NF-κB圈套策略抗大脑皮质神经元氧糖剥夺/复氧后炎性损伤及其机制的研究
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摘要
背景
缺血性脑血管病是最常见的危及生命的神经疾病,是继心脏和肿瘤之后的第三位致死性疾病,并且是老年人当中最主要的致残性疾病。目前国内外对该病作了大量实验与临床研究,使人们对脑缺血疾病的病理生理机制认识得到了不断深化,并且研发了大量新药,这给神经保护带来了希望,但是令人失望的是当这些神经保护药物应用于临床时其结果大多无效或因不良反应而被提前终止。目前t-PA是唯一一个被FDA批准的用于人类脑缺血治疗的药物,但众所周知t-PA治疗时间窗窄,对很大一部分脑缺血患者并不适用,另外一方面,溶栓再灌注时的炎症反应可能加剧继发性脑损害。
脑缺血/再灌注后的炎症反应是一把双刃剑,炎症反应的时间、炎症因子的类型、以及炎症反应的程度都决定了其对神经细胞的利弊。脑缺血/再灌注后大量巨噬细胞以及中性粒细胞的增加可以损伤血管内皮细胞导致神经细胞死亡;另一方面,小胶质细胞的活化有利于清除坏死组织、补体系统的激活可以启动局部炎症反应,增强神经元的存活或组织修复,但是不适当的炎症反应可加剧神经细胞的损伤。
神经元具有感受体内、外刺激,传导冲动和整合信息的能力,是神经系统的形态和功能单位。神经元数量庞大,约有10~(11)个,它们通过突触彼此连接,形成复杂的神经网络和通路。脑损伤等多种神经疾病均涉及到神经元的损伤,尤其在脑缺血时,在缺血中心区和半影区存在大量的神经元变性、死亡。神经元作为神经功能的主要承载者和体现者,保护其结构和功能是十分重要的。目前,脑缺血/再灌注后神经元损伤机制的研究主要集中在氧化应激、兴奋性氨基酸、细胞内钙超载、炎症反应和凋亡等,其中炎症反应是神经元凋亡的主要促发因素。因此,适当调控炎症因子表达,保护神经细胞的功能显得尤为重要。脑缺血/再灌注情况下,核因子NF-κB可以激活一系列细胞因子、酶类介质参与炎症反应,NF-κB在神经元存活与死亡过程中起着重要作用。研究表明,NF-κB可以通过激活大量的炎症因子促进神经元死亡,也可调控某些抗凋亡因子增强神经元的存活。因此适当调控NF-κB活化成为国内外研究的热点。
研究表明,NF-κB亚基二聚体对靶基因的DNA有不同亲合力,其不同的结合位点可以作用于不同靶基因的启动子,调控各种下游基因的表达。NF-κB对神经细胞的双重作用可能与不同的实验模型、不同的细胞组织类型有关,也与NF-κB不同亚基调控的不同下游因子表达关系密切。基于以上原理,人工合成不同的NF-κB圈套寡核苷酸(NF-κBdecoy oligodeoxynucleiotides即NF-κB decoy ODNs)——其是含有NF-κB不同结合位点的双螺旋核苷酸,当NF-κB从IκB复合物中解离出来以后NF-κB decoy ODNs即可与NF-κB上的不同部位结合,封闭NF-κB不同亚基活化,特异性地阻断下游因子的表达,因此圈套策略以其特异性及高效性有着其他策略不可比拟的优势成为一种新型基因治疗方案。
目的
1、探讨大鼠大脑皮质神经元的培养方法并对之进行形态学观察和鉴定;
2、建立一种方便、稳定、可靠的体外氧糖剥夺(oxygenglucosedeprivation,OGD)/复氧(reoxygenation)模型;
3、通过该模型,研究大脑皮质神经元在氧糖剥夺/复氧后NF-κB亚基P65、P50和c-Rel蛋白的表达、活化规律;
4、观察两种不同NF-κB decoy ODNs转染大脑皮质神经元在氧糖剥夺/复氧条件下对NF-κB亚基P65、P50和c-Rel蛋白的影响以及对下游因子TNF-α、IL-1α、Bcl-x_L mRNA及蛋白的影响,进一步探讨不同NF-κB亚基在大脑皮质神经元氧糖剥夺/复氧中的作用。
方法
1、取Wistar大鼠乳鼠脑组织,采用机械分离方法获取大脑皮质神经元并进行培养,通过倒置显微镜、免疫细胞化学进行鉴定;
2、采用细胞培养、MTT法、酶学检查、光学显微镜评价氧糖剥夺模型;
3、采用免疫细胞化学和Western Blot法检测大脑皮质神经元在氧糖剥夺/复氧后不同时间点NF-κB亚基P65、P50和c-Rel蛋白的动态表达规律;
4、大鼠大脑皮质神经元在氧糖剥夺、氧糖剥夺/复氧条件下转染不同的NF-κB哑铃形圈套IgG-κB decoy ODNs和Bcl-x decoy ODNs、杂合圈套后,采用Western Blot法检测不同组别NF-κB亚基P65、P50和c-Rel蛋白的表达,采用ELISA和RT-PCR法检测神经元TNF-α、IL-1α、Bcl-x_L mRNA及蛋白表达的水平。
结果
1、经形态学、免疫细胞化学鉴定所培养的细胞为大脑皮质神经元;
2、氧糖剥夺后大脑皮质神经元形态变化显著、LDH释放量、MTT检测显示:随着氧糖剥夺时间延长,氧糖剥夺前后神经元形态结构发生显著变化,神经元活性降低,MTT吸光度值逐渐降低,培养液中LDH释放逐渐增加,氧糖剥夺1h组、2h组与正常组无显著性差异(P＞0.05),氧糖剥夺4h组与正常组有显著性差异(P＜0.05)。
3、免疫细胞化学、Western Blot结果显示:正常组细胞内只有少量P65、P50及c-Rel蛋白表达,OGD4h组NF-κB P65和P50蛋白表达开始增加明显高于正常组(P＜0.05),OGD4h/2h组、OGD4h/6h组NF-κBP65和P50蛋白表达持续性升高,并在复氧后6h达高峰(P＜0.01),此后逐渐下降,至OGD4h/12h时与正常组有显著差异(P＜0.05),OGD4h/24h NF-κB P65和P50蛋白表达降低与正常组比较无统计学差异(P＞0.05);NF-κB亚基c-Rel蛋白表达在OGD4h组与正常组无差异(P＞0.05),OGD4h/R2h至OGD4h/R12h表达持续增加(P＞0.05),并在复氧后12h达高峰(P＜0.01),并且在OGD4h/24h时c-Rel蛋白表达仍未恢复到正常水平(P＞0.05)。
