白介素-10抑制神经炎症和多巴胺能神经元凋亡的研究

发布时间：2018-05-04 05:53

  本文选题：白介素-10 + 神经炎症　； 参考：《苏州大学》2016年博士论文

【摘要】：目的:白介素-10(interleukin-10,IL-10)是最重要和最有效的抗炎细胞因子之一。中枢神经系统中有多种细胞可产生IL-10或表达IL-10受体,这提示IL-10可以通过作用于脑内相关细胞调节神经炎症反应。而研究表明帕金森病与神经炎症之间存在重要关联。另外,IL-10也存在抗凋亡作用,那么我们推测IL-10是否可以其抗炎和抗凋亡作用对帕金森病进行防治。因此本研究观察IL-10在PD发生发展过程中对神经炎症和多巴胺(dopamine,DA)能神经元缺失的影响,并对其作用机制和信号通路进行初步探讨,以明确IL-10对DA能神经元的保护作用,从而发掘IL-10的治疗潜力,为防治PD提供实验依据。方法:1.细胞培养:(1)中脑腹侧细胞培养:取大鼠胚胎14±0.5天的中脑腹侧组织进行原代细胞培养,培养7天后备用。(2)中脑腹侧单纯神经元培养:取大鼠胚胎14±0.5天的中脑腹侧组织进行原代细胞培养,用阿糖胞苷抑制胶质细胞生长以获得神经元,培养7天后备用。(3)中脑腹侧神经元-小胶质细胞共培养:取新生大鼠大脑皮层组织进行原代细胞培养,12-14天后获取小胶质细胞加入已培养6天的神经元,24 h后备用。(4)中脑腹侧神经元-星形胶质细胞共培养:取新生大鼠大脑皮层组织进行原代细胞培养,12-14天后去除小胶质细胞,至少4次传代获取星形胶质细胞加入已培养6天的神经元,24 h后备用。2.上述四种培养体系中,IL-10抑制LPS诱导的神经炎症和DA能神经元缺失的检测:用IL-10(15,50或150 ng/m L)预处理培养物1 h后再加入LPS(50 ng/m L)作用8 h(检测凋亡相关蛋白表达)或24 h。应用免疫荧光细胞化学法检测DA能和非DA能神经元的数目,PCR和Western blot方法检测培养物中炎症介质(i NOS、COX-2、IL-1β和TNF-α)和神经营养因子(BDNF、IGF-1和GDNF)的水平,ELISA方法检测培养上清中IL-1β、TNF-α或IGF-1的浓度。另外,中脑腹侧细胞培养体系中,应用化学比色法检测上清中NO和H2O2的浓度;中脑腹侧单纯神经元培养体系中,应用免疫荧光双标法观察LPS受体TLR-4在神经元(Neu N+)上的定位情况,Western blot方法检测培养物中促凋亡酶(活化的caspase-3和caspase-9)的蛋白表达,原位末端标记法检测神经元凋亡率。3.中脑腹侧细胞培养体系中,IL-10抑制MPP+诱导的神经炎症和DA能神经元缺失的检测:用IL-10(15或50 ng/m L)预处理培养物1 h后再加入MPP+(5μM)作用24 h。应用免疫荧光细胞化学法检测DA能神经元的数目;Western blot方法检测培养物中炎症介质(i NOS、COX-2、IL-1β和TNF-α)和神经营养因子(BDNF、IGF-1和GDNF)的表达;ELISA方法检测培养上清中IL-1β、TNF-α和IGF-1的浓度。4.中脑腹侧单纯神经元培养体系中,IL-10抑制MPP+诱导的神经炎症和DA能神经元缺失的检测:用IL-10(15或50 ng/m L)预处理培养物1 h后再加入MPP+(5μM)作用8h或24h。应用免疫荧光细胞化学法检测DA能神经元的数目;Western blot方法检测培养物中活化的caspase-3和caspase-9的表达。我们设想IL-10可通过减少TNF-α,抑制细胞凋亡,减轻DA能神经元缺失,于是应用免疫荧光双标法确定TNF-α在受损DA能神经元的定位情况;Western blot方法检测培养物TNF-α的蛋白表达;ELISA方法检测培养上清中TNF-α的浓度。为确认TNF-α在MPP+诱导的神经炎症中的作用,我们进一步用中和性TNF-α抗体(1μg/m L)预处理培养物2h后,加或不加IL-10(50 ng/m L)处理1 h后,再加入MPP+(5μM)作用8 h或24 h。应用免疫荧光细胞化学法检测DA能神经元的数目;Western blot方法检测培养物中活化的caspase-3和caspase-9的表达。5.中脑腹侧单纯神经元培养体系中,IL-10通过神经元上IL-10受体和神经元内JAK-STAT3信号通路抑制LPS诱导的神经炎症和神经元凋亡的检测:首先,应用免疫荧光双标法确定IL-10Rα在神经元上的定位情况。其次,利用基因干扰技术沉默神经元上的IL-10Rα基因或用JAK抑制剂(0.5或1μM)预处理神经元1 h,之后IL-10(50 ng/m L)处理1 h后再加入LPS作用8 h或24 h。应用免疫荧光细胞化学法检测DA能神经元和非DA能神经元的数目;Western blot法检测培养物中COX-2、TNF-α、BDNF、GDNF、活化的caspase-3和caspase-9的表达;ELISA方法检测培养上清中TNF-α的浓度。另外,JAK抑制剂预处理的情况下,应用原位末端标记法检测神经元凋亡率;Western blot法检测磷酸化Stat3的水平。结果:1.中脑腹侧细胞培养体系中,IL-10减轻LPS诱导的神经炎症和DA能神经元缺失:LPS单独处理培养物可导致DA能和非DA能神经元数目减少,i NOS、COX-2、IL-1β和TNF-α水平升高,NO和H2O2浓度增加,BDNF表达降低,但IGF-1增加,GDNF变化不明显。IL-10预处理可明显减轻LPS产生的毒性作用。2.中脑腹侧细胞培养体系中,IL-10减轻MPP+诱导的神经炎症和DA能神经元缺失:MPP+单独处理培养物可导致DA能神经元数目减少,i NOS、COX-2、IL-1β和TNF-α水平升高,BDNF、IGF-1和GDNF水平降低。IL-10预处理可明显减轻MPP+产生的上述毒性作用。3.中脑腹侧神经元-小胶质细胞共培养体系中,IL-10减轻LPS诱导的神经炎症和DA能神经元缺失:LPS单独处理培养物可导致DA能和非DA能神经元数目减少,i NOS、COX-2、IL-1β和TNF-α水平升高,BDNF、IGF-1和GDNF水平降低。IL-10预处理可明显减轻LPS产生的上述毒性作用。4.中脑腹侧神经元-星形胶质细胞共培养体系中,IL-10减轻LPS诱导的炎症介质上调:LPS单独处理培养物,DA能和非DA能神经元数目变化不明显,i NOS、COX-2、IL-1β和TNF-α水平升高,同时BDNF基因、蛋白表达和IGF-1基因表达也增加。GDNF变化不明显,未检测到IGF-1的蛋白表达。IL-10预处理可明显抑制炎症介质的生成。5.中脑腹侧单纯神经元培养体系中,IL-10减轻MPP+诱导的神经炎症,DA能神经元凋亡和缺失:TNF-α与受损DA能神经元有共定位。MPP+单独处理培养物可导致DA能神经元数目减少,TNF-α、活化的caspase-3和caspase-9水平升高。IL-10预处理可明显减轻MPP+的上述毒性作用。中和性TNF-α抗体预处理培养物可降低培养物中促凋亡酶的表达,减轻DA能神经元缺失,但不能增强IL-10的作用。6.中脑腹侧单纯神经元培养体系中,IL-10通过与神经元上IL-10受体相互作用激活神经元内JAK-STAT3信号通路抑制LPS诱导的神经炎症和神经元凋亡:LPS受体TLR-4与Neu N有共定位,说明LPS可通过其受体直接作用于神经元。LPS单独处理培养物可导致DA能和非DA能神经元数目减少,COX-2基因表达、TNF-α水平、活化的caspase-3和caspase-9蛋白表达增加,BDNF、磷酸化的Stat3水平下降,神经元凋亡率升高,但COX-2蛋白表达和GDNF水平变化不明显。IL-10预处理可明显减轻LPS的上述毒性作用。未检测到i NOS、IL-1β和IGF-1的表达。IL-10Rα与Neu N有共定位。但沉默神经元上的IL-10Rα基因或JAK抑制剂预处理神经元后,IL-10不能减轻LPS诱导的神经元缺失,也不能减轻TNF-α和促凋亡酶的上调以及BDNF的下调。另外,JAK抑制剂预处理神经元后,IL-10也不能降低神经元凋亡率,不能减轻Stat3的磷酸化水平下调。结论:1.IL-10可通过作用于胶质细胞,下调炎症介质,上调神经营养因子,抑制神经炎症和多巴胺能神经元缺失,发挥保护神经元的作用。2.IL-10也可通过与神经元上IL-10Rα相互作用激活神经元内JAK-STAT3信号通路,下调炎症介质,上调神经营养因子,降低促凋亡酶水平,减少神经元凋亡,直接发挥保护神经元的作用。
