發(fā)布時間：2018-06-27 09:25

  本文選題：去甲茴三硫 + 神經(jīng)炎癥　； 參考：《蘇州大學(xué)》2016年碩士論文

【摘要】：目的:神經(jīng)炎癥與小膠質(zhì)細(xì)胞激活在帕金森病發(fā)病機制中起著非常重要的作用,可能引起多巴胺能神經(jīng)元的損傷和死亡。去甲茴三硫(ADT-OH)是一種能夠釋放H2S的物質(zhì),H2S是一種重要的氣體信號因子來調(diào)節(jié)腦內(nèi)穩(wěn)態(tài),有證據(jù)顯示H2S能夠抑制促炎性細(xì)胞因子的表達(dá)。本研究目的在于探究去甲茴三硫是否具有較好的抑制神經(jīng)炎癥及抗腫瘤效應(yīng),從而能夠?qū)ε两鹕〉闹委熖峁┮欢ǖ慕梃b意義。方法:體外培養(yǎng)小膠質(zhì)細(xì)胞系BV2及多巴胺能神經(jīng)元SH-SY5Y細(xì)胞系,利用LPS的刺激來建立小膠質(zhì)細(xì)胞持續(xù)過度激活模型,對細(xì)胞模型進(jìn)行加藥預(yù)處理,根據(jù)藥物處理的濃度梯度和時間梯度實驗結(jié)果確定最佳處理時間和有效濃度。采用Weston-blot檢測下游炎癥因子iNOS、COX2蛋白含量的改變,運用ELISA檢測細(xì)胞分泌到上清中的促炎性因子IL-6、TNF-α含量的變化。運用RT-PCR及Q-PCR檢測一系列炎癥因子轉(zhuǎn)錄水平的變化。此外利用Weston-blot及免疫熒光染色觀察加藥處理的細(xì)胞模型中NF-κB通路蛋白的磷酸化水平及核轉(zhuǎn)位現(xiàn)象。利用小膠質(zhì)細(xì)胞條件培養(yǎng)基處理SH-SY5Y,通過MTT法,PI染色,流式等來觀察去甲茴三硫是否能夠間接性的保護(hù)多巴胺能神經(jīng)元。利用MTT法檢測去甲茴三硫是否能夠抑制多種腫瘤細(xì)胞系的活性及增殖,利用流式細(xì)胞儀檢測腫瘤細(xì)胞系細(xì)胞周期的變化。結(jié)果:對小膠質(zhì)細(xì)胞過度激活模型加藥處理實驗結(jié)果表明ADT-OH能夠明顯的抑制LPS引起的BV2激活,并且ADT-OH本身對小膠質(zhì)細(xì)胞狀態(tài)沒有明顯的影響。ADT-OH的預(yù)處理能夠明顯抑制促炎性因子(如iNOS,COX2,IL-6,TNF-α等)的轉(zhuǎn)錄及蛋白水平的增加。Weston-bolt檢測NF-κB通路的IKK及其磷酸化活性和IκB家族蛋白水平,p65及其磷酸化水平的變化,發(fā)現(xiàn)LPS刺激30 min內(nèi),ADT-OH的處理能夠使IKK的磷酸化水平明顯下調(diào),同時p65的磷酸化也同樣被抑制。通過免疫熒光觀察到LPS刺激30 min內(nèi)ADT-OH的預(yù)處理能夠明顯抑制LPS引起的p65核轉(zhuǎn)位現(xiàn)象。制備不同處理條件的小膠質(zhì)細(xì)胞條件培養(yǎng)基,培養(yǎng)SH-SY5Y細(xì)胞48 h,PI/Hochest染色結(jié)果與MTT測試結(jié)果均表明ADT-OH能夠通過抑制膠質(zhì)細(xì)胞過度激活引起的神經(jīng)炎癥來間接的對多巴胺能神經(jīng)元起到保護(hù)作用。不同濃度的ADT-OH處理He La,A549等幾種腫瘤細(xì)胞系24 h或48 h,MTT細(xì)胞活性測試結(jié)果顯示ADT-OH能夠明顯地抑制腫瘤細(xì)胞的活性,同時流式細(xì)胞儀檢測細(xì)胞周期發(fā)現(xiàn)腫瘤細(xì)胞發(fā)生細(xì)胞周期阻滯現(xiàn)象。結(jié)論:去甲茴三硫能夠通過抑制p65的入核來緩解LPS引起的小膠質(zhì)細(xì)胞過度激活,并間接的保護(hù)多巴胺能神經(jīng)元。同時通過阻滯細(xì)胞周期對多種腫瘤細(xì)胞具有明顯的抑制效應(yīng)。
[Abstract]:Objective: neuroinflammation and microglia activation play a very important role in the pathogenesis of Parkinson's disease, which may cause damage and death of dopaminergic neurons. Three sulphur (ADT-OH) is a substance capable of releasing H2S, and H2S is an important gas signal factor to regulate homeostasis in the brain. There is evidence that H2S can be suppressed. The purpose of this study is to explore whether three sulphur has a better inhibition of neuroinflammation and antitumor effect, which can provide some reference for the treatment of Parkinson's disease. Methods: the microglia line BV2 and the SH-SY5Y cell line of dopaminergic neurons are cultured in vitro, and the stimulation of LPS is used. To establish the continuous overactivation model of microglia and preprocess the cell model, the optimal treatment time and effective concentration were determined according to the concentration gradient and the time gradient of the drug treatment. The changes of the downstream inflammatory