4、大鼠大脑皮质神经元在转染不同的NF-κB哑铃形圈套IgG-κBdecoy ODNs和Bcl-x decoy ODNs、杂合圈套、脂质体后,在氧糖剥夺、氧糖剥夺/复氧条件下,Western Blot法和免疫细胞化学结果显示:转染IgG-κB decoy ODNs后NF-κB亚基P65、P50蛋白表达显著降低(P＜0.05),而c-Rel蛋白的表达与OGD4h/R6h相比无差异(P＞0.05):转染Bcl-x decoy ODNs后NF-κB亚基P50、c-Rel蛋白表达较OGD4h/R6h组显著降低(P＜0.05),而P65蛋白的表达与OGD4h/R6h相比无显著改变(P＞0.05);脂质体组、无关decoy ODNs组对NF-κBP65、P50及c-Rel蛋白表达未见抑制(P＞0.05)。
5、RT-PCR和ELISA显示:氧糖剥夺/复氧后大脑皮质神经元内TNF-α、IL-1α、Bcl-x_L蛋白和mRNA表达明显升高(P＜0.05),转染IgG-κB decoy ODNs可显著抑制NF-κB下游基因IL-1a、TNF-a蛋白和mRNA表达(IgG-κB decoy ODNs组vs OGD4h/R6h,P＜0.05),对Bcl-xLmRNA表达未见抑制作用(P＞0.05);而Bcl-x decoy ODNs可显著抑制IL-1a、Bcl-xL蛋白和mRNA表达(Bcl-x decoy ODNs组vs OGD4h/R6h,P＜0.05),对TNF-a mRNA表达未见抑制作用(P＞0.05);而脂质体组、无关decoy ODNs组对IL-1a、TNF-a、Bcl-xL蛋白和mRNA表达未见抑制作用(P＞0.05)。
结论
1、我们成功培养出了原代大脑皮质神经元,该方法的建立为体外研究缺血性脑血管疾病提供了帮助;
2、建立了一种有效、方便、实用的体外模拟缺血性损伤模型,为神经元缺血性损伤机制的研究及临床治疗奠定基础;
3、正常情况下,大脑皮质神经元存在少量NF-κB P65、P50和c-Rel蛋白表达,免疫细胞化学和Western Blot显示氧糖剥夺/复氧后大脑皮质神经元中NF-κB亚基P65、P50和c-Rel蛋白表达增加,NF-κB P65和P50蛋白在氧糖剥夺4h开始增加,复氧后2-6h呈持续增加过程,在复氧6h表达到达高峰,延迟到复氧后12h与正常组比较仍有显著差异,24h时回复到正常水平。NF-κB c-Rel蛋白在复氧后2h才开始增加,并延迟到复氧后12h达高峰,其在复氧后24h表达仍高,与正常组比较有统计学意义。实验结果表明,NF-κB P65、P50和c-Rel蛋白在不同时间点表达规律存在差异,提示NF-κB亚基P65和P50可能在脑缺血再灌注后神经元内早、中期发挥作用,而NF-κB亚基P50和c-Rel主要在中晚期发挥重要作用,两者在时间上有交叉重叠;
4、氧糖剥夺/复氧可以诱导大脑皮质神经元大量表达IL-1a、TNF-a、Bcl-xL蛋白和mRNA,IgG-κB decoy ODNs可以显著抑制炎症因子IL-1a、TNF-a蛋白和mRNA表达,对Bcl-xL蛋白和mRNA表达无抑制作用;Bcl-x decoy ODNs可以抑制IL-1a、Bcl-xL蛋白和mRNA表达,对TNF-a蛋白和mRNA表达无显著抑制。
Backgroud
Cerebral ischemia is the most life-threatening neurological disease,and is the third leading cause of death after heart disease and cancer,and in the elderly it is a leading cause of long-term disability.Many studies have been done for the disease both at home and board,which gradually deepens the comprehension of the ischemic injury.Howere,an ideal curative effect has not been achieved as yet.So it is necessary to research for a new and a stronger pointed therapy strategy.
Now,the thrombolytic compound tissue plasminogen activator(t-PA) is the only Food and Drug Administration-approved agent for cerebral ischemia therapeutic window and is not suitable for many cerebral ischemia, in the other hand,many studies had showed that the inflammation response during reperfusion speeds the secondary brain damages after thromobolysis and play a key role in role in I/R.
Inflammation acts as a double-edged sword in I/R.The timming,type of inflammatory mediator and the extent of its stimulation determines the balance between the bad versus good effects of inflammation.Uncontrolled numbers of the infiltrated macrophages and neutrophils increases free radical generation which is detrimental to vascular integrity leading to neuron death.Howere,microglial responses after ischemia is necessary for scavenging the necrotic debris,and the complement system that gets activated in post-ischemic brain initiates local inflammatory responses that ultimately contribute to neuronal survival and tissue remodeling.Howere, complement activation to an inappropriate extent promotes inflammatory mediator release and tissue injury.
