摘要
缺血性脑血管病是神经内科临床最常见的神经系统疾病，缺血性脑损伤是其重要的病理及病理生理过程。近年来，细胞信号转导通路参与缺血性脑损伤与修复的机制研究已受到广泛关注，越来越多的信号通路被发现可参与脑缺血损伤过程。
Notch1信号通路是一个在进化过程中高度保守的信号转导通路。有研究表明，Notch1信号可调控动脉生成、参与缺血脑损伤后神经元新生过程[1]。但Notch1信号通路在缺血性脑损伤过程中的变化及其作用机制尚不清楚。本研究在体外建立脑缺血损伤模型，探讨Notch1信号通路在脑缺血损伤中作用及机制，为进一步揭示Notch1信号转导通路与脑缺血损伤的联系和内在机制研究奠定实验基础，为脑缺血损伤的治疗提供理论依据和可能的治疗靶点。
1. PC12细胞转化为神经元样细胞的培养、鉴定及其氧糖剥夺模型的制备
目的：体外培养PC12细胞，诱导其转化为神经元样细胞，行氧糖剥夺（OGD），建立体外脑缺血损伤模型。
方法：体外培养PC12细胞，NGF(终浓度150ng/ml)刺激诱导其分化为神经元样细胞，应用无糖培养基及缺氧培养箱行OGD；应用MTT、流式细胞术及免疫荧光染色法行模型评价。
结果：终浓度150ng/ml NGF的DMEM完全培养基培养PC12细胞6d，细胞呈典型的神经元形态，MAP2免疫荧光细胞化学染色呈强阳。OGD后神经元出现不同程度的损伤、坏死及凋亡。随OGD时间延长，存活率逐渐降低、凋亡率升高。
结论：（1）NGF诱导的神经元样PC12细胞，具有神经元特性，可应用于建立神经元缺血损伤模型；（2）成功建立神经元样PC12细胞氧糖剥夺模型，该模型可稳定模拟体内的神经元缺血损伤，可重复性高。
2. Notch1-siRNA靶向沉默Notch1基因及沉默效果检测
目的：对神经元样PC12细胞靶向沉默Notch1，检测沉默效果，筛选最有效干扰序列、最佳转染浓度和最佳转染时间。
方法：以Notch1基因为靶目标，设计合成三条Notch1-siRNA序列，合成基因片段，lipofecamine~(TM)2000转染细胞，用Realtime-PCR和Western blot技术检测不同沉默序列、浓度及时间对Notch1mRNA及蛋白表达的影响。
结果：（1）Notch1-siRNA2组序列片段经lipofecamine~(TM)2000转染神经元样PC12细胞后，Notch1mRNA及蛋白表达较对照组明显降低（P<0.05）；（2）Notch1-siRNA2干扰序列组，转染浓度2μg/ml、转染时间48h Notch1mRNA及蛋白表达较对其他（序列/浓度/时间）组别明显降低（P<0.05）。
结论：Lipofecamine~(TM)2000可成功将Notch1-siRNA基因序列片段转染至神经元样PC12细胞，应用Notch1-siRNA2干扰序列、2μg/mL浓度行48h转染可最有效沉默Notch1。
3. Notch1信号通路在神经元样PC12细胞OGD损伤后表达变化及Notch1-siRNA对其影响
目的：检测Notch1信号通路在神经元样PC12细胞OGD损伤及Notch1-siRNA后表达变化，探讨Notch1信号转导通路在脑缺血损伤中的信号转导模式及机制。
方法：在体外神经元样PC12细胞OGD12h模型基础上，对其行Notch1-siRNA，Realtime-PCR技术检测Notch1mRNA表达变化，Western-blot技术检测Notch1及Notch1胞内段（NICD）的蛋白表达变化。
结果：（1）氧糖剥夺（OGD12h）组Notch1mRNA表达较正常对照组显著升高，氧糖剥夺+Notch1-siRNA组Notch1mRNA较氧糖剥夺组显著降低（P<0.05）；（2）氧糖剥夺组Notch1及NICD蛋白表达较正常对照组均显著升高，氧糖剥夺+Notch1-siRNA组较氧糖剥夺组显著降低（P<0.05）。
结论：脑缺血损伤可诱导内源性Notch1信号转导通路激活，脑缺血损伤诱导Notch1高表达可能为Notch1信号通路激活的始动因素。
4. Notch1-siRNA对神经元样PC12细胞缺血性损伤的影响及机制
目的：检测Notch1-siRNA对缺血神经元样细胞凋亡率及凋亡相关基因IAP-1、Caspase-3表达的影响，探讨神经细胞缺血性损伤诱导Notch1信号转导通路活性变化的内源性作用及可能机制。
方法：在体外神经元样PC12细胞OGD12h模型基础上，流式细胞术检测Notch1-siRNA对神经元样细胞缺血损伤细胞凋亡率的变化的影响，Realtime-PCR及Western-blot方法检测凋亡相关基因IAP-1、Caspase-3的表达变化。
结果：（1）氧糖剥夺+Notch1-siRNA组细胞凋亡率较单纯氧糖剥夺组显著升高（P<0.05）；（2）氧糖剥夺组较正常对照组IAP-1表达显著降低，Caspase-3显著升高（P<0.05）；（3）氧糖剥夺+Notch1-siRNA组较氧糖剥夺组IAP-1表达显著降低，Caspase-3显著升高（P<0.05）。
结论：缺血损伤诱导的内源性Notch1信号通路激活，可能在脑缺血的特定时程通过上调IAP-1、抑制Caspase-3的表达发挥抗凋亡脑保护作用。
Ischemic cerebrovascular disease is the most common clinical neurologicaldisease, and ischemic cerebral injury is its important pathological and pathophysiolo-gical processes. In recent years, studies on the mechanism of cell signal transductionpathways involving in ischemic brain damage and repair have received extensiveattentions, and more and more signal pathways were found to be involved in theprocess of cerebral ischemia injury.