factor iNOS, the content of COX2 protein were detected by Weston-blot, and ELISA was used to detect the secretion of the cells to the supernatant. Changes in the levels of pro-inflammatory factors IL-6, TNF- alpha. The changes in the transcriptional level of a series of inflammatory factors were detected by RT-PCR and Q-PCR. In addition, Weston-blot and immunofluorescence staining were used to observe the phosphorylation level and nuclear transposition of NF- kappa B pathway protein in the treated cell model. SH-SY5Y was treated with microglia conditioned medium. The MTT method, PI staining, flow pattern and so on were used to observe whether the three sulfur can protect the dopaminergic neurons indirectly. Using the MTT method to detect the activity and proliferation of a variety of tumor cell lines by three sulfur removal, the changes in the cell cycle of the tumor cell lines were detected by flow cytometry. The experimental results showed that ADT-OH could obviously inhibit the activation of BV2 induced by LPS, and ADT-OH itself had no obvious effect on the state of microglia, and the preconditioning of.ADT-OH could obviously inhibit the transcription of pro-inflammatory factors (such as iNOS, COX2, IL-6, TNF- alpha, etc.) and the increase of protein level by.Weston-bolt to detect the NF- kappa B pathway As well as its phosphorylation activity, I kappa B family protein level, p65 and its phosphorylation level changes, it is found that LPS stimulates 30 min, ADT-OH can reduce the phosphorylation level of IKK obviously, and the phosphorylation of p65 is also inhibited. The pretreatment of ADT-OH in the LPS stimulus 30 min can obviously inhibit LPS caused by the immunofluorescence. 5 nucleation translocation phenomenon. Preparation of microglia conditioned medium with different treatment conditions, culture of SH-SY5Y cells 48 h, PI/Hochest staining results and MTT test results all showed that ADT-OH could protect the dopaminergic neurotransmitter indirectly by inhibiting neuroglia induced by glial activation. Different concentrations of ADT-OH treatment He La, A549 and other tumor cells were 24 h or 48 h. The results of MTT cell activity test showed that ADT-OH could obviously inhibit the activity of tumor cells, and the flow cytometer detected the cell cycle arrest in the cell cycle. Conclusion: the three sulfur of the deannel can pass the nucleation of p65 to alleviate the microglia caused by LPS. Excessive activation of cells and indirect protection of dopaminergic neurons, while blocking cell cycle has a significant inhibitory effect on a variety of tumor cells.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R96

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