Brain ischemia/reperfusion has relationship with many factor,such as free radicals,the excitotoxicity,calcium over-loading,the dysfunction of mitchondria,inflammation and apotosis,among them,inflammation is a known precipitator of neuronal death after cerebral ischemia.Neurons' injury and dysfunction is a key event early in the pathogenesis of cerebrovascular disease,so it is important to protect the function and structure of neurons from injury.Degradation ofⅠκB induces NF-κB activation leading to the coordinated induction of multiple genes is involved in many inflammatory and immune cascades in I/R.NF-κB play a dynamic role in the survival and death of neuron in I/R.Much research has established that the activation of NF-κB enhances neuronal survival by preventing apoptosis.In contrast to the anti-apoptosis action of NF-κB,controversial results have appeared regarding the causative role of NF-κB to excitotoxicity.
NF-κB dimer species exhibit different binding affinities to the different DNA sequences,the different protein-DNA complexes act on different gene promoters with different NF-κB binding sites.All of these differing conclusions were drawn from different experiment models including cell lines and tissues under different stimuli.NF-κB decoy strategy has been a powerful tool for gene therapy and gene regulation in the study of transcriptional rehulation.NF-κB decoy ODNs in dumbbell shape with the nuclear sequence which was similar to NF-κB cis-elements were synthetized in our study.Oliogodeoxynucleotides with high affinity for transcription factors may be used to bind the transcription factors and block the activation of target gene.
Objectives
(1) To culture and observe the morphology of primary cortical neurons, the cells were affirmed.
(2) To establish a simple,stable and reliable model of oxygen glucose deprivation/reoxygenation(OGD/R) in primary cortical neurons.
(3) To investigate the protein expression and the activation of NF-κB P65,P50 and c-Rel following OGD/R of primary cortical neurons in vitro.
(4) To study the therapeutic effects of two different decoys:IgG-κB decoy ODNs and Bcl-x decoy ODNs on NF-κB downstream genes TNF-α, Bcl-xL,IL-1αand cell protection in primary cultured cortical neurons in an oxygen glucose deprivation(OGD) model
Methods
(1) Primary neuronal cultures of cerebral cortex were obtained from neonatal Wistar rat(＜24h),and neurons were affirmed by immunocytochemical method,morphologies under Light Microscope.
