本文選題：硫化氫 + 丙烯腈�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:硫化氫(hydrogen sulfide,H_2S)作為繼CO、NO后的第三種氣體信號(hào)分子,有著重要的神經(jīng)保護(hù)性和神經(jīng)調(diào)節(jié)作用,在保護(hù)化學(xué)物和藥物的神經(jīng)損傷的應(yīng)用中有著廣闊前景。丙烯腈(acrylonitrile,AN)是一種高毒的有機(jī)腈,易經(jīng)呼吸道、皮膚和胃腸道侵入人體,主要侵害神經(jīng)系統(tǒng)。我們前期研究已經(jīng)發(fā)現(xiàn)AN神經(jīng)毒性與硫化氫、谷胱甘肽等含硫化合物代謝密切相關(guān),因此,本實(shí)驗(yàn)以大鼠原代星形膠質(zhì)細(xì)胞為研究對(duì)象,硫氫化鈉(NaHS)為外源性H_2S供體:1.初步探索外源性H_2S對(duì)AN誘導(dǎo)的星形膠質(zhì)細(xì)胞毒性的影響;2.進(jìn)一步探索外源性H_2S對(duì)AN誘導(dǎo)的細(xì)胞毒性影響的調(diào)控機(jī)制;3.為研發(fā)新的丙烯腈預(yù)防和治療藥物提供科學(xué)依據(jù),以期尋找新的解毒方向。方法:1.采用AN處理原代培養(yǎng)的星形膠質(zhì)細(xì)胞,NaHS為外源性H_2S供體預(yù)處理細(xì)胞1 h。MTT法檢測(cè)細(xì)胞活力和LDH漏出率檢測(cè)細(xì)胞毒性評(píng)價(jià)NaHS對(duì)AN誘導(dǎo)的細(xì)胞毒性的影響。檢測(cè)GSH和ROS含量的變化評(píng)價(jià)細(xì)胞氧化應(yīng)激水平的改變。2.單純應(yīng)用NaHS處理細(xì)胞,檢測(cè)自噬相關(guān)蛋白Beclin1、SQSTM1/P62、Atg5表達(dá)變化,免疫熒光方法觀察星形膠質(zhì)細(xì)胞中自噬顆粒變化,吖啶橙染色檢測(cè)細(xì)胞酸性自噬泡,自噬雙標(biāo)腺病毒檢測(cè)自噬流等方法檢測(cè)NaHS對(duì)原代星形膠質(zhì)細(xì)胞自噬水平的影響。3.單獨(dú)應(yīng)用NaHS處理細(xì)胞,采用細(xì)胞核質(zhì)蛋白分離,免疫印跡法檢測(cè)Nrf2、HO-1、γ-GCS的表達(dá)以及免疫熒光標(biāo)記Nrf2觀察其核轉(zhuǎn)位情況來(lái)綜合評(píng)價(jià)NaHS對(duì)細(xì)胞Nrf2/ARE信號(hào)通路的影響。4.通過自噬抑制劑和siRNA轉(zhuǎn)染的方法抑制自噬或Nrf2的表達(dá),MTT法和LDH漏出率法檢測(cè)NaHS對(duì)AN誘導(dǎo)的細(xì)胞毒性效應(yīng)的改變,探索調(diào)控自噬或Nrf2/ARE信號(hào)通路對(duì)NaHS誘導(dǎo)的保護(hù)效應(yīng)的影響。結(jié)果:1.采用NaHS(0、200、400、800μM)預(yù)處理細(xì)胞1 h,與AN單獨(dú)處理組相比,細(xì)胞活力明顯升高,LDH滲出率下降。同時(shí),細(xì)胞抗氧化物還原性GSH含量升高,氧化應(yīng)激產(chǎn)物ROS水平降低。2.NaHS單獨(dú)處理細(xì)胞1h后,自噬相關(guān)蛋白Beclin1,Atg5表達(dá)明顯升高,而SQSTM1/P62表達(dá)下降,并具有濃度依賴性。同時(shí)單獨(dú)NaHS處理組星形膠質(zhì)細(xì)胞內(nèi)LC3綠色熒光斑點(diǎn)明顯增多,AO染色結(jié)果顯示酸性自噬泡(紅色熒光斑點(diǎn))也明顯增多,采用自噬雙標(biāo)腺病毒檢測(cè)結(jié)果提示自噬流增強(qiáng),說明NaHS可誘導(dǎo)星形膠質(zhì)細(xì)胞自噬。3.單純NaHS處理細(xì)胞,與對(duì)照組相比,細(xì)胞核內(nèi)Nrf2分布增多,Nrf2/ARE通路下游蛋白HO-1、γ-GCS表達(dá)升高,說明外源性NaHS可引起Nrf2發(fā)生核轉(zhuǎn)位。4.分別用藥理學(xué)和基因?qū)W手段抑制自噬或Nrf2信號(hào)后,NaHS對(duì)AN毒性的拮抗效應(yīng)減弱。結(jié)論:1.NaHS預(yù)處理對(duì)AN誘導(dǎo)的星形膠質(zhì)細(xì)胞毒性有拮抗效應(yīng),并可改善AN誘導(dǎo)的氧化應(yīng)激損傷。2.單純NaHS處理可誘導(dǎo)細(xì)胞自噬的活化,同時(shí)也可誘導(dǎo)Nrf2發(fā)生核轉(zhuǎn)位,激活Nrf2/ARE信號(hào)通路。3.抑制自噬或Nrf2表達(dá),NaHS對(duì)AN毒性的拮抗效應(yīng)均減弱,說明NaHS對(duì)AN毒性的保護(hù)效應(yīng)與其對(duì)自噬以及Nrf2/ARE信號(hào)通路的激活均有一定的關(guān)聯(lián)。這提示H_2S可能成為一種新的丙烯腈中毒治療藥物。
[Abstract]:Objective: hydrogen sulfide (H_2S), a third gas signal molecule following CO and NO, has an important neuroprotective and neuromodulation effect. It has a broad prospect in the application of protecting chemicals and drugs for nerve damage. Acrylonitrile (acrylonitrile, AN) is a highly toxic organic nitrile, easy to pass through the respiratory tract, skin, and gastrointestinal tract. We have found that AN neurotoxicity is closely related to the metabolism of sulfur compounds, such as hydrogen sulfide, glutathione, and so on. Therefore, this experiment takes the primary astrocytes of rats as the research object, sodium thiohydrate (NaHS) is a exogenous H_2S donor: 1. preliminary exploration of exogenous H_2S to AN induced stars The effect of the toxicity of glial cells; 2. further explore the regulatory mechanism of exogenous H_2S on the cytotoxicity induced by AN; 3. to provide scientific basis for the development of new prophylaxis and treatment of drugs, in order to find new detoxification directions. Method: 1. using AN to treat primary cultured astrocytes, and NaHS as a exogenous H_2S donor Detection of cell viability and LDH