【作者】 张晓英；

【导师】 杨于嘉；

【作者基本信息】 中南大学 ， 儿科学， 2011， 博士

【摘要】 缺氧缺血性脑病(hypoxic-ischemic encephalopathy, HIE)是指围产期窒息导致脑的缺氧缺血性损伤,而出现的一系列中枢神经系统异常的表现,是新生儿时期脑损伤的常见原因,也是导致儿童发生严重神经系统后遗症的主要原因。2005年WHO资料显示,全球每年有400万新生儿死亡,与窒息相关者占23%,欧美等发达国家,中重度新生儿HIE的死亡和严重伤残率高达53%～61%,而发展中国家的状况远甚于此。目前临床上对于HIE的发病机制的研究虽然取得了很大进展,但仍缺乏特效治疗方法。长期以来,人们认为中枢神经系统损伤后不能修复,但近年来在对神经干细胞(neural stem cells, NSCs)的研究中,人们发现在大脑的某些部位如侧脑室管膜下(subventricular zone, SVZ)和海马齿状回(hippocampus dentate gyrus, DG)终身存在着内源性的NSCs,脑损伤可刺激这些NSCs增殖、分化,进行自我修复。已经有文献报道,新生大鼠在缺氧缺血性脑损伤(hypoxic-ischemic brain damage, HIBD)后,脑内源性NSCs有增殖和分化现象。但内源性NSCs不仅数量有限,而且受到神经营养因子缺乏、轴突生长抑制因子存在等因素影响,大脑的这种自生修复潜能有限,因此,能够找到一种将大脑自生修复功能放大的方法将为HIE的治疗带来广阔的前景。高压氧(hyperbaric oxygen HBO)应用于新生儿HIE的治疗已经有数十年的历史。我国从上世纪90年代开始应用HBO治疗HIE以来,大部分文献报道认为HBO可减轻患儿症状,降低病死率和伤残率。我们最近的研究也已证实,HBO不仅可促进HIBD新生大鼠内源性NSCs的增殖,并且还可促进增殖的内源性NSCs向损伤的大脑皮层迁移并分化为成熟的神经元。近来,国外有学者回顾分析近15年来NCBI(National Center for Biotechnology Information)关于HBO治疗脑缺血的文章,有六十多个关于HBO治疗动物及人脑缺血性损伤的研究,结果显示,HBO可以改善局灶性脑缺血、全脑缺血、新生儿HIE、蛛网膜下腔出血的预后,HBO可以用于治疗人中枢神经系统缺血性损伤HBO可以减轻HIBD大鼠的神经损伤并促进其恢复,HBO治疗HIBD新生大鼠时,可促进脑内NSCs的增殖,其机制可能与Wnt信号的活化有关。过去几年中,一系列在体与离体的研究已经显示Wnt信号分子对组织干细胞包括NSCs的增殖分化及命运决定起着重要调控作用。β-catenin是Wnt信号通路中关键分子,缺血性脑损伤时,β-catenin在调节神经干细胞的增殖、分化、移位过程中起着关键作用。β-catenin在神经前体细胞(neural precursor cells, NPCs)的高表达会引起大脑皮质神经前体细胞的增殖,相反,如果NPCsβ-catenin缺失,将会导致神经元损伤,细胞增殖减少,并引起细胞凋亡。研究显示,β-catenin激活后转入胞核内,与TCF结合为复合体(β-catenin/TCF)后,激活神经元素(neurogenins, Ngns)和细胞周期蛋白(CyclinD1), Ngn1作为转录激活剂,使下游的分化基因NeuroD, Math2等增多,诱导晚期NPCs分化为神经元。NPCs在分化成神经元的过程中,骨形成蛋白(bone morphogenetic protein, BMP)的表达日益增多,BMP则反馈抑制NPCs向神经元分化,而促进其向星型胶质细胞分化。那么,HBO治疗新生儿HIBD时Wnt/β-catenin信号系统是如何调控NSCs增殖分化的?目前仍不是十分清楚。鉴于Wnt信号通路在NSCs的增殖、迁移和分化中的重要地位,我们对HBO能够促进HIBD新生大鼠NSCs增殖这一现象提出如下工作假设：HBO→Wnt→β-catenin进入细胞核→形成β-catenin /TCF→转录Ngn1基因→诱导NPCs向神经元分化(?)BMP→诱导NPCs向星形胶质细胞分化故本课题旨在离体细胞水平,进一步探讨HBO促HIBD新生大鼠NSCs增殖分化的机制,以期为HBO治疗HIBD的临床应用提供有力的理论依据。为此,我们参照文献分别将NSCs在正常脑组织匀浆及HIBD脑组织匀浆中培养,以模拟正常及HIBD时脑内微环境,再给予HBO处理,通过将β-catenin siRNA转染到NSCs内来验证这一假说：离体细胞水平,β-catenin调控了HBO引起的HIBD新生大鼠NSCs的增殖分化。本研究分三部分：一、构建靶向大鼠β-catenin基因的shRNA的真核表达质粒,并筛选出沉默β-catenin基因效果最明显的shRNA表达质粒目的构建靶向大鼠β-catenin基因的shRNA的真核表达质粒,并筛选出沉默β-catenin基因效果最明显的shRNA表达质粒。方法设计3对针对β-catenin基因不同位点的shRNA片段,构建携带此shRNA片段的真核表达质粒(shRNA1-3)并行测序分析。然后通过电穿孔法将重组质粒转染到神经干细胞中,48 h后测定转染率,并分别采用RT-PCR及Western blot检测β-catenin mRNA和β-catenin蛋白表达情况。结果靶向大鼠β-catenin基因的shRNA真核表达重组质粒pGCPU6/GFP/Neo shRNA-1,2,3经测序证实构建成功。质粒在神经干细胞中的转染率约为62.63%±15.9%。shRNA1,shRNA2,shRNA3转染入神经干细胞48 h后,β-catenin RNA水平和蛋白水平均明显低于阴性对照组和空白对照组(0.17±0.01、0.09±0.01、0.08±0.00vs 0.75±0.01,0.74±0.02；0.21±0.16.0.19±0.12.0.12±0.10, vs 0.56±0.02,0.65±0.01,均P<0.01)。与shRNA1和shRNA2相比,shRNA3对β-catenin mRNA和蛋白的抑制作用均最强(89.37% vs 77.08%,88.02%；77.95% vs 61.31%、65.65%).结论成功构建并筛选出的靶向大鼠β-catenin基因的shRNA真核表达质粒对神经干细胞的β-catenin表达具有明显抑制作用。为研究Wnt/β-catenin信号途径在NSCs生长分化中的作用奠定基础。二、HBO对NSCs增殖分化的影响目的探讨HBO和脑组织匀浆上清液对离体SD大鼠NSCs分化的影响。方法传至2-3代的SD大鼠NSCs,随机分为九个组,未转染质粒者设为空白对照组(CON),其余8组根据转染阴性对照质粒和转染β-catenin shRNA真核表达质粒及给予后续处理的不同分组,九组细胞在同一时间行免疫荧光染色,分别进行NSE、GFAP、04染色,荧光显微镜下计数各组NSCs分化为NSE阳性细胞、GFAP阳性细胞和O4阳性细胞的百分率,并进行比较。结果与CON组相比,转染阴性对照质粒的NSCs(ncNSCs)中：HBO组,N组,HIBD组分化为神经元和少突胶质细胞的百分比明显增加(P<0.05),其中HIBD组增加最多,在此基础上再给予HBO处理,ncNSCs向神经元的分化会进一步增加,少突胶质细胞的分化也相应增加；N组,HIBD组,HIBD+HBO组分化为星形胶质细胞的百分比明显减少了(P<0.05)。