(2) Primary neuronal cultures of cerebral cortex were exposed to oxygen glucose deprivation(OGD) for various time(control group,OGD2h group,OGD4h group,OGD6h group),The neuronal injury was detected by the lactate dehydrogenase(LDH) relase and the tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H tetrazolium bromide(MTT), and morphologic changes were studied under Light Microscopeto evaluate model of oxygen/glucose deprivation.
(3) The protein level of NF-κB subunit P65,P50 and c-Rel in primary neuron was detected by Western Blot and immunocytochemistry after OGD/R
(4) We applied different NF-κB decoy ODNs(IgG-κB decoy ODNs, Bcl-x decoy and scramble decoy ODNs) into primary cotical neurons,and then Western Blotting was used to detect the expression of NF-κB P65,P50 and c-Rel protein in neurons.The protein level of TNF-α,IL-1αin primary neuron was detected by ELISA,and the expression of TNF-αBcl-xL,IL-1αmRNA was detected by RT-PCR.
Results
(1) The characterization of primary cortical neuron was comfirmed based on the morphology and positive by the detetion of NSE andβ-tubulin 3.
(2) The MTT and the LDH showed primary corticai neurons activity decreased and changed morphology and the mount of LDH release from injured neurons was increased significantly in OGD 4h group than in control group(P＜0.05).
(3) The result of immunocytochemical method and Western Blotting indicated that the expression of P65,P50 protein gradually increased,there was significant different between OGD4h and control group(P＜0.05),and it was significantly highest on the OGD4h/R6h(P＜0.01).The expression of c-Rel protein begin increased on OGD4h/R2h,and it was significantly highest on the OGD4h/R12h(P＜0.05),and then it gradually decreased,but was still higher than that of the control group(P＜0.05).
(4) We applied different NF-κB decoy ODNs(IgG-κBdecoy ODNs, Bcl-x decoy and scramble decoy ODNs into primary cotical neurons.The result of immunocytochemical method and Western Blot showed that IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein (P＜0.05),but no effect on c-Rel(P＞0.05),while Bcl-x decoy reduced the expression of NF-κB c-Rel and P50 protein(P＜0.05) but no effect on P65, scrambled decoy ODNs and Reagent have no effect on NF-κB subunit (P＞0.05).
(5) The result of RT-PCR and ELISA showed that the mRNA and protein levels of TNF-α,Bcl-xL,IL-1αwere markedly elevated after OGD (P＜0.05),IgG-κB decoy ODNs decreased TNF-α,IL-1αmRNA and protein expressions in neurons(P＜0.05),but no effect on Bcl-xL mRNA (P＞0.05),Bcl-xL decoy ODNs suppressed Bcl-xL,IL-1αmRNA and protein expressions(P＜0.05),but no effect on TNF-αmRNA and protein (P＞0.05),scrambled decoy ODNs and Reagent have no effect on IL-1a、TNF-a、Bcl-xL protein and mRNA(P＞0.05)
Conclusions
(1) We isolated and cultured primary cortical neuron in vitro successfully.The method provided a useful way to study the cerebrovascular diseases in viro.
(2) In vitro cerebral ischemia/reperfusion model may be induced easily by OGD in cultured neurons may be used to further study the mechanism of ischemic neuronal cell injury and its protection.
(3) There are a small quantity of NF-κB P65,P50 and c-Rel protein expressions in control group,western Blotting and immune cytochemistry has shown that the NF-κB P65,P50 and c-Rel protein expressions increased after oxygen glucose deprivation/reoxygenation.The NF-κB P65,P50 protein begins to increase at 4h after oxygen/glucose deprivatio and keeps increasing at 2-6h after reoxygenation,reaches the summit at 6h after reoxygenation,and returns to the normal level at 24h,while the NF-κB c-Rel protein begins to increase at 2h after reoxygenation,reaches the summit at 12h after reoxygenation and still has a high expression at 24h after reoxygenation.The experimental results reveal that the expression pattern of NF-κB P65,P50 and c-Rel protein varies with different time points,which indicates that NF-κB P65,P50 may have an effect on the neuron in the early and middle stage after the cerebral ischemia reperfusion and NF-κB c-Rel primarily plays an important role in the middle and late stage.
(4) OGD induced TNF-α,Bcl-xL,IL-1αmRNA and protein expression in primary cortical neurons.IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein,decreased TNF-α,IL-1αand Bcl-xL mRNA and protein expressions in neurons,while Bcl-x decoy ODNs reduced the expression of NF-κB c-Rel and P50 protein,suppressed Bcl-xL,IL-1αmRNA and protein.
