【作者】 钟海星；

【导师】 熊利泽； 王强；

【作者基本信息】 第四军医大学 ， 麻醉学， 2013， 博士

【摘要】 中风是世界第二位、我国第一位的致死性疾病，更是成年人致残的首要病因。而中风后神经元坏死和凋亡导致的神经元缺失(loss of neuron)以及突触功能异常则为中风后神经功能障碍的最根本原因。目前除了再灌注治疗（t-PA，血管再通术）可临床应用、造福患者，抗兴奋性毒性、抗氧化、抗凋亡、抗炎等新治疗措施均在临床实验中因为效果不佳或毒副作用而失败。因此，寻找能够有效减轻脑缺血后神经元损伤、抑制神经元凋亡，促进神经发生并重建神经环路的新治疗靶点或药物，成为了中风治疗的重点和难点。成年哺乳动物神经发生(neurogenesis)的发现，为中风后神经功能康复带来了曙光。而神经源素2（Ngn2）作为bHLH转录因子家族的成员，在成年哺乳动物神经发生的调节中发挥着极为重要的作用。研究发现，Ngn2不仅是启动神经发生的重要因子，还参与调节神经元的终末分化，调控神经前体细胞的迁移与成熟，影响轴突投射和树突形成。不仅如此，单纯应用Ngn2就可诱导胚胎干细胞、星形胶质细胞和少突胶质细胞前体细胞分化为具有相应结构和功能的成熟谷氨酸能神经元；而转染Ngn2还可促进移植后神经前体细胞的存活、分化、迁移与成熟，参与损伤神经环路和网络的修复与重建，提示Ngn2可能成为中风治疗尤其是康复治疗的重要作用靶点。然而，Ngn2因分子量过大，难以通过血脑屏障，限制了其在中枢神经系统疾病中的应用。而我们前期合成的具有血脑屏障通透性和损伤区域靶向性的TAT-LBD-Ngn2融合蛋白，为进一步明确Ngn2对缺血性脑中风的治疗作用及其机制提供了研究基础。因此，本课题以原代培养的海马神经元氧糖剥夺（OGD）模型和小鼠全脑缺血（GCI）模型为对象，应用神经行为学、免疫组织化学和western blot等技术，观察TAT-LBD-Ngn2对缺血性脑损伤的短期和长期神经保护作用，明确其对神经发生的促进作用，并初步阐明TAT-LBD-Ngn2神经保护作用的机制，旨在为缺血性脑中风治疗提供新的干预靶点和治疗药物，并为Ngn2神经保护作用的机制研究提供新的思路。实验一TAT-LBD-Ngn2对氧糖剥夺原代神经元的保护作用及其机制研究目的：通过离体研究，明确TAT-LBD-Ngn2对氧糖剥夺的原代培养神经元是否具有保护作用，并初步探讨其机制。方法：采用原代培养的小鼠海马神经元，以125μg/L浓度的Ngn2、TAT-LBD、TAT-LBD-Ngn2分别孵育神经元6h，通过免疫细胞化学观察各药物对细胞膜的通透性，并进一步明确各药物对原代培养海马神经元神经突生长的影响。再分为5组：Control组、OGD组、OGD+TAT-LBD-Ngn2（62.5μg/L）组、OGD+TAT-LBD-Ngn2（125μg/L）组和OGD+TAT-LBD-Ngn2（250μg/L）组。OGD复氧后24h后，应用MTT比色法检测神经元细胞活力，应用LDH释放试验检测神经元损伤程度，应用TUNEL染色观察神经元的凋亡情况，并进一步应用Elisa和Western Blot检测凋亡相关蛋白Caspase-3、Bcl-2和Bax的表达水平。结果：125μg/L的TAT-Ngn2和TAT-LBD-Ngn2均可通过细胞膜，并促进原代培养海马神经元的神经突生长（P<0.05）。OGD模型24h后，TAT-LBD-Ngn2组的细胞活力显著提高（P<0.01），LDH释放减少（P<0.01），并且细胞活力的改善与TAT-LBD-Ngn2的药物浓度呈剂量依赖关系，250μg/L的TAT-LBD-Ngn2具有最大的神经保护作用。同时，TAT-LBD-Ngn2组TUNEL阳性神经元的数量明显减少（P<0.05）；促凋亡蛋白Caspase-3和Bax的表达明显受到抑制（P<0.01），而抗凋亡蛋白Bcl-2水平显著增高（P<0.01），TAT-LBD-Ngn2的抗凋亡作用同样具有剂量依赖性，于250μg/L时达到最大保护效果。结论：TAT-LBD-Ngn2可透过神经元细胞膜，促进神经突生长；还可通过抑制内源性和外源性的凋亡通路，减少神经元凋亡，减轻缺血再灌注损伤。实验二TAT-LBD-Ngn2对脑缺血损伤短期保护作用及其机制的研究目的：通过在体研究，明确TAT-LBD-Ngn2对小鼠全脑缺血损伤是否具有保护作用，并初步探讨其神经保护作用的机制。方法：采用C57BL/6小鼠，行双侧颈总动脉结扎（BCCAO）术建立GCI模型，随机分为Control组（PBS200μL, qd ip, n=30）和TAT-LBD-Ngn2组（TAT-LBD-Ngn2250μg/kg, qd ip, n=30），再灌注即刻首次给药，此后每天进行药物腹腔注射。首次药物注射24h后通过免疫组织化学和Western Blot观察药物通过血脑屏障和细胞膜的情况。通过1d、2d和3d的总体运动评分（TMS）观察BCCAO小鼠术后的神经行为能力。应用TUNEL染色观察并计数BCCAO后3d神经元的凋亡情况；并应用免疫组织化学观察神经营养因子（BDNF，NGF）在海马和皮层的表达情况。结果：在成功建立的全脑缺血模型小鼠中，TAT-LBD-Ngn2可通过血脑屏障进入脑组织，并表达于海马和皮层的神经元和其他神经细胞。同时，TAT-LBD-Ngn2因对层粘连蛋白的亲和力和靶向性，在脑内尤其是海马区域的表达显著高于TAT-Ngn2（P<0.01）；TAT-LBD-Ngn2治疗可改善BCCAO术后1d、2d和3d小鼠的TMS评分（P<0.05）。TAT-LBD-Ngn2治疗还可显著减少BCCAO术后3d海马CA1区TUNEL阳性神经元数目（P<0.05）；诱导皮层神经元BDNF的表达，但对NGF的表达则无明显影响。结论：TAT-LBD-Ngn2可透过血脑屏障，聚集于损伤区域；还可通过促进BDNF表达，抑制神经元凋亡，减轻脑缺血再灌注损伤，改善运动功能。实验三TAT-LBD-Ngn2对脑缺血损伤长期保护作用及其机制研究目的：观察TAT-LBD-Ngn2对小鼠全脑缺血损伤后神经功能恢复的影响，并初步探讨神经发生在其中发挥的作用。方法：采用C57BL/6小鼠，行BCCAO术建立GCI模型，随机分为Control组（PBS200μL, qd ip, n=30）和TAT-LBD-Ngn2组（TAT-LBD-Ngn2250μg/kg, qd ip,n=30），于再灌注时开始给药，持续14或28d。于BCCAO后14、28d通过旷场试验观察小鼠的运动功能，通过Morris水迷宫检测TAT-LBD-Ngn2对小鼠空间学习和记忆功能的影响。通过免疫组织化学，观察并计数BCCAO后14、28d海马CA1区神经元数量；观察海马齿状回双皮质素（DCX）和溴脱氧尿苷（BrdU）阳性的新生神经元数目；检测突触素（SYN）在脑内的表达情况以明确新生神经元是否建立了功能性突触。结果：两组间小鼠的运动功能无统计学差异。但Morris水迷宫结果显示，TAT-LBD-Ngn2组小鼠在14d和28d的逃避潜伏期显著缩短（P<0.05），空间探索试验中的穿台次数和目标象限停留时间也明显优于Control组（P<0.05），具有较好的空间学习和记忆功能。免疫组织化学结果显示250μg/kg TAT-LBD-Ngn2可显著提高海马CA1区神经元的数量（P<0.05）；并增加海马齿状回BrdU和DCX阳性神经元的数目（P<0.05），这些新生神经元主要位于海马齿状回的颗粒下层；此外，TAT-LBD-Ngn2还增加了皮层和海马SYN的表达。结论：TAT-LBD-Ngn2治疗可促进缺血易损区海马CA1区神经元的存活，显著改善BCCAO小鼠的空间学习和记忆功能，其作用可能是通过促进神经发生，促进突触结构和功能的重建实现的。小结：通过上述研究证实，TAT-LBD-Ngn2可透过血脑屏障和细胞膜，富集于损伤区神经元内；TAT-LBD-Ngn2可通过诱导BDNF的表达，抑制神经元凋亡，减轻脑缺血早期损伤；TAT-LBD-Ngn2还可通过促进神经发生、重建突触结构和功能，促进脑缺血性损伤后神经功能的恢复，提示TAT-LBD-Ngn2可能开发为临床治疗脑中风的候选药物。本研究不仅为大分子多肽治疗脑损伤开辟了新方向，也为临床中风药物的研发提供了新的作用靶点和研究基础，有望从短期减轻损伤，长期促进功能恢复两个方面双管齐下造福脑中风患者。更多还原

