【摘要】：目的神經(jīng)退行性疾病是一類復(fù)雜的疾病類型,而神經(jīng)細(xì)胞受損是導(dǎo)致該類疾病的一個(gè)主要誘因。近年來(lái),紅景天苷因其廣泛的藥理活性而倍受關(guān)注,而它在神經(jīng)細(xì)胞保護(hù)方面的作用也日益引起人們的重視。紅景天苷的藥理作用縱然廣泛,然而它的藥效卻始終存在不盡如人意之處,其親水性太強(qiáng),不容易透過(guò)細(xì)胞質(zhì)膜和血腦屏障,從而影響其發(fā)揮作用�，F(xiàn)階段對(duì)紅景天苷類似物發(fā)揮神經(jīng)細(xì)胞保護(hù)的報(bào)道較少,在前期研究中本實(shí)驗(yàn)室合成了一系列紅景天苷類似物,本研究通過(guò)建立神經(jīng)細(xì)胞損傷模型,模擬體內(nèi)神經(jīng)細(xì)胞損傷狀態(tài)并將紅景天苷類似物運(yùn)用至其中,以探明它們?cè)谏窠?jīng)細(xì)胞保護(hù)方面發(fā)揮的作用。結(jié)果表明,與紅景天苷相比較,MADP和化合物6的親酯性有了很大提高,其細(xì)胞保護(hù)作用比紅景天苷更強(qiáng),該研究結(jié)果為今后進(jìn)一步開(kāi)發(fā)防治神經(jīng)系統(tǒng)退行性疾病的新藥提供參考依據(jù)。方法本研究采用兩種細(xì)胞損傷模型,在第一種模型中,預(yù)先給予海馬神經(jīng)元以MADP(120,240μM)和Sal(120,240μM)預(yù)保護(hù)24 h,再以125μM谷氨酸作用海馬神經(jīng)元15 min,細(xì)胞繼續(xù)培養(yǎng)24 h后,光鏡下觀察海馬神經(jīng)元形態(tài)變化,通過(guò)3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽(MTT)及乳酸脫氫酶(LDH)釋放量評(píng)估細(xì)胞活力,同時(shí)采用Hoechst 33342、TUNEL、Annexin V-FITC/PI染色檢測(cè)神經(jīng)元凋亡情況,通過(guò)Fluo-4/AM檢測(cè)[Ca2+]i動(dòng)態(tài)變化,最后通過(guò)QPCR和Western blot實(shí)驗(yàn)分別檢測(cè)與凋亡相關(guān)的基因和蛋白的表達(dá)情況。在第二種模型中,預(yù)先給予PC12細(xì)胞以化合物6,7,11,12(75,150,300μg/mL)和Sal(300μg/mL)預(yù)保護(hù)24 h,再以低糖低血清損傷細(xì)胞24 h,通過(guò)MTT實(shí)驗(yàn)檢測(cè)細(xì)胞活力,光鏡觀察細(xì)胞形態(tài)變化,采用Hoechst 33342染色檢測(cè)細(xì)胞凋亡率,最后通過(guò)Western blot和Caspase-3活性檢測(cè)試劑盒分別檢測(cè)Bcl-2/Bax蛋白的表達(dá)和Caspase-3的活性。結(jié)果谷氨酸損傷模型中的MTT和LDH釋放測(cè)定實(shí)驗(yàn)結(jié)果表明,相同條件下MADP組中細(xì)胞活力高于Sal組;Hoechst 33342、TUNEL、Annexin V-FITC/PI染色揭示了MADP組中細(xì)胞凋亡率低于Sal組;Fluo-4/AM熒光染色結(jié)果顯示Sal組中[Ca2+]i熒光強(qiáng)度強(qiáng)于MADP組;Sal組的Caspase-3 mRNA表達(dá)量低于MADP組;Western blot結(jié)果顯示兩個(gè)類別組中Bcl-2/Bax以及p-ERK蛋白表達(dá)量均未發(fā)生變化,而MADP組中p-Akt和p-JNK的表達(dá)量高于Sal組。在低糖低血清損傷模型中,MTT實(shí)驗(yàn)的結(jié)果表明大多數(shù)受試化合物都發(fā)揮了對(duì)PC12細(xì)胞的保護(hù)作用,尤以化合物6的保護(hù)作用最強(qiáng);化合物6的預(yù)保護(hù)能改善受損的PC12細(xì)胞形態(tài);Hoechst 33342染色、Caspase-3活性測(cè)定以及Western blot實(shí)驗(yàn)結(jié)果均揭示了化合物6可劑量依賴性的抑制低糖低血清損傷誘導(dǎo)的PC12細(xì)胞凋亡,化合物6在300μg/mL時(shí)發(fā)揮的保護(hù)作用最佳。結(jié)論紅景天苷類似物MADP和化合物6較紅景天苷能更好的發(fā)揮對(duì)海馬神經(jīng)元和PC12細(xì)胞的保護(hù)作用。
[Abstract]:Objective Neurodegenerative diseases are a kind of complex diseases, and nerve cell damage is one of the main causes of neurodegenerative diseases. In recent years, salidroside has attracted much attention because of its wide range of pharmacological activities, and its role in the protection of nerve cells has attracted more and more attention. Although salidroside has a wide range of pharmacological effects, its pharmacodynamics is always unsatisfactory, and its hydrophilicity is too strong to penetrate the cytoplasmic membrane and blood-brain barrier. At present, there are few reports that salidroside analogues play a role in nerve cell protection. A series of salidroside analogues were synthesized in our laboratory in previous studies. To study the role of salidroside analogue in neuronal protection by simulating neuronal injury in vivo. The results showed that the