缺血性脑血管病是最常见的危及生命的神经疾病,是继心脏和肿瘤之后的第三位致死性疾病,并且是老年人当中最主要的致残性疾病。目前国内外对该病作了大量实验与临床研究,使人们对脑缺血疾病的病理生理机制认识得到了不断深化,并且研发了大量新药,这给神经保护带来了希望,但是令人失望的是当这些神经保护药物应用于临床时其结果大多无效或因不良反应而被提前终止。目前t-PA是唯一一个被FDA批准的用于人类脑缺血治疗的药物,但众所周知t-PA治疗时间窗窄,对很大一部分脑缺血患者并不适用,另外一方面,溶栓再灌注时的炎症反应可能加剧继发性脑损害。
脑缺血/再灌注后的炎症反应是一把双刃剑,炎症反应的时间、炎症因子的类型、以及炎症反应的程度都决定了其对神经细胞的利弊。脑缺血/再灌注后大量巨噬细胞以及中性粒细胞的增加可以损伤血管内皮细胞导致神经细胞死亡;另一方面,小胶质细胞的活化有利于清除坏死组织、补体系统的激活可以启动局部炎症反应,增强神经元的存活或组织修复,但是不适当的炎症反应可加剧神经细胞的损伤。
神经元具有感受体内、外刺激,传导冲动和整合信息的能力,是神经系统的形态和功能单位。神经元数量庞大,约有10~(11)个,它们通过突触彼此连接,形成复杂的神经网络和通路。脑损伤等多种神经疾病均涉及到神经元的损伤,尤其在脑缺血时,在缺血中心区和半影区存在大量的神经元变性、死亡。神经元作为神经功能的主要承载者和体现者,保护其结构和功能是十分重要的。目前,脑缺血/再灌注后神经元损伤机制的研究主要集中在氧化应激、兴奋性氨基酸、细胞内钙超载、炎症反应和凋亡等,其中炎症反应是神经元凋亡的主要促发因素。因此,适当调控炎症因子表达,保护神经细胞的功能显得尤为重要。脑缺血/再灌注情况下,核因子NF-κB可以激活一系列细胞因子、酶类介质参与炎症反应,NF-κB在神经元存活与死亡过程中起着重要作用。研究表明,NF-κB可以通过激活大量的炎症因子促进神经元死亡,也可调控某些抗凋亡因子增强神经元的存活。因此适当调控NF-κB活化成为国内外研究的热点。
研究表明,NF-κB亚基二聚体对靶基因的DNA有不同亲合力,其不同的结合位点可以作用于不同靶基因的启动子,调控各种下游基因的表达。NF-κB对神经细胞的双重作用可能与不同的实验模型、不同的细胞组织类型有关,也与NF-κB不同亚基调控的不同下游因子表达关系密切。基于以上原理,人工合成不同的NF-κB圈套寡核苷酸(NF-κBdecoy oligodeoxynucleiotides即NF-κB decoy ODNs)——其是含有NF-κB不同结合位点的双螺旋核苷酸,当NF-κB从IκB复合物中解离出来以后NF-κB decoy ODNs即可与NF-κB上的不同部位结合,封闭NF-κB不同亚基活化,特异性地阻断下游因子的表达,因此圈套策略以其特异性及高效性有着其他策略不可比拟的优势成为一种新型基因治疗方案。
目的
1、探讨大鼠大脑皮质神经元的培养方法并对之进行形态学观察和鉴定;
2、建立一种方便、稳定、可靠的体外氧糖剥夺(oxygenglucosedeprivation,OGD)/复氧(reoxygenation)模型;
3、通过该模型,研究大脑皮质神经元在氧糖剥夺/复氧后NF-κB亚基P65、P50和c-Rel蛋白的表达、活化规律;
4、观察两种不同NF-κB decoy ODNs转染大脑皮质神经元在氧糖剥夺/复氧条件下对NF-κB亚基P65、P50和c-Rel蛋白的影响以及对下游因子TNF-α、IL-1α、Bcl-x_L mRNA及蛋白的影响,进一步探讨不同NF-κB亚基在大脑皮质神经元氧糖剥夺/复氧中的作用。
方法
1、取Wistar大鼠乳鼠脑组织,采用机械分离方法获取大脑皮质神经元并进行培养,通过倒置显微镜、免疫细胞化学进行鉴定;
2、采用细胞培养、MTT法、酶学检查、光学显微镜评价氧糖剥夺模型;
3、采用免疫细胞化学和Western Blot法检测大脑皮质神经元在氧糖剥夺/复氧后不同时间点NF-κB亚基P65、P50和c-Rel蛋白的动态表达规律;
4、大鼠大脑皮质神经元在氧糖剥夺、氧糖剥夺/复氧条件下转染不同的NF-κB哑铃形圈套IgG-κB decoy ODNs和Bcl-x decoy ODNs、杂合圈套后,采用Western Blot法检测不同组别NF-κB亚基P65、P50和c-Rel蛋白的表达,采用ELISA和RT-PCR法检测神经元TNF-α、IL-1α、Bcl-x_L mRNA及蛋白表达的水平。
结果
1、经形态学、免疫细胞化学鉴定所培养的细胞为大脑皮质神经元;
2、氧糖剥夺后大脑皮质神经元形态变化显著、LDH释放量、MTT检测显示:随着氧糖剥夺时间延长,氧糖剥夺前后神经元形态结构发生显著变化,神经元活性降低,MTT吸光度值逐渐降低,培养液中LDH释放逐渐增加,氧糖剥夺1h组、2h组与正常组无显著性差异(P＞0.05),氧糖剥夺4h组与正常组有显著性差异(P＜0.05)。
3、免疫细胞化学、Western Blot结果显示:正常组细胞内只有少量P65、P50及c-Rel蛋白表达,OGD4h组NF-κB P65和P50蛋白表达开始增加明显高于正常组(P＜0.05),OGD4h/2h组、OGD4h/6h组NF-κBP65和P50蛋白表达持续性升高,并在复氧后6h达高峰(P＜0.01),此后逐渐下降,至OGD4h/12h时与正常组有显著差异(P＜0.05),OGD4h/24h NF-κB P65和P50蛋白表达降低与正常组比较无统计学差异(P＞0.05);NF-κB亚基c-Rel蛋白表达在OGD4h组与正常组无差异(P＞0.05),OGD4h/R2h至OGD4h/R12h表达持续增加(P＞0.05),并在复氧后12h达高峰(P＜0.01),并且在OGD4h/24h时c-Rel蛋白表达仍未恢复到正常水平(P＞0.05)。
4、大鼠大脑皮质神经元在转染不同的NF-κB哑铃形圈套IgG-κBdecoy ODNs和Bcl-x decoy ODNs、杂合圈套、脂质体后,在氧糖剥夺、氧糖剥夺/复氧条件下,Western Blot法和免疫细胞化学结果显示:转染IgG-κB decoy ODNs后NF-κB亚基P65、P50蛋白表达显著降低(P＜0.05),而c-Rel蛋白的表达与OGD4h/R6h相比无差异(P＞0.05):转染Bcl-x decoy ODNs后NF-κB亚基P50、c-Rel蛋白表达较OGD4h/R6h组显著降低(P＜0.05),而P65蛋白的表达与OGD4h/R6h相比无显著改变(P＞0.05);脂质体组、无关decoy ODNs组对NF-κBP65、P50及c-Rel蛋白表达未见抑制(P＞0.05)。
