【作者】 梅春丽；

【导师】 徐忠信；

【作者基本信息】 吉林大学 ， 神经病学， 2012， 博士

【摘要】 缺血性脑卒中是危害人类健康的严重疾病，其防治方法和发病机制一直是国内外研究的热点。缺血性脑损伤可发生复杂的信号转导通路的变化同时产生一定的生物学作用，因此，阐明与缺血性脑损伤有关的信号转导通路的变化，寻找通路中的关键作用靶点和可应用于临床的神经保护物质，进而在神经细胞缺血损伤后促进神经细胞功能的恢复，对缺血性脑卒中的治疗具有重要意义。Smad7属于I-Smad家族，在TGF-β信号通路中发挥抑制作用。Smad7是TGF-β作用的靶基因之一，可反馈调节TGF-β/Smad信号通路，维持该通路的平衡，Smad7能够与R-Smad（Smad2、Smad3）竞争性地结合被激活的TGF-β受体，从而阻止Smad2、Smad3与TGF-β受体结合及其随后的磷酸化过程。目前研究表明，Smad7蛋白的高表达与胰腺癌、肝癌密切相关，还有研究表明转染Smad7基因可明显抑制肾间质纤维化的进程，另有研究表明，TGF-β超家族成员激活素A（ActA）/Smads通路的激活具有神经保护作用。Smad7作为一种信号蛋白在中枢神经系统存在其表达，Smad7在缺血性脑损伤中的变化规律和作用尚不明确，国内外未见报道。本研究通过体外实验，探讨了Smad7对脑缺血损伤的作用和对ActA/Smads信号转导通路的影响。首先在体外建立由PC12细胞诱导的神经细胞氧糖剥夺（OGD）模型，同时靶向沉默Smad7，应用免疫荧光、Real time PCR和Westernblot技术对沉默效果进行检测，并应用Real time PCR，Western blot技术检测Smad7-siRNA对神经细胞缺血损伤时ActA/Smads信号转导通路的影响，应用DAPI、FCM、Western blot等技术检测并观察Smad7-siRNA对神经细胞缺血损伤细胞凋亡的影响。结果显示，PC12成功诱导分化为神经细胞，OGD模型成功制备；靶向沉默Smad7，缺血神经细胞中ActA表达增加，其下游转录共激活因子p300表达增加，ActA/Smads通路激活；Smad7-siRNA后神经细胞缺血损伤细胞凋亡减少。提示Smad7在脑缺血损伤中具有重要作用，Smad7-siRNA可增强ActA/Smads信号转导通路，降低神经细胞缺血损伤的细胞凋亡率。本研究首次探讨了Smad7在神经细胞缺血损伤中的作用及分子机制，为研究ActA/Smads信号转导通路在脑缺血损伤中的分子机制奠定基础，ActA/Smads信号转导通路在脑缺血损伤中具有重要作用，通路中主要位点及其调节蛋白可能成为缺血脑损伤机制研究和治疗的新靶点。1. PC12细胞诱导的神经细胞OGD模型的制备和鉴定目的：制备神经细胞OGD模型，为后续实验奠定基础。方法：利用PC12细胞，经NGF（100ng/mL）刺激诱导1，2，3，4，5，6d后向神经细胞分化，6d后对分化细胞进行OGD（5%CO2and95%N2，1mmol/LNa2S2O4in sugar-free DMEM)3，6，9，12，16，24h，建立神经细胞OGD模型。采用形态学，免疫荧光，免疫印迹，FCM，DAPI等技术对此模型进行鉴定。结果：NGF（100ng/mL）体外诱导PC12细胞分化为神经细胞，MAP2蛋白表达呈动态变化；在体外成功建立神经细胞的OGD模型，细胞凋亡率和HIF-1基因表达随OGD时间呈动态变化。结论：NGF（100ng/mL）诱导PC12细胞6d，OGD（5%CO2and95%N2，1mmol/L Na2S2O4in sugar-free DMEM)16h可建立神经细胞OGD模型。2.靶向沉默Smad7基因及沉默效果的检测目的：对PC12细胞分化的神经细胞靶向沉默Smad7，同时进行沉默效果检测，筛选出最有效干扰序列，测试出最佳转染浓度和最佳的作用时间。方法：以Smad7基因为靶目标，设计合成3条siRNA序列，进行细胞转染，利用Real time-PCR和Western blot技术检测沉默效果，筛选出最有效的干扰序列，同时检测出最佳的转染浓度和转染时间。结果：针对Smad7基因设计合成及筛选靶向沉默Smad7基因的干扰序列（siRNA1）；siRNA1的最佳转染浓度是4μg/mL；siRNA1的最佳转染时间是24h；siRNA1对Smad7的抑制效果优于其它干扰序列。结论：siRNA1能有效沉默Smad7基因；lipofecta-mineTM2000可成功将siRNA1转染至神经细胞，转染效率较高，利用siRNA技术能有效抑制神经细胞Smad7基因表达。3. Smad7-siRNA对ActA/Smads信号转导通路的影响目的：探讨Smad7基因对ActA/Smads信号转导通路的影响。方法：在细胞培养，细胞分化6d，细胞转染（Smad7-siRNA）和OGD16h的基础上，采用Real time-PCR和Westhern blot技术检测ActA/Smads信号转导通路中上游信号蛋白ActA和下游信号蛋白转录共激活因子p300mRNA水平和蛋白水平的表达变化情况，观察Smad7基因对ActA/Smads信号转导通路的影响。结果：神经细胞缺血损伤后ActA，p300表达增加；Smad7-siRNA，神经细胞缺血损伤后ActA，p300蛋白因子表达进一步增加；Smad7-siRNA，可以增强ActA/Smads信号转导通路。结论：Smad7-siRNA后激活缺血神经细胞的ActA/Smads信号转导通路，其上游信号蛋白ActA和下游信号蛋白转录共激活因子p300表达均增加。4. Smad7-siRNA对神经细胞缺血细胞凋亡的影响目的：探讨Smad7基因对神经细胞缺血损伤细胞凋亡的影响。方法：在细胞培养、细胞分化6d、细胞转染（Smad7-siRNA）、OGD16h的基础上，采用FCM、DAPI、免疫印迹方法观察Smad7-siRNA对神经细胞缺血损伤后细胞凋亡率的变化、凋亡小体的变化和活化前凋亡蛋白（Procaspase-3）蛋白表达的变化，探讨Smad7基因对神经细胞缺血损伤细胞凋亡的影响。结果：Smad7-siRNA可以降低神经细胞缺血损伤的细胞凋亡率；Smad7-siRNA可上调缺血性神经细胞中的Procaspase-3蛋白表达。结论：Smad7-siRNA可通过Smads信号转导通路，影响细胞凋亡及凋亡调控基因的表达对脑缺血损伤具有保护作用。更多还原