【Abstract】 Stroke is the second fatal disease in the world and the leading cause of death inChina. Moreover, stroke remains to be the most important cause of adult disability.The damage of synapses and the loss of neurons are the crucial reasons ofneurological dysfunction after stroke. Up to now, there is no safety and effectivetreatment for stroke, except for reperfusion therapy (t-PA, vascular recanalization).And new strategies including anti-excitatory toxicity, anti-oxidant, anti-apoptosis andanti-inflammion treatments were all failed in clinical translation because ofineffectiveness or side effects. Thus, development of novel treatment strategies forstroke, especially in suppression apoptosis, prompting neurogenesis and rebuildingneural circuits, is in high priority.The scientific discovery of neurogenesis in adult mammalian is believed to be amilestone in improving neurological recovery after stroke. And as a member of bHLH family, Ngn2plays an important role in neurogenesis of adult mammalian.Many studies have demonstrated that Ngn2is not only the initiation factor ofneurogenesis, but also the regulator of neuronal differentiation, subtype specification,axons projection, dendrites formation, maturation and integration in various regionsof brain. Moreover, latest researches have found that Ngn2is sufficient to stimulateembryonic stem cells, astrocytes, and oligodendrocytes precursor cells differentiationinto glutamate neurons. Meanwhile, Ngn2promotes the translated neural precursorcells to survive, differentiate, migrate and mature. These results indicate that Ngn2may be an attractive candidate for the treatment of stroke, especially in rehabilitationtreatment. However, as a macromolecular peptide, Ngn2can not cross the bloodbrain barrier (BBB), that limits the use of Ngn2in the treatment of central nervoussystem diseases. In the past, we have fused Ngn2, protein transduction domain (PTD)of TAT protein and LBD (laminin binding domain from the first135amino acids ofagrin) to TAT-LBD-Ngn2protein, which may cross the cell membrane and BBB, toaggregate at lesion regions. Moreover, the TAT-LBD-Ngn2fusion protein provides afoundation for the further study on the neuroprotective effect on ischemic stroke.Cultured hippocampal neurons exposed to oxygen-glucose deprivation(OGD),mice global cerebral ischemia model, neurological behavior, immunohistochemistry,and western blot techniques were used in this study to investigate the short-term andlong term neuroprotective effects of TAT-LBD-Ngn2, to confirm the effect ofTAT-LBD-Ngn2on neurogenesis, and to clarify the underlying mechanisms. Thepresent study is aimed to represent a promising therapeutic target and candidate drugfor treatment