5、RT-PCR和ELISA显示:氧糖剥夺/复氧后大脑皮质神经元内TNF-α、IL-1α、Bcl-x_L蛋白和mRNA表达明显升高(P＜0.05),转染IgG-κB decoy ODNs可显著抑制NF-κB下游基因IL-1a、TNF-a蛋白和mRNA表达(IgG-κB decoy ODNs组vs OGD4h/R6h,P＜0.05),对Bcl-xLmRNA表达未见抑制作用(P＞0.05);而Bcl-x decoy ODNs可显著抑制IL-1a、Bcl-xL蛋白和mRNA表达(Bcl-x decoy ODNs组vs OGD4h/R6h,P＜0.05),对TNF-a mRNA表达未见抑制作用(P＞0.05);而脂质体组、无关decoy ODNs组对IL-1a、TNF-a、Bcl-xL蛋白和mRNA表达未见抑制作用(P＞0.05)。
结论
1、我们成功培养出了原代大脑皮质神经元,该方法的建立为体外研究缺血性脑血管疾病提供了帮助;
2、建立了一种有效、方便、实用的体外模拟缺血性损伤模型,为神经元缺血性损伤机制的研究及临床治疗奠定基础;
3、正常情况下,大脑皮质神经元存在少量NF-κB P65、P50和c-Rel蛋白表达,免疫细胞化学和Western Blot显示氧糖剥夺/复氧后大脑皮质神经元中NF-κB亚基P65、P50和c-Rel蛋白表达增加,NF-κB P65和P50蛋白在氧糖剥夺4h开始增加,复氧后2-6h呈持续增加过程,在复氧6h表达到达高峰,延迟到复氧后12h与正常组比较仍有显著差异,24h时回复到正常水平。NF-κB c-Rel蛋白在复氧后2h才开始增加,并延迟到复氧后12h达高峰,其在复氧后24h表达仍高,与正常组比较有统计学意义。实验结果表明,NF-κB P65、P50和c-Rel蛋白在不同时间点表达规律存在差异,提示NF-κB亚基P65和P50可能在脑缺血再灌注后神经元内早、中期发挥作用,而NF-κB亚基P50和c-Rel主要在中晚期发挥重要作用,两者在时间上有交叉重叠;
4、氧糖剥夺/复氧可以诱导大脑皮质神经元大量表达IL-1a、TNF-a、Bcl-xL蛋白和mRNA,IgG-κB decoy ODNs可以显著抑制炎症因子IL-1a、TNF-a蛋白和mRNA表达,对Bcl-xL蛋白和mRNA表达无抑制作用;Bcl-x decoy ODNs可以抑制IL-1a、Bcl-xL蛋白和mRNA表达,对TNF-a蛋白和mRNA表达无显著抑制。
Backgroud
Cerebral ischemia is the most life-threatening neurological disease,and is the third leading cause of death after heart disease and cancer,and in the elderly it is a leading cause of long-term disability.Many studies have been done for the disease both at home and board,which gradually deepens the comprehension of the ischemic injury.Howere,an ideal curative effect has not been achieved as yet.So it is necessary to research for a new and a stronger pointed therapy strategy.
Now,the thrombolytic compound tissue plasminogen activator(t-PA) is the only Food and Drug Administration-approved agent for cerebral ischemia therapeutic window and is not suitable for many cerebral ischemia, in the other hand,many studies had showed that the inflammation response during reperfusion speeds the secondary brain damages after thromobolysis and play a key role in role in I/R.
Inflammation acts as a double-edged sword in I/R.The timming,type of inflammatory mediator and the extent of its stimulation determines the balance between the bad versus good effects of inflammation.Uncontrolled numbers of the infiltrated macrophages and neutrophils increases free radical generation which is detrimental to vascular integrity leading to neuron death.Howere,microglial responses after ischemia is necessary for scavenging the necrotic debris,and the complement system that gets activated in post-ischemic brain initiates local inflammatory responses that ultimately contribute to neuronal survival and tissue remodeling.Howere, complement activation to an inappropriate extent promotes inflammatory mediator release and tissue injury.
