養(yǎng)的PC12細(xì)胞相比,10-6β-細(xì)辛醚和10-7M丁香酚組細(xì)胞存活率最高； 2.RT-PCR技術(shù)檢測(cè)凋亡相關(guān)基因,顯示Aβ1-42寡聚體作用于PC12細(xì)胞后,促凋亡蛋白Bax表達(dá)開(kāi)始增加,抗凋亡蛋白Bcl-2表達(dá)逐漸降低； 3.10-10-10-8Mp-細(xì)辛醚和丁香酚可明顯拮抗Aβ1-42寡聚體引起的PC12細(xì)胞促凋亡基因Bax表達(dá)上調(diào)以及抗凋亡基因Bcl-2表達(dá)下降； 4.TUNEL方法檢測(cè)PC12細(xì)胞凋亡：β-細(xì)辛醚、丁香酚及其混合物的預(yù)孵育組,與Aβ1-42寡聚體對(duì)照組相比較,陽(yáng)性細(xì)胞數(shù)量明顯減少。p-細(xì)辛醚聯(lián)合丁香酚用藥組優(yōu)于單體的單獨(dú)作用組。 結(jié)論： 1.Aβ1-42寡聚體能顯著抑制神經(jīng)元的再生,這種毒性效應(yīng)可能與其促進(jìn)凋亡相關(guān)因子的表達(dá)有關(guān)。 2.β-細(xì)辛醚和丁香酚能明顯拮抗Aβ1-42寡聚體引發(fā)的神經(jīng)元(PC12細(xì)胞)細(xì)胞凋亡,這可能與其能抑制凋亡相關(guān)因子的表達(dá)有關(guān)。 3.對(duì)于Aβ1-42寡聚體誘導(dǎo)的原代皮層神經(jīng)元和PC12細(xì)胞損傷模型的保護(hù)作用,β-細(xì)辛醚聯(lián)合丁香酚組明顯優(yōu)于單體的單獨(dú)作用組。這為石菖蒲用于抗老年性癡呆藥物的研發(fā)提供了科學(xué)的依據(jù)。
[Abstract]:Research background:
Alzheimer's Disease (AD) is the most common degenerative disease of the central nervous system and is the most important type of dementia. Now the "A beta hypothesis" is widely accepted as the main pathogenesis of AD, that is, the main component of the senile plaque, beta amyloid (amyloid- beta, AB), is the central machine for the pathogenesis of AD. In a specific brain area, it is aggregated in a specific brain area, triggering a corresponding neurotoxic effect, causing synaptic damage and causing death of neurons, resulting in the resulting symptoms of memory decline and cognitive impairment in AD patients.
At present, the treatment research strategy of AD mainly includes three aspects: first, the development of new low toxic quasi choline drugs; two is the use of neurotrophic substances, including direct ventricle perfusion neurotrophic factor and neurotrophic factor gene modified cell transplantation in the brain; three is stem cell transplantation treatment. But these treatments are basically To slow down the degeneration of neurons or accelerate the production of neurons, it does not fundamentally prevent the disease of AD patients. The prospect of long-term treatment is not optimistic. The clinical manifestation of traditional Chinese medicine indicates that the prescription of Acorus calamus is the basic structure for the treatment of AD prescription, which is the first place of the single drug use frequency, but the effect of Acorus calamus on AD The mechanism of "A beta" against AD has not been clarified, which has affected the further improvement of the efficacy of Acorus calamus against AD.
The early work of my room has proved that the volatile oil parts of Acorus calamus have a significant effect on the learning and memory ability of AD model mice. The mechanism may be related to its antagonism to the expression of apoptosis related factors, suggesting that the part of the volatile oil may be used for the prevention and control of AD. To further discuss the anti AD and the neuroprotective effect of the volatile oil from the calamus The effective components, which provide a scientific basis for the anti AD of Acorus calamus and the research and development of neuroprotective drugs, first optimized the primary cultured cortical neurons in vitro by A beta 1-42 oligomer, and established the neuron damage model of A beta 1-42 oligomers. Asarone
The protective effect and mechanism of CO incubation with A beta 1-42 induced neuron damage model can provide a scientific basis for the anti AD of Acorus calamus and the research and development of neuroprotective drugs.
