【摘要】：目的:1.在Aβ誘導(dǎo)的星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞的氧化應(yīng)激損傷模型中,初步探討Nrf2對(duì)Aβ誘導(dǎo)的氧化應(yīng)激的抑制作用及DMF對(duì)Nrf2及其下游抗氧化應(yīng)激因子如Nqo1和HO-1的調(diào)節(jié)作用。2.通過對(duì)星形膠質(zhì)細(xì)胞Keap1、HDAC2因子和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞Keap1、CX3CL1因子的檢測(cè),初步探討DMF在Aβ誘導(dǎo)的氧化應(yīng)激條件下對(duì)Nrf2作用機(jī)制。方法:1.將大鼠星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞分別與Aβ25-35共培養(yǎng),誘導(dǎo)氧化應(yīng)激反應(yīng)。在大鼠星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞中分別設(shè)立Aβ組、Aβ+DMF組、Aβ+Si-nrf2組和Aβ+Si-nrf2+DMF組。Aβ組細(xì)胞給予Nrf2-conRNA轉(zhuǎn)染。Aβ+Si-nrf2組細(xì)胞給予Nrf2-siRNA轉(zhuǎn)染以敲低Nrf2基因的表達(dá)。以DMF(4uM)分別對(duì)Aβ組、Aβ+Si-nrf2兩組細(xì)胞進(jìn)行干預(yù),干預(yù)后的細(xì)胞分別為Aβ+DMF組、Aβ+Si-nrf2+DMF組。然后采用RT-PCR分別檢測(cè)兩種細(xì)胞中各Aβ組、Aβ+DMF組、Aβ+Si-nrf2組和Aβ+Si-nrf2+DMF組的Nrf2、Nqo1、HO-1的mRNA表達(dá)水平。2.采用RT-PCR檢測(cè)星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞各Aβ組、Aβ+DMF組、Aβ+Si-nrf2組和Aβ+Si-nrf2+DMF組的Keap1 mRNA表達(dá)水平。采用Western blot檢測(cè)星形膠質(zhì)細(xì)胞各組的HDAC2蛋白表達(dá)水平和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞各組的CX3CL1蛋白表達(dá)水平。結(jié)果:1.在星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞中,Aβ+Si-nrf2組較Aβ組、Aβ+Si-nrf2+DMF組較Aβ+DMF組Nrf2、Nqo1和HO-1 mRNA表達(dá)水平顯著降低(P0.05)。Aβ+DMF組較Aβ組Nrf2、Nqo1和HO-1 mRNA表達(dá)顯著升高(P0.05)。2.在星形膠質(zhì)細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y中,Aβ+DMF組較Aβ組、Aβ+Si-nrf2+DMF組較Aβ+Si-nrf2組Keap1 m RNA表達(dá)顯著降低(P0.05)。在星形膠質(zhì)細(xì)胞中,Aβ+DMF組較Aβ組、Aβ+Si-nrf2+DMF組較Aβ+Si-nrf2組的HDAC2蛋白表達(dá)水平均明顯下降(P0.05)。在人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞中,Aβ+DMF組較Aβ組、Aβ+Si-nrf2+DMF組較Aβ+Si-nrf2組的CX3CL1蛋白表達(dá)水平均明顯升高(P0.05)。結(jié)論:1.Nrf2對(duì)Aβ誘導(dǎo)的氧化應(yīng)激具有抑制作用。在Aβ誘導(dǎo)氧化應(yīng)激的條件下,DMF可提高Nrf2的表達(dá),增加抗氧化應(yīng)激因子的表達(dá)。2.DMF可能通過抑制HDAC2的表達(dá),提高Nrf2水平,從而減弱Aβ誘導(dǎo)的大鼠星形膠質(zhì)細(xì)胞的氧化應(yīng)激反應(yīng)。DMF可能通過增加CX3CL1和減少Keap1的表達(dá)雙重途徑共同提高Nrf2水平,從而減弱Aβ誘導(dǎo)的人神經(jīng)母細(xì)胞瘤SH-SY5H細(xì)胞的氧化應(yīng)激反應(yīng)。
[Abstract]:Objective: 1. In A 尾 -induced oxidative stress injury model of astrocytes and human neuroblastoma SH-SY5Y cells, To investigate the inhibitory effect of Nrf2 on A 尾 -induced oxidative stress and the regulatory effect of DMF on Nrf2 and its downstream antioxidant stress factors such as Nqo1 and HO-1. 