Brain ischemia/reperfusion has relationship with many factor,such as free radicals,the excitotoxicity,calcium over-loading,the dysfunction of mitchondria,inflammation and apotosis,among them,inflammation is a known precipitator of neuronal death after cerebral ischemia.Neurons' injury and dysfunction is a key event early in the pathogenesis of cerebrovascular disease,so it is important to protect the function and structure of neurons from injury.Degradation ofⅠκB induces NF-κB activation leading to the coordinated induction of multiple genes is involved in many inflammatory and immune cascades in I/R.NF-κB play a dynamic role in the survival and death of neuron in I/R.Much research has established that the activation of NF-κB enhances neuronal survival by preventing apoptosis.In contrast to the anti-apoptosis action of NF-κB,controversial results have appeared regarding the causative role of NF-κB to excitotoxicity.
NF-κB dimer species exhibit different binding affinities to the different DNA sequences,the different protein-DNA complexes act on different gene promoters with different NF-κB binding sites.All of these differing conclusions were drawn from different experiment models including cell lines and tissues under different stimuli.NF-κB decoy strategy has been a powerful tool for gene therapy and gene regulation in the study of transcriptional rehulation.NF-κB decoy ODNs in dumbbell shape with the nuclear sequence which was similar to NF-κB cis-elements were synthetized in our study.Oliogodeoxynucleotides with high affinity for transcription factors may be used to bind the transcription factors and block the activation of target gene.
Objectives
(1) To culture and observe the morphology of primary cortical neurons, the cells were affirmed.
(2) To establish a simple,stable and reliable model of oxygen glucose deprivation/reoxygenation(OGD/R) in primary cortical neurons.
(3) To investigate the protein expression and the activation of NF-κB P65,P50 and c-Rel following OGD/R of primary cortical neurons in vitro.
(4) To study the therapeutic effects of two different decoys:IgG-κB decoy ODNs and Bcl-x decoy ODNs on NF-κB downstream genes TNF-α, Bcl-xL,IL-1αand cell protection in primary cultured cortical neurons in an oxygen glucose deprivation(OGD) model
Methods
(1) Primary neuronal cultures of cerebral cortex were obtained from neonatal Wistar rat(＜24h),and neurons were affirmed by immunocytochemical method,morphologies under Light Microscope.