与CON组相比,转染β-catenin shRNA的4组NSCs(siNSCs),分化为神经元的百分比都明显减少了(P<0.01),分化为星形胶质细胞的百分比都明显增加了(P<0.01)；少突胶质细胞的分化增加,但少于ncNSCs(P<0.01),其中HIBD组分化为神经元和少突胶质细胞较多,在此基础上再给予HBO处理,神经元和少突胶质细胞的分化进一步增加。结论(1)HBO可促进HIBD大鼠ncNSCs分化为神经元和少突胶质细胞而抑制其向星形胶质细胞分化。(2)β-catenin shRNA抑制了siNSCs向神经元分化而促进了其向星形胶质细胞的分化,这一作用虽可被HBO弱化但不能被HBO逆转。(3)β-catenin参与了HBO引起的HIBD大鼠ncNSCs向神经元分化。三、HBO对NSCs Ngn1, BMP4表达的影响目的探讨HBO和脑组织匀浆上清液对离体SD大鼠神经干细胞Ngn1, BMP4表达的影响。方法传至2-3代的SD大鼠NSCs,随机分为九个组,未转染质粒者设为空白对照组(CON),其余8组根据转染阴性对照质粒和转染β-catenin shRNA真核表达质粒及给予后续处理的不同分组,在同一时间收集上述9组细胞,分别提取细胞总RNA和总蛋白,行RT-PCR和Western blot,以测定Ngn1, BMP4 mRNA和Ngn1, BMP4蛋白的表达情况,并进行比较。结果与CON组相比,4组ncNSCs的Ngn1 mRNA和Ngn1蛋白的表达明显增加(P<0.01)而BMP4 mRNA和BMP4蛋白的表达明显减少(P<0.01)；在siNSCs,与CON组和ncNSCs组相比,4个siNSCs组的Ngn1 mRNA和Ngn1蛋白的表达明显减少(P<0.01), BMP4 mRNA和BMP4蛋白的表达明显增加(P<0.01),4个siNSCs组间Ngn1和BMP4的表达无统计学差异。结论(1)HBO可促进ncNSCs Ngnl的表达,抑制BMP4的表达,(2)β-catenin基因被抑制后,siNSCs的Ngn1蛋白和mRNA的表达被抑制,BMP4蛋白和mRNA的表达被促进,(3) BMP4和Ngn1在大鼠NSCs的增殖分化中起着重要的协调作用,Ngn1可能是一种潜在的神经元促成剂。综上所述,本研究结论如下：1、成功构建并筛选出的靶向大鼠β-catenin基因的shRNA真核表达质粒对大鼠神经干细胞的β-catenin表达具有明显抑制作用。2、HBO可促进离体HIBD大鼠NSCs分化为神经元和少突胶质细胞而抑制其向星形胶质细胞分化。3、HBO促进离体大鼠神经干细胞分化为神经元与β-catenin的激活有关4、离体细胞水平,HBO是通过激活β-catenin上调Ngn 1的表达促进NSCs分化为神经元,通过下调BMP4的表达减少NSCs分化为星形胶质细胞。更多还原

【Abstract】 Hypoxic ischemic encephalopathy (HIE) is the major recognized perinatal cause of neurological morbidity in full-term newborns and can result in mental impairment, seizures and permanent motor deficits, such as cerebral palsy. At present, there are no effective measures of treating HIBD (hypoxic-ischemic brain damage, HIBD)Ever since a long time ago, people considered that the central lesion could not be recovered. But now we have found that there are many endogenous neural stem cells(NSCs) at subventricular zone (SVZ) and hippocampus dentate gyrus(DG) lifetimes these cells can proliferate, differentiate by stimulation of pathologic impairments. It is found that NSCs were capable of being activated to proliferation and differentiation. However the capacity for recruiting endogenous NSCs is limited.Thus,other strategies used to modify endogenous neurogenesis after ischemic brain gamage have been described.Study showed there were proliferation of endogenous neural stem cells in hypoxic-ischemic neonate rats . Hyperbaric oxygen therapy has been used in HIE for decades, many studies have reported improved neurological outcomes with hyperbaric oxygen therapy. We also have found HBO therapy promoted the proliferation of endogenous neural stem cells in hypoxic-ischemic neonate rats. In a review, about hyperbaric oxygen therapy and cerebral ischemia, with an emphasis on the mechanisms of hyperbaric oxygen therapy-related neuroprotection, relevant literature was located in the National Library of Medicine and National Institutes of Health Database, and from bibliographies of articles reviewed. In the last 15 years,there have been no fewer than 65 animal and human studies of hyperbaric oxygen therapy in cerebral ischemic injury. Numerous studies have demonstrated a protective effect of hyperbaric oxygen therapy in experimental ischemic brain injury, and many physiological and molecular mechanisms of hyperbaric oxygen therapy-related neuroprotection have