本文選題：莫諾苷 + SK-N-SH細(xì)胞　； 參考：《蚌埠醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的:建立SK-N-SH細(xì)胞的過(guò)氧化氫(hydrogen peroxide,H2O2)的氧化損傷模型,初步探討莫諾苷對(duì)細(xì)胞氧化損傷的保護(hù)作用。方法:(1)采用對(duì)數(shù)生長(zhǎng)期的人神經(jīng)母細(xì)胞瘤(SK-N-SH)細(xì)胞,給予不同濃度的莫諾苷(1μM,10μM和100μM)預(yù)處理24 h,加入H2O2(200-400μM)作用24 h以誘導(dǎo)細(xì)胞損傷。(2)在倒置相差顯微鏡下觀察各組SK-N-SH細(xì)胞的生長(zhǎng)和形態(tài)學(xué)變化。(3)用MTT法檢測(cè)細(xì)胞存活率;乳酸脫氫酶(LDH)細(xì)胞毒性檢測(cè)試劑盒檢測(cè)細(xì)胞內(nèi)LDH的釋放量;脂質(zhì)氧化(MDA)檢測(cè)試劑盒檢測(cè)細(xì)胞脂質(zhì)氧化水平;超氧化物歧化酶(SOD)測(cè)定試劑盒檢測(cè)細(xì)胞內(nèi)SOD的含量。(4)以活性氧(ROS)檢測(cè)試劑盒檢測(cè)細(xì)胞內(nèi)ROS的生成;超氧化物陰離子熒光探針(Dihydroethidium)檢測(cè)細(xì)胞內(nèi)超氧化物陰離子的水平。(5)應(yīng)用線粒體膜電位檢測(cè)試劑盒(JC-1)檢測(cè)線粒體膜電位(△Ψm)的變化;采用分光光度法檢測(cè)細(xì)胞內(nèi)Caspase-3活性。(6)通過(guò)流式細(xì)胞術(shù)(FCM)、Hoechst染色分析細(xì)胞凋亡。(7)利用Western blot檢測(cè)細(xì)胞凋亡相關(guān)蛋白的表達(dá)變化。(8)利用RT-PCR檢測(cè)凋亡相關(guān)基因的表達(dá)。結(jié)果:1.MTT細(xì)胞活力檢測(cè)結(jié)果顯示,H2O2可降低SK-N-SH細(xì)胞活力,并呈現(xiàn)出劑量和時(shí)間依賴(lài)的關(guān)系。經(jīng)過(guò)H2O2(200μM)處理24 h后,細(xì)胞活力較對(duì)照組顯著降低,而莫諾苷能明顯抑制H2O2導(dǎo)致的SK-N-SH細(xì)胞活力下降。2.形態(tài)學(xué)結(jié)果顯示,經(jīng)過(guò)H2O2(200μM)處理24 h后,SK-N-SH細(xì)胞可出現(xiàn)突起消失、胞體腫脹圓縮、部分細(xì)胞發(fā)生聚集并破裂成細(xì)胞碎片等顯著的形態(tài)學(xué)變化;而莫諾苷的預(yù)處理能夠減輕這一損傷變化。3.LDH細(xì)胞毒性檢測(cè)結(jié)果表明,莫諾苷可減少胞內(nèi)酶LDH的釋放。4.流式細(xì)胞術(shù)和Hoechst染色的結(jié)果表明,莫諾苷可顯著抑制H2O2誘導(dǎo)的SK-N-SH細(xì)胞凋亡。5.DCFH-DA和Dihydroethidium熒光探針檢測(cè)結(jié)果顯示,莫諾苷可有效抑制H2O2誘導(dǎo)的細(xì)胞內(nèi)ROS蓄積。6.脂質(zhì)氧化檢測(cè)結(jié)果表明,莫諾苷可抑制細(xì)胞內(nèi)的脂質(zhì)發(fā)生過(guò)氧化;SOD的檢測(cè)表明,莫諾苷可顯著抑制H2O2誘導(dǎo)的SOD含量的下降。7.JC-1熒光探針和分光光度法的結(jié)果顯示,莫諾苷可顯著抑制H2O2誘導(dǎo)的SK-N-SH細(xì)胞線粒體膜電位的下降以及Caspase-3的激活。8.Western blot和RT-PCR的結(jié)果顯示,莫諾苷可有效抑制H2O2誘導(dǎo)的BAX的表達(dá)上調(diào),并促進(jìn)Bcl-2的表達(dá)。結(jié)論:莫諾苷可抑制H2O2誘導(dǎo)的SK-N-SH細(xì)胞氧化損傷,其機(jī)制可能與抑制細(xì)胞內(nèi)氧化應(yīng)激、抑制H2O2誘導(dǎo)的BAX的表達(dá)上調(diào),并促進(jìn)Bcl-2的表達(dá)、阻斷線粒體凋亡途徑密切相關(guān)。
[Abstract]:Aim: to establish the oxidative damage model of SK-N-SH cell line hydrogen peroxide-H _ 2O _ 2 and to explore the protective effect of monoside on cell oxidative damage. Methods: SK-N-SH1 cells of human neuroblastoma in logarithmic phase were used. The cells were pretreated with different concentrations of Monoside 1 渭 M 10 渭 M and 100 渭 M for 24 h, and added H2O2(200-400 渭 M for 24 h to induce cell injury. (2) the growth and morphological changes of SK-N-SH cells in each group were observed under inverted phase contrast microscope. The survival rate of SK-N-SH cells was measured by MTT method. Lactic dehydrogenase (LDH) cytotoxicity assay kit was used to detect the amount of LDH released in the cells, and the lipid oxidation assay kit was used to detect the lipid oxidation level of the cells. Superoxide dismutase (SOD) assay kit was used to detect the content of SOD in cells. (4) reactive