[Abstract]:Objective: -10 (interleukin-10, IL-10) is one of the most important and most effective anti-inflammatory cytokines. There are many cells in the central nervous system that produce IL-10 or express IL-10 receptors. This suggests that IL-10 can regulate neuroinflammatory responses in the brain related cells. The study shows that there is a heavy weight between Parkinson's disease and neuro inflammation. In addition, IL-10 also has anti apoptosis effect, then we speculate whether IL-10 can prevent and control Parkinson's disease with its anti-inflammatory and anti apoptotic effects. Therefore, this study observed the effects of IL-10 on neuroinflammation and the loss of neurons in the neuroinflammation and dopamine (dopamine, DA) neurons in the process of PD development, and on its mechanism and signaling pathway. To clarify the protective effect of IL-10 on DA neurons in order to explore the therapeutic potential of IL-10 and provide experimental basis for the prevention and control of PD. Methods: 1. cells culture: (1) medium ventral ventral cell culture of the middle brain: cultured the ventral mesencephalon of the rat embryo for 14 + 0.5 days and culture for 7 days. (2) the simple neuron culture of the ventral ventral ventral culture of the middle brain Culture: the mesencephalic ventral tissue of the rat embryo was cultured for 14 + 0.5 days, and the cells were cultured with cytarabine to inhibit the growth of glial cells for 7 days. (3) the ventral ventral neurons of the middle brain - microglia were cultured in the middle brain. The primary cells were cultured in the cerebral cortex of the newborn rats. After 12-14 days, the microglia was added to the microglia. The neurons were trained for 6 days and 24 h later. (4) the ventral ventral neurons and astrocytes were cultured in the middle brain. The primary cultured rat cerebral cortex was cultured, and the microglia were removed for 12-14 days. At least 4 times, astrocytes were obtained and cultured for 6 days, and 24 h after 24 h. IL-10 inhibits the detection of LPS induced neuroinflammation and DA neuron loss: using IL-10 (15,50 or 150 ng/m L) preconditioning culture 1 h, and then adding LPS (50 ng/m L) action 8 h (detection of apoptosis related protein expression) or 24 applied immunofluorescence cytochemical method for detecting the number of neurons and non reactive neurons The levels of the inflammatory mediators (I NOS, COX-2, IL-1 beta and TNF- alpha) and neurotrophic factors (BDNF, IGF-1 and GDNF) in the culture were detected by ELISA method to detect the concentration of IL-1 beta, TNF- alpha or IGF-1 in the culture supernatant. In the system, the localization of LPS receptor TLR-4 on the neuron (Neu N+) was observed by immunofluorescence double labeling method. The protein expression of the apoptotic enzyme (activated caspase-3 and caspase-9) was detected by Western blot method. In situ terminal labeling method was used to detect the neuron apoptosis rate in the culture system of ventral ventral cells in the neuronal apoptosis rate, and IL-10 inhibited