lipophilic properties of MADP and compound 6 were greatly improved compared with salidroside, and their cell protection was stronger than salidroside. The results provide a reference for the further development of new drugs for the prevention and treatment of neurodegenerative diseases. Methods in the first model, hippocampal neurons were preprotected with MADP (120240 渭 M) and Sal (120240 渭 M) for 24 h, then treated with 125 渭 M glutamic acid for 15 min. The morphological changes of hippocampal neurons were observed under light microscope. The cell viability was evaluated by the release of 3- (4-dimethylthiazol-2) -2-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH), and the apoptosis of neurons was detected by Hoechst 33342Tunel Annexin V-FITCP-PI staining. The dynamic changes of [Ca 2] I were detected by Fluo-4 / AM, and the expression of apoptosis-related genes and proteins were detected by QPCR and Western blot, respectively. In the second model, PC12 cells were pretreated with compounds 6C71111C12 (75150300 渭 g / mL) and Sal (300 渭 g / mL) for 24 h, then the cells were injured with low glucose and low serum for 24 h. The cell viability was detected by MTT assay, the morphological changes of cells were observed by light microscope, and the apoptosis rate was detected by Hoechst 33342 staining. Finally, the expression of Bcl-2 / Bax protein and the activity of Caspase-3 were detected by Western blot and Caspase-3 assay kit. Results MTT and LDH release assay in glutamate injury model showed that, The cell viability in MADP group was higher than that in Sal group by Hoechst33342Tunel Annexin V-FITC / Pi staining under the same condition. The results showed that the apoptotic rate in MADP group was lower than that in Sal group Fluo-4 / AM fluorescence staining showed that [Ca 2] I fluorescence intensity in Sal group was stronger than that in Sal group and Caspase-3 mRNA expression in Sal group was lower than that in MADP group. The results of Western blot showed that the expression of Bcl-2 / Bax and p-ERK protein did not change in both groups. The expression of p-Akt and p-JNK in MADP group was higher than that in Sal group. The results of MTT assay showed that most of the tested compounds had protective effect on PC12 cells, especially compound 6 had the strongest protective effect on PC12 cells. The preprotection of compound 6 could improve the activity of Caspase-3 in damaged PC12 cells by Hoechst 33342 staining and the results of Western blot assay showed that compound 6 could inhibit apoptosis of PC12 cells induced by low glucose and low serum levels in a dose-dependent manner. Compound 6 had the best protective effect at 300 渭 g / mL. Conclusion salidroside analogues MADP and compound 6 can protect hippocampal neurons and PC12 cells better than salidroside.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741
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本文編號(hào)：2124976