2. Through the detection of astrocyte Keap1,HDAC2 factor and Keap1,CX3CL1 factor of human neuroblastoma SH-SY5Y cells, the mechanism of DMF acting on Nrf2 under A 尾 -induced oxidative stress was preliminarily investigated. Methods: 1. Rat astrocytes and human neuroblastoma SH-SY5Y cells were co-cultured with A 尾 25-35 to induce oxidative stress. In SH-SY5Y cells of rat astrocytes and human neuroblastoma, A 尾 group and A 尾 DMF group were established respectively. In A 尾 Si-nrf2 group and A 尾 Si-nrf2 DMF group, A 尾 cells were transfected with Nrf2-conRNA, and A 尾 Si-nrf2 cells were transfected with Nrf2-siRNA to lower the expression of Nrf2 gene. The cells of A 尾 group and A 尾 Si-nrf2 group were treated with DMF (4uM) respectively. The cells after intervention were A 尾 DMF group and A 尾 Si-nrf2 DMF group respectively. Then RT-PCR was used to detect the mRNA expression of Nrf2,Nqo1,HO-1 in A 尾 group, A 尾 DMF group, A 尾 Si-nrf2 group and A 尾 Si-nrf2 DMF group. The expression of Keap1 mRNA in astrocytes and human neuroblastoma SH-SY5Y cells was detected by RT-PCR in A 尾 group, A 尾 DMF group, A 尾 Si-nrf2 group and A 尾 Si-nrf2 DMF group. Western blot was used to detect the expression of HDAC2 protein in astrocytes and CX3CL1 protein in SH-SY5Y cells of human neuroblastoma. The result is 1: 1. In astrocytes and human neuroblastoma SH-SY5Y cells, A 尾 Si-nrf2 group was significantly lower than A 尾 group, A 尾 Si-nrf2 DMF group was significantly lower than A 尾 DMF group Nrf2,Nqo1 and HO-1 mRNA expression level (P0.05). A 尾 DMF group than A 尾 group Nrf2,) The expression of Nqo1 and HO-1 mRNA increased significantly (P0.05). In SH-SY5Y of astrocytes and human neuroblastoma, the expression of Keap1 m RNA in A 尾 DMF group was significantly lower than that in A 尾 group and A 尾 Si-nrf2 DMF group (P0.05). In astrocytes, the expression of HDAC2 protein in A 尾 DMF group was significantly lower than that in A 尾 group and A 尾 Si-nrf2 DMF group compared with A 尾 Si-nrf2 group (P0.05). In SH-SY5Y cells of human neuroblastoma, the expression of CX3CL1 protein in A 尾 DMF group was significantly higher than that in A 尾 group and A 尾 Si-nrf2 DMF group compared with A 尾 Si-nrf2 group (P0.05). Conclusion: 1.Nrf2 can inhibit the oxidative stress induced by A 尾. Under the condition of A 尾 -induced oxidative stress, DMF could increase the expression of Nrf2 and the expression of antioxidant stress factor. 2.DMF might increase the level of Nrf2 by inhibiting the expression of HDAC2. Thus attenuating the oxidative stress response of astrocytes induced by A 尾. DMF may increase the level of Nrf2 by increasing CX3CL1 and decreasing the expression of Keap1. Thus, the oxidative stress response of human neuroblastoma SH-SY5H cells induced by A 尾 was attenuated.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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