the MPP+ induced God. Detection of inflammation and DA neuron loss: the number of DA neurons was detected by IL-10 (15 or 50 ng/m L) pre treated culture 1 h and then MPP+ (5 mu M) used by 24 h. to detect the number of DA neurons. Western blot method was used to detect the inflammatory mediators in the culture. The expression of IL-1 beta, TNF- alpha and IGF-1 in the culture supernatant of the supernatant in the culture system of simple neuron culture in.4., IL-10 inhibits the detection of neuroinflammation and DA neuron loss induced by MPP+: pre treated with IL-10 (15 or 50 ng/m L) after 1 h, and then add (5 mu) to immunofluorescence The number of DA neurons was detected by the method of learning, and the expression of activated caspase-3 and caspase-9 in the culture was detected by the Western blot method. We conceived that IL-10 could reduce the loss of DA energy by reducing TNF- a, inhibiting apoptosis and reducing the loss of DA neurons. Therefore, the localization of TNF- alpha in the damaged DA energy neurons was determined by the double immunofluorescence method, and the Western blot method was used. The protein expression of TNF- alpha was detected and the concentration of TNF- alpha in the culture supernatant was detected by ELISA method. In order to confirm the role of TNF- alpha in MPP+ induced neuritis, we further used neutralized TNF- alpha antibody (1 mu g/m L) to pretreat the culture, plus or without IL-10 (50 ng/m L) treatment 1, and then add (5 mu) to 8 or 24 applications. The number of DA neurons was detected by immunofluorescence cytochemistry, and the Western blot method was used to detect the activation of Caspase-3 and caspase-9 in the culture medium.5. in the simple neuron culture system in the ventral ventral region of the brain. IL-10 suppressed neuro inflammation and neuron apoptosis induced by LPS through the IL-10 receptor on the neuron and the JAK-STAT3 signaling pathway in the neuron. Detection: first, the localization of IL-10R alpha on neurons was determined by immunofluorescence double labeling. Secondly, the gene interference technique was used to silence the IL-10R alpha gene on the neuron or the neuron 1 h was pretreated with JAK inhibitor (0.5 or 1 M), and then IL-10 (50 ng/m L) was treated after 1 h and then added to LPS to act 8 h or 24 h. to apply immunofluorescence cytochemistry. The number of DA neurons and non DA neurons was detected by the method. The expression of COX-2, TNF- a, BDNF, GDNF, activated caspase-3 and caspase-9 were detected by Western blot, and ELISA method was used to detect the concentration of TNF- alpha in the culture supernatant. The lot method was used to detect the level of phosphorylated Stat3. Results: in 1. midbrain ventral cell culture system, IL-10 alleviated LPS induced neuroinflammation and DA energy loss. LPS alone treated culture could lead to the decrease in the number of DA and non DA neurons, I NOS, COX-2, IL-1 beta and alpha levels increased, but the expression decreased, but increased Addition, the changes of GDNF were not obvious,.IL-10 preconditioning significantly alleviated the toxicity of LPS in the culture system of ventral ventral cells in.2., IL-10 alleviated MPP+ induced neuroinflammation and the deletion of DA neurons: MPP+ alone treated culture could lead to a decrease in the number of DA neurons, I NOS, COX-2, beta