程序性细胞坏死调控的TNF-α诱导的神经毒性独立于ROS和钙离子通道

发布时间：2018-02-02 00:03

本文关键词： 细胞坏死 TNF-α 海马神经元 RIP3 ROS 钙离子通道神经毒性　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：【研究背景和意义】肿瘤坏死因子(TNF-)在调节神经炎症方面是一个重要的促炎症因子。在各种神经退行性疾病中，例如阿尔茨海默病（即老年痴呆症）和帕金森氏病中可以看到TNF-的上调。然而，TNF-诱导的神经毒性这条信号通路仍然不清楚。【研究目的】确定RIP3介导的程序性细胞坏死（又称necroptosis）是否参与TNF-和TRAIL诱导的神经细胞毒性。【研究方法】野生型小鼠和RIP3缺陷型小鼠分别注射PBS和不同浓度肿瘤坏死因子（TNF-）,通过小鼠大脑组织切片染色观察小鼠大脑海马区细胞损伤情况以及检测TNF-刺激后海马组织中细胞坏死关键调控蛋白的表达情况；利用普遍用于研究的海马神经元细胞系HT-22深入研究TNF-诱导的细胞死亡机制,首先检测HT-22细胞对TNF-诱导的细胞凋亡（常规诱导体系为TNF-或者TNF-Smac mimetic）和TNF-诱导的细胞坏死(常规诱导体系为TNF-/z-VAD）的敏感性，同时也检测了细胞对TNF家族的另一重要因子TRAIL诱导的细胞凋亡和坏死的敏感性；应用RNA干扰技术分别沉默程序性细胞坏死通路中的相关蛋白，检测蛋白沉默后对TNF-/z-VAD或TRAIL/z-VAD诱导的神经毒性的影响；应用蛋白免疫印迹方法检测沉默后关键蛋白的表达情况；应用慢病毒感染的方法构建稳定表达野生型RIP3(WT)，激酶突变的RIP3(RIP3-K51A)和RIP同源相互作用域突变的RIP3(RIP3-RHIM Mut)的HT-22的细胞系检测关键蛋白RIP3在TNF-/z-VAD或TRAIL/z-VAD诱导的细胞坏死中的作用；利用特异性化学抑制剂分别干预ROS以及钙离子通道，从而检测这些步骤是否参与TNF-诱导的HT-22细胞坏死。【研究结果】（1）小鼠脑室内注射TNF-后，发现在野生型小鼠海马区细胞出现损伤，而且呈现剂量依赖性，但在RIP3缺陷的小鼠中并没有出现明显的海马细胞损伤；应用蛋白免疫印迹发现注射TNF-的野生型小鼠海马组织中RIP3和RIP1蛋白表达呈现明显上调。这些结果表明RIP3-依赖性细胞坏死参与TNF-诱导小鼠海马神经细胞毒性。（2）我们选择了小鼠海马神经细胞HT-22用于深入研究TNF-诱导的海马神经毒性的分子机制。我们发现HT-22细胞对常规的TNF-诱导的细胞凋亡体系不敏感，只有在凋亡抑制剂z-VAD的存在下才对TNF-表现出敏感性，同时还发现只在z-VAD的存在下细胞对TRAIL也表现出敏感性下提示这些表明HT-22细胞更倾向于发生TNF-以及TRAIL诱导的细胞坏死而不是凋亡。（3）通过RNAi干扰技术，沉默RIP3蛋白的表达，发现RIP3的缺陷能抑制TNF-/z-VAD和TRAIL/z-VAD诱导的海马神经细胞死亡，这说明TNF-/z-VAD和TRAIL/z-VAD诱导的细胞死亡是RIP3介导的程序性细胞坏死。（4）进一步沉默RIP1或者CYLD（RIP1的去泛素化酶）,同样能够抑制这种细胞死亡，也说明这种细胞坏死依赖于RIP1和CYLD。（5）接着我们又沉默了RIP3下游蛋白MLKL,该蛋白也是TNF-/z-VAD和TRAIL/z-VAD诱导的细胞死亡。以上结果说明了TNF-诱导的海马细胞坏死是由CYLD-RIP1-RIP3-MLKL信号通路所介导发生的。（6）ROS以及钙离子通道被报道在一些细胞株中参与TNF-α诱导的细胞坏死，我们利用了两种ROS抑制剂（BHA和NAC）以及钙离子抑制剂（LaCl3）干预这些步骤，，发现这些抑制剂并不能有效阻止TNF-诱导的HT-22细胞坏死，表明TNF-诱导的海马神经细胞坏死不依赖于ROS以及钙离子通道。【结论】综上所述，这些结果不仅首次提供了RIP3在体内参与TNF-诱导的海马神经细胞毒性的证据，并且揭示了TNF-可诱导海马神经细胞发生CYLD-RIP1-RIP3-MLKL介导的细胞坏死，而且其下游信号不依赖于ROS和钙离子通道。
[Abstract]:[tumor] research background and significance of necrosis factor (TNF-) in the regulation of nerve inflammation is an important proinflammatory factor. In a variety of neurodegenerative diseases, such as Alzheimer's disease (Alzheimer's) and Parkinson's disease can be seen in the up regulation of TNF-. However, TNF- neurotoxicity induced by this pathway is still not clear.