【Abstract】 Ischemic cerebrovascular disease, such as cerebral thrombosis, cerebral infaction,and so on, are the most important causes of morbidity, and the third most commoncause of death in elderly patients in the world. Effective methods of preventing andcontrolling ischemic cerebrovascular disease have been a topic of great interest.Ischemic damage of nerve cells leads to a series of complex signaling pathways thatproduce corresponding biological functions. A better understanding of the role of thesignal transduction mechanisms that underlie brain ischemic injury could identify keytargets for neuroprotective substances.Ischemic stroke occurs when the blood supply to the brain is obstructed.Accumulating evidence suggests that the cell death observed during the first fewhours of cerebellar ischemia is a result of apoptosis as opposed to necrosis, which wasconsidered the predominant form of cerebellar damage generated by ischemia.Moreover, Effective methods of preventing and controlling ischemic cerebrovasculardisease have been a topic of great interest. The ischemic damage of nerve cells leadsto the disruption of a series of complex signaling pathways that produces an effect oncorresponding biological functions and affects the function of the brain. A betterunderstanding of the role of the signal transduction mechanisms that underlie brainischemic injury could identify key targets for neuroprotective substances. Some datasuggest that Smad7can also have proinflammatory effects. Smad7overexpression hasbeen noted in mucosa and mucosal T cells from patients with inflammatory boweldisease, and blockade of Smad7restores TGF-β signaling and reduces proinflammatory cytokine generation in this context. Overexpression of Smad7in mouse Tcells promotes airway hyperreactivity and enhances airway inflammation, whereasinhibition of Smad7suppresses autoimmune encephalitis. Thus, it appears that therole of Smad7in the regulation of inflammation and immunity is complex, is possiblydependent on cell lineage, context, and duration of inflammatory response, and needsto be more completely defined before overexpression of Smad7really becomes viableas a therapeutic goal. For expounding Actin/Smads pathways about ischemic brain injury and signaltransduction mechanism and looking for nerve protective substances that can beapplied to clinical practise and key targets of Actin/Smads pathways. A stable in vitroneuronal ischemia model had been established. Nerve growth factor (NGF) caninduce the differentiation of PC12cells into neuron-like cells. The sympatheticneuron-like cells are characterized by electrical excitability, expression of neuronspecific genes and neurite outgrowth. PC12cells were cultured in different oxygenconditions. The metabolic activity of PC12cells was measured in the final4h prior tocellular characterization using an alamar blue assay following the manufacturer’sinstructions. A neuronal ischemia model was conducted on research the molecularlevel.The experiment small interference targeting Smad7on in vitro neuronal ischemiamodel was taken and observed the changes of cell apoptosis. For study the role ofSmad7on stroke and signal transduction mechanism. And look for nerve protectivesubstances that can be applied for clinic in the furture and key targets in ActA/smadspathways. Protecting and promoting neurological recovery, it will bring the new hopefor the treatment of ischemic cerebrovascular disease.1. NGF combined with OGD induces neural ischemia tolerance in PC12cellsObjective: Ischemic