oxygen species (Ros) assay kit was used to detect the production of ROS in cells. Superoxide anion fluorescence probe (Dihydroethidium) was used to detect the level of superoxide anion in cells. The mitochondrial membrane potential detection kit (JC-1) was used to detect the changes of mitochondrial membrane potential (蠄 m). Caspase-3 activity in cells was detected by spectrophotometry.) apoptosis was analyzed by flow cytometry (FCM) Hoechst staining. The expression of apoptosis-related protein was detected by Western blot. 8) the expression of apoptosis-related genes was detected by RT-PCR. Results: 1. The results of cell viability test showed that H _ 2O _ 2 could decrease the activity of SK-N-SH cells in a dose-dependent and time-dependent manner. After treatment with H2O2(200 渭 M for 24 h, the cell viability was significantly lower than that in the control group, while Monoside could significantly inhibit the decrease of SK-N-SH cell viability induced by H2O2. The morphological results showed that after treatment with H2O2(200 渭 M for 24 h, the SK-N-SH cells showed obvious morphological changes, such as the disappearance of the processes, the swelling and contraction of the cell bodies, the aggregation of some cells and the fragmentation of the fragments of the SK-N-SH cells. The results of cytotoxicity test showed that monoside could reduce the release of intracellular enzyme LDH. 4. The results of flow cytometry and Hoechst staining showed that Monoside could significantly inhibit the apoptosis of SK-N-SH cells induced by H2O2. 5. DCFH-DA and Dihydroethidium fluorescence probe assay showed that Monoside could effectively inhibit the intracellular ROS accumulation induced by H2O2. The results of lipid oxidation test showed that Monoside could inhibit lipid peroxidation in cells. The results showed that Monoside could significantly inhibit the decrease of SOD content induced by H2O2. 7. 7. JC-1 fluorescence probe and spectrophotometry showed that Monoside could inhibit the decrease of SOD content. Monoside could significantly inhibit the decrease of mitochondrial membrane potential induced by H2O2 and the activation of Caspase-3. 8. The results of Western blot and RT-PCR showed that Monoside could effectively inhibit the up-regulation of BAX expression induced by H2O2 and promote the expression of Bcl-2. Conclusion: Monoside can inhibit the oxidative damage of SK-N-SH cells induced by H2O2, and its mechanism may be related to the inhibition of intracellular oxidative stress, the up-regulation of BAX expression induced by H2O2, the promotion of Bcl-2 expression and the blocking of mitochondrial apoptosis pathway.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446

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