去甲茴三硫抗神经炎症及抗肿瘤药理活性的研究

发布时间：2018-06-27 09:25

本文选题：去甲茴三硫 + 神经炎症　；参考：《苏州大学》2016年硕士论文

【摘要】：目的:神经炎症与小胶质细胞激活在帕金森病发病机制中起着非常重要的作用,可能引起多巴胺能神经元的损伤和死亡。去甲茴三硫(ADT-OH)是一种能够释放H2S的物质,H2S是一种重要的气体信号因子来调节脑内稳态,有证据显示H2S能够抑制促炎性细胞因子的表达。本研究目的在于探究去甲茴三硫是否具有较好的抑制神经炎症及抗肿瘤效应,从而能够对帕金森病的治疗提供一定的借鉴意义。方法:体外培养小胶质细胞系BV2及多巴胺能神经元SH-SY5Y细胞系,利用LPS的刺激来建立小胶质细胞持续过度激活模型,对细胞模型进行加药预处理,根据药物处理的浓度梯度和时间梯度实验结果确定最佳处理时间和有效浓度。采用Weston-blot检测下游炎症因子iNOS、COX2蛋白含量的改变,运用ELISA检测细胞分泌到上清中的促炎性因子IL-6、TNF-α含量的变化。运用RT-PCR及Q-PCR检测一系列炎症因子转录水平的变化。此外利用Weston-blot及免疫荧光染色观察加药处理的细胞模型中NF-κB通路蛋白的磷酸化水平及核转位现象。利用小胶质细胞条件培养基处理SH-SY5Y,通过MTT法,PI染色,流式等来观察去甲茴三硫是否能够间接性的保护多巴胺能神经元。利用MTT法检测去甲茴三硫是否能够抑制多种肿瘤细胞系的活性及增殖,利用流式细胞仪检测肿瘤细胞系细胞周期的变化。结果:对小胶质细胞过度激活模型加药处理实验结果表明ADT-OH能够明显的抑制LPS引起的BV2激活,并且ADT-OH本身对小胶质细胞状态没有明显的影响。ADT-OH的预处理能够明显抑制促炎性因子(如iNOS,COX2,IL-6,TNF-α等)的转录及蛋白水平的增加。Weston-bolt检测NF-κB通路的IKK及其磷酸化活性和IκB家族蛋白水平,p65及其磷酸化水平的变化,发现LPS刺激30 min内,ADT-OH的处理能够使IKK的磷酸化水平明显下调,同时p65的磷酸化也同样被抑制。通过免疫荧光观察到LPS刺激30 min内ADT-OH的预处理能够明显抑制LPS引起的p65核转位现象。制备不同处理条件的小胶质细胞条件培养基,培养SH-SY5Y细胞48 h,PI/Hochest染色结果与MTT测试结果均表明ADT-OH能够通过抑制胶质细胞过度激活引起的神经炎症来间接的对多巴胺能神经元起到保护作用。不同浓度的ADT-OH处理He La,A549等几种肿瘤细胞系24 h或48 h,MTT细胞活性测试结果显示ADT-OH能够明显地抑制肿瘤细胞的活性,同时流式细胞仪检测细胞周期发现肿瘤细胞发生细胞周期阻滞现象。结论:去甲茴三硫能够通过抑制p65的入核来缓解LPS引起的小胶质细胞过度激活,并间接的保护多巴胺能神经元。同时通过阻滞细胞周期对多种肿瘤细胞具有明显的抑制效应。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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