leakage rate by cell 1 h.MTT assay the effect of NaHS on the cytotoxicity of AN induced cytotoxicity. Detection of changes in GSH and ROS content and evaluation of changes in oxidative stress levels of cells.2. simply applied NaHS treated cells to detect the changes of Beclin1, SQSTM1/P62, Atg5 expression of autophagy related proteins and immunofluorescence methods The changes of autophagic particles in astrocytes, acridine orange staining and detection of autophagic vesicles, autophagy detection of autophagy by autophagy double labeled adenovirus, the effect of NaHS on the autophagy level of primary astrocytes was detected by.3. alone with NaHS cells, cell nuclear protein separation, and Western blot to detect the expression of Nrf2, HO-1, and gamma -GCS And immunofluorescent labeling Nrf2 observation of its nuclear transposition to evaluate the effect of NaHS on cell Nrf2/ARE signaling pathway,.4. inhibits autophagy or Nrf2 expression through autophagy inhibitors and siRNA transfection. MTT and LDH leaks detect the changes in the cytotoxic effects of NaHS on AN induced by AN, and explore the regulation of autophagy or Nrf2/ARE signaling pathway. The effects on the protective effect induced by NaHS. Results: 1. the cells were pretreated with NaHS (0200400800 M) for 1 h, compared with the AN alone treatment group, the cell viability was significantly increased and the LDH exudation rate decreased. At the same time, the reduced GSH content of the cell antioxidants was increased, the ROS level of the oxidative stress products was reduced by.2.NaHS alone, and the autophagy associated protein Be was processed. The expression of clin1, Atg5 was significantly increased, while the expression of SQSTM1/P62 decreased and was dependent on concentration. At the same time, the LC3 green fluorescence spots in astrocytes were significantly increased in the NaHS treatment group, and the results of AO staining showed that the acid autophagic vesicles (red fluorescent spots) were also significantly increased. The autophagic double standard adenovirus detection results suggested the enhancement of autophagic flow, indicating N. AHS can induce the autophagy of astrocytes to autophagy.3. NaHS cells. Compared with the control group, the distribution of Nrf2 in the nucleus increased, the downstream protein HO-1, and the expression of gamma -GCS in the Nrf2/ARE pathway increased, indicating that the exogenous NaHS could cause the Nrf2 occurrence of nuclear transposition.4. to inhibit the autophagy or Nrf2 signal by pharmacological and genetic hand segments, and the antagonism of NaHS to toxicity. The effect is weakened. Conclusion: 1.NaHS pretreatment can antagonize the toxicity of AN induced astrocytes, and improve the oxidative stress induced by AN, which can induce the activation of autophagy in.2. alone, and also induce the nuclear transposition of Nrf2, and activate the Nrf2/ARE signaling pathway to inhibit autophagy or Nrf2 expression by.3., and the antagonism of NaHS to AN toxicity. The effects were weakened, indicating that the protective effect of NaHS on the toxicity of AN and the activation of the autophagy and the Nrf2/ARE signaling pathway were related to a certain extent. This suggests that H_2S may be a new drug for the treatment of acrylonitrile poisoning.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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