of ischemic stroke, and provide novel evidence and insights on theneuroprotective effect of Ngn2.Experiment1The protective effect of TAT-LBD-Ngn2againstOGD damage in cultured neuronsPurpose: To investigate the neuroprotective effects of TAT-LBD-Ngn2onprimary neurons exposed to OGD, and to preliminarily clarify the mechanisms in vitro.Methods: The primary cultured hippocampal neurons were respectivelycultured in the presence of TAT-Ngn2, TAT-LBD and TAT-LBD-Ngn2, at aconcentration of125μg/L. Six hours later, the transduction ability and neurite growthwere investigated by immunofluoresence analysis. Then the neurons were dividedinto5groups: Control group, OGD group, OGD+TAT-LBD-Ngn2(62.5μg/L) group,OGD+TAT-LBD-Ngn2(125μg/L) group and OGD+TAT-LBD-Ngn2(250μg/L)group. At24h after OGD, the cell viability was assessed by MTT assay, and neuralinjury was evaluated by LDH release. To determine whether TAT-LBD-Ngn2canreduce neural apoptosis induced by OGD, TUNEL staining was performed. At thesame time, Elisa and western blot were conducted to investigate the expression ofapoptosis-related proteins, such as Caspase-3, Bcl-2and Bax.Results: We found that both TAT-LBD-Ngn2and TAT-Ngn2could cross the cellmembrane into hippocampal neurons, and promote neurite growth (P<0.05) in vitro.At24h after reperfusion, the cell viability significantly increased (P<0.01), and theLDH release decreased (P<0.01) in TAT-LBD-Ngn2group, which showed adose-dependent effect. TAT-LBD-Ngn2also statistically reduced the number ofTUNEL labeling-positive neurons (P<0.05). Meanwhile, the expression ofpro-apoptotic protein(Caspase-3and Bax) decrease (P<0.01), and the expression ofanti-apoptotic protein Bcl-2increased(P<0.01) in TAT-LBD-Ngn2groups. Theanti-apoptotic effect of TAT-LBD-Ngn2was also strengthened with the increasedosage.Conclusion: TAT-LBD-Ngn2could cross the cell membrane, and promoteneurite growth. In addition, TAT-LBD-Ngn2could reduce neurons apoptosis andalleviate ischemia reperfusion injury by suppressing the intrinsic and extrinsicapoptotic pathways.Experiment2The neuroprotective effects of TAT-LBD-Ngn2oncerebral ischemic injury in vivo Purpose: To investigate the neuroprotective effect of TAT-LBD-Ngn2onglobal cerebral ischemia injury, and to preliminarily clarify the anti-apoptoticmechanisms in vivo.Methods: BCCAO was used as a model of global cerebral ischemia (GCI).Male C57BL/6mice were randomly divided into two groups: control group (PBS200μL, qd ip, n=30) and TAT-LBD-Ngn2(TAT-LBD-Ngn2250μg/kg, qd ip, n=30).At24h after injection of medicines, fluorescent staining and western blot wereperformed to investigate whether TAT-LBD-Ngn2could cross BBB and cellmembrane. The total motor score (TMS)(9possible points) was calculated to detectmotor deficits at1,2,3d after BCCAO. In addition, TUNEL staining was conductedto determine the neuron apoptosis at3d after BCCAO. At the same time, theexpression of neurotrophic factors including BDNF and NGF were investigated byimmunohistochemistry.Results: The model of GCI was successfully established. We found thatTAT-LBD-Ngn2could cross the BBB and transduce into