The Notch1signal is a highly conserved signal transduction pathway in theevolution process. Research has shown that Notch1signal can regulate the generationof arteries and involve in the neonatal process of neurons after ischemic cerebralinjury[1], but it is still not clear as to the changes of Notch1signaling pathway and itsmechanism in the process of cerebral ischemic injury. This study established themodel of cerebral ischemia injury in vitro, explored the early effects and mechanismof Notch1signal pathway in cerebral ischemic injury, which lays a foundation for theresearch on the mechanism of Notch1signaling pathway in cerebral ischemic injury.Furthermore, the research revealed the relationship and internal mechanism betweenNotch1signal transduction pathway and cerebral ischemic injury, which may providetheoretical basis and therapeutic target for the therapy of cerebral ischemic injury.
1.Model of neuron-like PC12cells subjected to oxygen-glucose deprivation invitro
Objective: To induce and cultivate neuron-like PC12cells by NGF(150ng/ml)stimulation, perform oxygen and glucose deprivation (OGD), and build cerebralischemic injury model in vitro.
Methods: Sugar-free culture medium and hypoxia box were used to OGD; theOGD model was evaluated by MTT and immunofluorescence staining method.
Results: PC12cells were cultured6d by NGF with the final concentration of150 ng/ml, which manifested a typical neuronal morphology. MAP2immunofluorescencestaining showed strong positive. After OGD neurons manifested injury, necrosis andapoptosis in different degree. With the extension of OGD, the survival rate decreased,and apoptosis rate increased.
Conclusions:(1) Neuron-like PC12cells induced by NGF has the properties ofneurons, and can be applied to establish a model of neuron ischemia injury;(2)Successfully established the neuron-like PC12cells model of oxygen glucosedeprivation（OGD） model, the OGD model can be stable analogy ischemic neuronaldamage in vivo, and has high repeatability.
2. Notch1-siRNA silenced Notch1mRNA and detected silence effects
Objective: To silence targeting Notch1to neuron-like PC12cells, detect silenceeffects, and select the most efficient interfere sequence, the best transfectionconcentration and the best transfection time.
Methods: We took the Notch1gene as a target, designed and synthesized threesiRNA sequences, transfected cells by lipofecamine~(TM)2000, and detected the effectsof different silence sequences, different concentration and time on the expression ofNotch1mRNA and protein by Realtime-PCR and Western blot technology.
Results:(1) After Notch1-siRNA were transfected into neuron-like PC12cellsby lipofecamine~(TM)2000, the expression of Notch1mRNA and protein weresignificantly decreased than those in the control group (P<0.05);(2) The expression ofNotch1mRNA and protein decreased significantly (sequence/time/concentration) inNotch1-siRNA2group, transfection concentration2μg/ml group, transfection time48h group compared with other groups（P<0.05）.
Conclusion: Lipofecamine~(TM)2000can successfully transfect Notch1-siRNA intoneuron-like PC12cells, and the Notch1gene was most effectively silenced throughNotch1-siRNA2interference sequence and2μg/ml concentration and48h transfectiontime.
3. The expression changes of Notch1signaling pathway in neuron-like PC12cellssubjected to OGD damage and the impact of Notch1-siRNA
Objective: To detect the expression changes of Notch1signaling pathway inneuron-like PC12cells subjected to OGD and Notch1-siRNA injury, and explore themode and mechanism of Notch1signal transduction pathway in cerebral ischemic injury.
Methods: On the basis of neuron-like PC12cells subjected to OGD12h model invitro, We treated it with Notch1siRNA, detected the expression of Notch1mRNA byRealtime-PCR technology, and detected the expression of Notch1and Notch1intracellular domain (NICD) protein by Western-blot technology.
Results:(1) The expression of Notch1mRNA was significantly increased inOGD group when compared with the control group, and was dramatically decreasedin OGD+Notch1-siRNA group when compared with OGD group（P<0.05）;(2) Theexpression of Notch1and NICD protein was significantly increased in OGD groupwhen compared with the control group, and was dramatically decreased inOGD+Notch1-siRNA group when compared with OGD group（P<0.05）.
Conclusion: Cerebral ischemic injury can induce the activation of endogenousNotch1signaling pathway, and the high expression of Notch1induced by cerebralischemic injury may be initiating factors for the activation of Notch1signalingpathway.
4. Effects and mechanism of Notch1-siRNA on neuron-like PC12cells subjectedto ischemic injury
Objective: To detect the effects of Notch1-siRNA on ischemia-induced nervecells apoptosis and apoptosis-related gene IAP-1and Caspase-3expression, and toexplore endogenous effects and possible mechanism of Notch1signal pathwayliveness induced by the nerve cells subjected to ischemic injury.
Methods: FCM was used to identify apoptosis rate. Realtime-PCR and Westernblot were used to detect the expression changes of the apoptosis related genes IAP-1and Caspase-3.
Results:(1)The apoptosis rate was significantly increased in Notch1-siRNA+OGDgroup when compared with OGD group (P<0.05);(2) The expression of IAP-1mRNAand protein was significantly decreased and Caspase-3was significantly increased inOGD group when compared with the control group（P<0.05）;(3) The expression ofIAP-1mRNA and protein was significantly decreased and Caspase-3was significantlyincreased in OGD+Notch1-siRNA group when compared with OGD group（P<0.05）.
Conclusions: The activation of endogenous Notch1signaling pathway induced byischemia may be exert anti-apoptotic neuron-protective effects in the specific process of the cerebral ischemia through the up-regulation of IAP-1and inhibition ofCaspase-3expression.
引文
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