(2) Primary neuronal cultures of cerebral cortex were exposed to oxygen glucose deprivation(OGD) for various time(control group,OGD2h group,OGD4h group,OGD6h group),The neuronal injury was detected by the lactate dehydrogenase(LDH) relase and the tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H tetrazolium bromide(MTT), and morphologic changes were studied under Light Microscopeto evaluate model of oxygen/glucose deprivation.
(3) The protein level of NF-κB subunit P65,P50 and c-Rel in primary neuron was detected by Western Blot and immunocytochemistry after OGD/R
(4) We applied different NF-κB decoy ODNs(IgG-κB decoy ODNs, Bcl-x decoy and scramble decoy ODNs) into primary cotical neurons,and then Western Blotting was used to detect the expression of NF-κB P65,P50 and c-Rel protein in neurons.The protein level of TNF-α,IL-1αin primary neuron was detected by ELISA,and the expression of TNF-αBcl-xL,IL-1αmRNA was detected by RT-PCR.
Results
(1) The characterization of primary cortical neuron was comfirmed based on the morphology and positive by the detetion of NSE andβ-tubulin 3.
(2) The MTT and the LDH showed primary corticai neurons activity decreased and changed morphology and the mount of LDH release from injured neurons was increased significantly in OGD 4h group than in control group(P＜0.05).
(3) The result of immunocytochemical method and Western Blotting indicated that the expression of P65,P50 protein gradually increased,there was significant different between OGD4h and control group(P＜0.05),and it was significantly highest on the OGD4h/R6h(P＜0.01).The expression of c-Rel protein begin increased on OGD4h/R2h,and it was significantly highest on the OGD4h/R12h(P＜0.05),and then it gradually decreased,but was still higher than that of the control group(P＜0.05).
(4) We applied different NF-κB decoy ODNs(IgG-κBdecoy ODNs, Bcl-x decoy and scramble decoy ODNs into primary cotical neurons.The result of immunocytochemical method and Western Blot showed that IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein (P＜0.05),but no effect on c-Rel(P＞0.05),while Bcl-x decoy reduced the expression of NF-κB c-Rel and P50 protein(P＜0.05) but no effect on P65, scrambled decoy ODNs and Reagent have no effect on NF-κB subunit (P＞0.05).
(5) The result of RT-PCR and ELISA showed that the mRNA and protein levels of TNF-α,Bcl-xL,IL-1αwere markedly elevated after OGD (P＜0.05),IgG-κB decoy ODNs decreased TNF-α,IL-1αmRNA and protein expressions in neurons(P＜0.05),but no effect on Bcl-xL mRNA (P＞0.05),Bcl-xL decoy ODNs suppressed Bcl-xL,IL-1αmRNA and protein expressions(P＜0.05),but no effect on TNF-αmRNA and protein (P＞0.05),scrambled decoy ODNs and Reagent have no effect on IL-1a、TNF-a、Bcl-xL protein and mRNA(P＞0.05)
Conclusions
(1) We isolated and cultured primary cortical neuron in vitro successfully.The method provided a useful way to study the cerebrovascular diseases in viro.
(2) In vitro cerebral ischemia/reperfusion model may be induced easily by OGD in cultured neurons may be used to further study the mechanism of ischemic neuronal cell injury and its protection.
(3) There are a small quantity of NF-κB P65,P50 and c-Rel protein expressions in control group,western Blotting and immune cytochemistry has shown that the NF-κB P65,P50 and c-Rel protein expressions increased after oxygen glucose deprivation/reoxygenation.The NF-κB P65,P50 protein begins to increase at 4h after oxygen/glucose deprivatio and keeps increasing at 2-6h after reoxygenation,reaches the summit at 6h after reoxygenation,and returns to the normal level at 24h,while the NF-κB c-Rel protein begins to increase at 2h after reoxygenation,reaches the summit at 12h after reoxygenation and still has a high expression at 24h after reoxygenation.The experimental results reveal that the expression pattern of NF-κB P65,P50 and c-Rel protein varies with different time points,which indicates that NF-κB P65,P50 may have an effect on the neuron in the early and middle stage after the cerebral ischemia reperfusion and NF-κB c-Rel primarily plays an important role in the middle and late stage.
(4) OGD induced TNF-α,Bcl-xL,IL-1αmRNA and protein expression in primary cortical neurons.IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein,decreased TNF-α,IL-1αand Bcl-xL mRNA and protein expressions in neurons,while Bcl-x decoy ODNs reduced the expression of NF-κB c-Rel and P50 protein,suppressed Bcl-xL,IL-1αmRNA and protein.
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