been identified. Hyperbaric oxygen therapy has been shown to ameliorate brain injury in a variety of animal models including focal cerebral ischemia, global cerebral ischemia, neonatal hypoxia-ischemia and subarachnoid hemorrhage.Human clinical studies have shown hyperbaric oxygen therapy may be beneficial in chronic cerebral vascular disease or in the setting of cardiopulmonary bypass to date.Recent studies have shown that HBO therapy may alleviate neuronal injury and promote the recovery of HIBD in rats an effect that is correlated with Wnt-3 protein. Several studies have shown that Wnt signaling is involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system, the Wnt signaling plays a key role on modulating cell differentiation or proliferation states.β-Catenin is a critical downstream mediator of the canonical Wnt pathway, which functions in gene transcription and cell adhesion, and plays vital roles in the proliferation and differentiation of neural progenitor cells. Overexpression ofβ-catenin in cortical neural precursors leads to expansion of the precursor population and cortical overgrowth.On the contrary, conditional deletion ofβ-catenin from cortical neural precursors results in delamination of neuroepithelial cells, loss of adherens junctions, impaired radial migration of neurons, decreased cell proliferation. Studies have shown that in the presence of Wnt signals, (3-catenin is stabilized and translocates to the nucleus,where it interacts with transcription factors of the LEF/TCF(lymphoid enhancer-binding factor/T cell-specific tran-scription factor) family to induce changes in gene expression, theβ-catenin/TCF complex appears to directly regulate the promoter of neurogenin1(Ngn1), a gene implicated in cortical neuronal differentiation. Ngn1 promotes neural progenitor cell differentiation into neurons. Bone morphogenetic protein 4(BMP4) belongs tothe TGF-b (transforming growth factor-b) superfamily. Study has shown that BMP and Wnt signaling could antagonize each other for self-renewal or differentiation of stem cells at specific tissues and times, and BMP4 has a potential to repress neurogenesis and induce astrocyto-genesis of neuroepithelial cells .Therefore, transfectingβ-catenin siRNA into NSCs and then treatting with HBO after cultured with HIBD brain tissue extract conditioned cultures and normal brain tissue extract conditioned cultures respectively as previously described in vitro, we tested the hypothesis thatβ-catenin regulated the proliferation of rats NSCs after HBO therapy.Our investigation was divided into the follwing three parts. PartⅠConstruction and screening of eukaryotic expression plasmids containing short hairpin RNA targeting at the ratβ-catenin geneObjective To construct eukaryotic expression plasmids containing short hairpin RNA (shRNA) that target at the neural stem cells (NSCs)β-catenin gene, and to select the plasmids that silenceβ-catenin gene most efficiently.Methods Three pairs of shRNAs that target atβ-catenin gene were designed. The eukaryotic expression plasmids (named shRNA1-3) were constructed and identified sequencing analysis. The plasmids were then transfected into NSCs by