The purpose of the study is:
1. the effects of beta asarone and eugenol on the morphology and cell viability of primary cortical neurons and PC12 cells in normal state were observed, and the neuroprotective effects of beta asarone and eugenol were preliminarily understood.
2. the protective effect of p- asarone, eugenol and p- asarone combined with eugenol on the primary cortical neurons and PC12 cell damage models induced by A beta 1-42 oligomers was observed and the mechanism of its action was discussed.
Research methods:
Experiment 1
1. first, the cortical tissues of fetal rats were cultured in primary cortical neurons for sixth days to identify neurons by immunocytochemical staining.
2. A beta 1-42 oligomer induced cortical neuron damage model was established, and ELISA was used to detect the release level of related factors; 3. the effects of beta asarone and eugenol on the cell viability of cortical neurons were observed.
4. the primary cultured cortical neurons were cultured for sixth days by adding A beta 1-42 oligomer to establish cell model. After 24 hours, beta asarone and eugenol were used to act on the cells. After three days, the protein was detected for the detection of apoptosis related factors.
Experiment two. The effects of 1. asarone and eugenol on the morphology and cell viability of PC12 cells under normal conditions.
2. to establish PC12 cell induced A beta 1-42 oligomer induced injury model.
3.PC12 cells were cultured for sixth days to add A beta 1-42 oligomer to establish cell model. After 24 hours, p- asarone and eugenol were used to act on the cells. After three days, RNA was detected for the detection of apoptosis related genes.
4. to explore the protective mechanism of p- asarone and eugenol on PC12 cells induced by A beta 1-42 oligomers by Tunel.
The results of the study:
Experiment 1
1. the neurites of the primary cortical neurons cultured for up to sixth days were obvious. Immunocytochemical staining showed that the neurons were positive.
2. to detect the release level of TNF- alpha by ELISA, and establish a cortical neuron injury model induced by A beta 1-42 oligomers.
3. the cortical neurons cultured for third days incubated 24h with 10-10-10-8Mp- asarone, eugenol and p- asarone combined with eugenol. Compared with the normal cultured neurons, the survival rate of 10-6 beta asarine and 10-7M eugenol group was the highest.
4.Western blotting technique detected apoptosis related proteins, which showed that after A beta 1-42 oligomer acted on 6h of cortical neurons, the expression of apoptotic protein Bax began to increase and reached a higher level to 24h. On the contrary, the expression of anti apoptotic protein Bcl-2 gradually decreased with the prolongation of the action time of A beta 1-42 oligomer.
5.10-10-10-8Mp- asarone and eugenol can significantly antagonize upregulated expression of Pro apoptotic protein Bax and decrease the expression of anti apoptotic protein Bcl-2 in A beta 1-42 oligomers.
Experiment two 1. PC12 cells cultured to third days were incubated with 10-10-10-8M beta asarone and eugenol respectively for 24h. Compared with normal cultured PC12 cells, the survival rate of 10-6 beta asarone and 10-7M eugenol group was the highest.
Apoptosis related genes were detected by 2.RT-PCR technology, which showed that A beta 1-42 oligomer acted on PC12 cells, and the expression of apoptotic protein Bax began to increase, and the expression of anti apoptotic protein Bcl-2 decreased gradually.
3.10-10-10-8Mp- asarone and eugenol could obviously antagonize the up regulation of Bax expression of PC12 cell apoptosis gene Bax and the decrease of Bcl-2 expression of anti apoptotic gene induced by A beta 1-42 oligomer.
4.TUNEL method was used to detect the apoptosis of PC12 cells: pre incubating group of beta asarone, eugenol and its mixture, compared with A beta 1-42 oligomer control group, the number of positive cells significantly reduced the group of.P- asarone combined with eugenol group better than the single body.
Conclusion:
1.A beta 1-42 oligomers can significantly inhibit the regeneration of neurons. This toxic effect may be related to the expression of apoptosis related factors.
2. beta asarone and eugenol can obviously antagonize the apoptosis of neurons (PC12 cells) induced by A beta 1-42 oligomers, which may be related to the inhibition of the expression of apoptosis related factors.
3. for the protective effect of A beta 1-42 oligomer induced primary cortical neurons and PC12 cell damage models, beta asarone combined with eugenol group is obviously better than the single action group. This provides a scientific basis for the development of Acorus calamus for the development of anti Alzheimer's drugs.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332;R749.16

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