[Objective] to determine whether RIP3 mediated programmed cell necrosis (also called necroptosis) is involved in the neurotoxicity induced by TNF- and TRAIL.
[Methods] wild type mice and RIP3 deficient mice were injected with different concentrations of PBS and tumor necrosis factor (TNF-), the expression of cell necrosis in hippocampus of mice hippocampus and brain cell injury after TNF- stimulation in the detection of key regulatory proteins in mouse brain slices stained by using; commonly used in the hippocampal neuronal cell line HT-22 study of TNF- induced cell death mechanism research, the first detection of HT-22 cells to TNF- induced apoptosis (conventional induction system for TNF- or TNF-Smac mimetic) and TNF- induced cell necrosis (conventional induction system TNF-/z-VAD) sensitivity, but also examined the sensitivity of cells to TNF family is another important factor induced by TRAIL the cell apoptosis and necrosis were silenced by RNA interference; related protein programmed cell death pathway, detection of eggs Effect of induction of TNF-/z-VAD or TRAIL/z-VAD white silence neurotoxicity; expression of proteins used by Western blotting after silencing the key protein; build a stable method using lentivirus infection expression of wild type RIP3 (WT), RIP3 kinase (RIP3-K51A) mutation and RIP homologous interaction domain mutations in RIP3 (RIP3-RHIM Mut RIP3) the key protein in HT-22 cell line detection in TNF-/z-VAD or TRAIL/z-VAD induced cell necrosis in action; using specific chemical inhibitors were the intervention of ROS and calcium ion channel, so as to detect these steps are involved in HT-22 cell necrosis induced by TNF-.
[results] (1) the mouse brain after injection of TNF-, found in the injury of hippocampus neurons in wild-type mice, and showed dose dependent, but there were no obvious damage of hippocampal cells in RIP3 deficient mice; Western blot using protein found significantly increased the expression of RIP3 protein and RIP1 injection of TNF- wild type of mouse hippocampus. These results suggest that RIP3- dependent cell necrosis in TNF- induced neurotoxicity in mouse hippocampus. (2) we chose the nerve cells in hippocampus of HT-22 mice for further study of molecular mechanism of TNF- induced hippocampal neurotoxicity. We found no sensitive system on the apoptosis of HT-22 cells induced by conventional TNF-, only in the the presence of apoptosis inhibitor z-VAD on TNF- showed sensitivity, also found only in the presence of z-VAD cells to TRAIL also showed the sensitivity that These indicate that HT-22 cells are more prone to TNF- and TRAIL induced cell necrosis but not apoptosis. (3) by RNAi interference technology to silence the expression of RIP3 protein, found in hippocampal neurons of RIP3 defects can inhibit TNF-/z-VAD and TRAIL/z-VAD induced cell death, indicating that TNF-/ z-VAD and TRAIL/z-VAD induced cell death is programmed cell necrosis RIP3 mediated silencing of RIP1. (4) further or CYLD (deubiquitinating enzyme RIP1), can also inhibit the cell death, also shows that the cell necrosis depends on RIP1 and CYLD. (5) then we silenced the RIP3 downstream protein MLKL, the protein is TNF-/z-VAD and TRAIL/z-VAD induced cell death. Results show that the hippocampus cell necrosis induced by TNF- is mediated by CYLD-RIP1-RIP3-MLKL signaling pathway occurs. (6) ROS and calcium ion channels are reported to participate in the TNF- in some cell lines Alpha induced cell necrosis, we use two kinds of ROS inhibitors (BHA and NAC) and calcium ion inhibitor (LaCl3) intervention in these steps, found that HT-22 cell necrosis and can not effectively prevent these inhibitors induced by TNF-, suggesting that TNF- induced hippocampal neuronal necrosis does not depend on the ROS and calcium ion channel.
[Conclusion] in conclusion, these results not only provide the first in vivo toxicity in RIP3 TNF- induced hippocampal neural cells and evidence reveals that TNF- could induce CYLD-RIP1-RIP3-MLKL mediated cell death of hippocampal neural cells and its downstream signal does not depend on ROS and calcium channels.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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