hippocampal and corticalneurons. Moreover, Ngn2distributed much more in hippocampus and cortex inTAT-LBD-Ngn2group(P<0.01), due to the laminin binding domains which inducedNgn2aggregation in the cerebral lesion regions. TAT-LBD-Ngn2treatmentsignificantly improved TMS scores (P<0.05) after GCI injury. Meanwhile,TAT-LBD-Ngn2could attenuate the number of TUNEL labeling-positive cells(P<0.05) in the hippocampal CA1subregion, and improve the expression of BDNFrather than NGF in cortex.Conclusion: TAT-LBD-Ngn2could cross BBB and aggregated at lesion regions.Moreover, TAT-LBD-Ngn2could alleviate neuron I/R injury and improveneurological function by increasing BDNF expression and suppressing neuronapoptosis.Experiment3The effect of TAT-LBD-Ngn2on neurogenesis aftercerebral ischemia Purpose: To investigate the long-term neuroprotective effect ofTAT-LBD-Ngn2, especially the recovery of neurological function. Moreover, topreliminary discuss the effect of neurogenesis in the neuroprotection..Methods: BCCAO was used as a model of GCI. Male C57BL/6mice wererandomly divided into two groups: control group (PBS200μL, qd ip, n=30) andTAT-LBD-Ngn2(TAT-LBD-Ngn2250μg/kg, qd ip, n=30). At14,28d after BCCAO,Morris water maze was conducted to determine the spatial learning and memoryfunction. Fluorescent staining was performed to investigate the number andmorphology of neurons in hippocampal CA1subregion, the new born neurons werelabeled by BrdU and DCX in DG, and functional synaptic connections were labeledby SYN.Results: The escape latency got shortened (P<0.05), and the number ofplatform-site crossover and time spent in target quandrant increased (P<0.05) inTAT-LBD-Ngn2group at14and28d after BCCAO, indicating a better spatial learningand memory functions. In addition, TAT-LBD-Ngn2, at a concentration of250μg/kg,could significantly increase the number of neuron in hippocampal CA1subregion(P<0.05), the numbers of BrdU and DCX labeling-positive neuron in DG (P<0.05).Meanwhile, the expression of SYN increased in TAT-LBD-Ngn2group compared withControl group in cortex and hippocampus.Conclusion: TAT-LBD-Ngn2could promote the survival of neurons inhippocampal CA1subregion. TAT-LBD-Ngn2also improves spatial learning andmemory functions after I/R injury by promoting neurogenesis and rebuilding thesynaptic structure.Summary: According to these results, TAT-LBD-Ngn2could cross blood brainbarrier and cell membrane, and gather into the neurons in the lesion regions.Moreover, TAT-LBD-Ngn2could increase the expression of BDNF, suppress theneuron apoptosis to alleviate early stage ischemic cerebral injury. TAT-LBD-Ngn2also enhances neurogenesis, and rebuilds synaptic structure to promote neurologicalrecovery after ischemic cerebral injury. All these results indicate that TAT-LBD-Ngn2can be a candidate drug for clinicalstroke treatment, which could alleviate cerebral injury in short-term, and promoteneurological recovery in long-term. The present study not only develops a newdirection of macromolecular peptides treatment for stroke, but also provides a newdrug target and research basis in drugs development of stroke.更多还原