cerebrovascular disease is a major disease in humans. Tobetter study this disease, a good ischemia model of nerve cells is needed.Methods: NGF（100ng/mL）and OGD（5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM) were used to stimulate PC12cells and converted them intoneurons in order to establish an ischemia model. After stimulation with NGF (100ng/mL for6d), PC12cells show a neuron-like function as measured by physiologyand biochemistry. After6d of NGF stimulation, we performed OGD（5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM) treatment for16h to establish anoxygen glucose deprivation model.Results: PC12cells transformed into cells that looked like neurons and thatMAP2was up-regulated in NGF-treated PC12cells. Cell apoptosis was found to beup-regulated after NGF（100ng/mL）stimulation and OGD (5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM for16h). A western blot analysis showed that OGD treatment increased the expression of HIF-1. The apoptosis rate after16hof OGD was19.44%.Conclusions: NGF（100ng/mL）treatment can be combined with OGD(5%CO2and95%N2,1mmol/L Na2S2O4in sugar-free DMEM for16h) to establish an in vitromodel of acute ischemic brain damage.2. Effects of RNA interference targeting Smad7on nerve cells ischemic injuryinduced in PC12cellsObjective: In order to further illustrate the role of Smad7genes in ischemiccerebral injury.Methods: Real time-PCR and Western blot were used to determinate the mRNAand protein expression of Smad7by Smad7-siRNA.Results: Smad7-siRNA can be translated into PC12cells by LipofectamineTM2000. The efficiency was higher.Conclusions: LipofectamineTM2000can be used in Smad7-siRNA in PC12cells.Smad7-siRNA can effectively silence Smad7gene expression. The experiment smallinterference targeting Smad7is an effect method in researching signal transductionpathways.3. Effects of RNA interference targeting Smad7on ActA/Smads signallingpathwayObjective: This study was mainly discussed the protein expression levels ofActA and p300in ischemic cerebral injury by targeted silence Smad7.Methods: Real time-PCR was used to detect the mRNA of ActA and p300.Western blot was used to detect the protein expression levels of ActA and p300.Results: The mRNA of ActA and p300were higher by Smad7-siRNA.Theprotein expression of ActA and p300were also higher by Smad7-siRNA in ischemiccerebral injury.Conclusions: The results showed that Smad7-siRNA could sitimute theActA/smads signaling pathways on acute ischemic injury.4. The apoptosis change by Smad7-siRNA on nerve cells ischemic injuryObjective: This experiment is mainly discussed the apoptosis in ischemiccerebral injury by targeted silence Smad7. Methods: FCM and DAPI were used to identify apoptosis rate. Western blot wasused to detect Procaspase-3protein expression.Results: The results showed that OGD16h apoptosis rate is19.54%. OGD16hcombined Smad7-siRNA apoptosis rate is12.34%.Conclusions: The results showed that the apoptosis rate is decreasing inischemic injury by Smad7-siRNA. At the same time, it provide the reference forfurther study ActA/Smads signaling pathways on acute ischemic injury.更多还原