and alpha levels. The reduction of.IL-10 preconditioning significantly alleviated the above-mentioned toxic effect of MPP+ in the co culture system of ventral ventral neurons - microglia in.3., and IL-10 alleviated LPS induced neuroinflammation and DA energy loss. LPS alone treated culture could lead to a decrease in the number of DA and non DA neurons, I NOS, COX-2, beta and alpha levels increased. F, IGF-1 and GDNF levels reduced.IL-10 preconditioning significantly alleviated the above-mentioned toxic effects of LPS in the co culture system of ventral ventral neuron - astrocytes in.4., and IL-10 alleviated the up regulation of the inflammatory mediators induced by LPS: LPS alone, the number of DA energy and non DA neurons did not change obviously. At the same time, the BDNF gene, the protein expression and the expression of IGF-1 gene also increased the change of.GDNF, and the.IL-10 preconditioning of the protein expression of IGF-1 could obviously inhibit the formation of the ventral ventral neuron culture system in the inflammatory mediator, IL-10 alleviated MPP+ induced neuritis, DA neuron apoptosis and deletion: TNF- alpha and damaged DA energy. The number of DA neurons was reduced by a co location of.MPP+ in neurons, and the number of DA neurons decreased. TNF- alpha, activated caspase-3 and caspase-9 levels increased the toxicity of MPP+. Neutralized TNF- a antibody preconditioning could reduce the expression of apoptotic enzymes in the culture and reduce the loss of DA neurons. But it can not enhance the role of IL-10 in the.6. ventral ventral simple neuron culture system, IL-10 can inhibit LPS induced neuroinflammation and neuronal apoptosis by activating the JAK-STAT3 signaling pathway of IL-10 receptor on the neuron on the neuron. The LPS receptor TLR-4 and Neu N have co localization, indicating that LPS can directly act on the neurons through its receptors. LPS alone can reduce the number of DA and non DA neurons, COX-2 gene expression, TNF- alpha level, activated caspase-3 and caspase-9 protein expression, BDNF, phosphorylation Stat3 level decreased, neuron apoptosis rate increased, but COX-2 protein expression and GDNF water level changes are not obvious.IL-10 preconditioning can significantly reduce the upper level The expression of I NOS, IL-1 beta and IGF-1 expressed.IL-10R alpha with Neu N. But after the IL-10R a gene or JAK inhibitor pretreated neurons on the silent neuron, IL-10 did not reduce the LPS induced neuron loss, nor could it lessen the regulation of TNF- alpha and apoptotic enzymes and down regulation. IL-10 can not reduce the apoptosis rate of neurons and can not reduce the rate of apoptosis of Stat3. Conclusion: 1.IL-10 can be used in glial cells, down regulation of inflammatory mediators, up regulation of neurotrophic factors, inhibition of neuroinflammation and dopaminergic neuron loss, and the role of.2.IL-10 to protect neurons through neurons and neurons. The interaction of IL-10R alpha activates the JAK-STAT3 signaling pathway in the neuron, down regulate the inflammatory mediators, up-regulation the neurotrophic factors, reduce the level of apoptotic enzymes, reduce the apoptosis of neurons, and play the role of protecting neurons directly.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.5
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本文编号：1841857