electroporation. The transfection rate of recombinant plasmids was measured 48 h after transfection, andβ-catenin mRNA and protein expression was determined using reverse transcriptase-polymerase chain reaction and Western blotting.Results The expression plasmids were confirmed by sequencing analysis. The transfection rate of recombinant plasmids in NSCs was approximately 62.63%±15.9%. Forty-eight hours after transfection,theβ-catenin mRNA and protein levels of shRNA1-3 group were tested.Conclusions The shRNA eukaryotic expression plasmid targeting atβ-catenin gene is constructed and selected successfully. Theβ-catenin mRNA and protein expression was suppressed significantly in NSCs by this given plasmid.we can use it to study the role of the wnt/β-catenin signaling in the development of NSCs.PartⅡInfluence of HBO on NSCs differentiationObjective To investigate the influences of HBO and rat brain extracts on proliferation of rats neural stem cells in vitroMethods Immunocytochemical staining was performed simultaneously on the 9 groups of NSCs on precoated chamber slides. The NSCs were processed for immunofluorescent labeling of FITC and CY3. The nucleus were counterstained with Hoechst 33258. The percentages of FITC-positive and CY3-positive cells were determined. NSE-positive NSCs,O4-positive NSCs and GFAP-positive NSCs were counted. The data represent the mean±SD of six separate experiments.Results In vitro HBO alone promoted ncNSCs (NSCs infected with negative control siRNA) differentiate into neurons and oligodendrocytes but not depressed astrogliosis;HIBD or normal brain tissue extract cultures promoted ncNSCs differentiate into neurons and oligodendrocytes but depressed astrogliosis, the effectus of HIBD brain extract cultures was superior to the latter and could be further increased by HBO.β-catenin siRNA decreased the NSE-positive neurons and increased GFAP-positive astrocytes in the siNSCs (NSCs infected withβ-catenin siRNA) in vitro,the effectus can not be inversed by HBO, though can be alleviate.Conclusions (1) HBO could promote ncNSCs differentiate into neuronal or Oligodendrocyte, and inhibited ncNSCs differentiate into astrocytes (2)β-catenin siRNA depressed neurogenesis, and promoted astrogliosis. (3) HBO therapy promotes the proliferation of NSCs in vitro, an effect that is correlated withβ-catenin protein. PartⅢInfluence of HBO on the expression of Ngn1 and BMP4 in the NSCsObjective To investigate the influences of HBO and rat brain extracts on the expression of Ngn1 and BMP4 in the NSCsMethods Quantitative RT-PCR was used to detect the relative contents of Ngn1 mRNA and BMP4 mRNA in the 9 groups of NSCs. Western blot was used to detect the relative contents of Ngnl protein and BMP4 protein in the 9 groups NSCs.Results HBO alone increased the level of Ngn1 mRNA and decreased BMP4 mRNA of ncNSCs; HIBD and normal brain tissue extract cultures increased the level of Ngn1 mRNA and decreased BMP4 mRNA of ncNSCs, the effect of HIBD brain extract cultures is superior to the latter and treatment with HBO further increased it; transfection ofβ-catenin siRNA could down-regulate the expression of Ngn1 mRNA and up-regulate BMP4 mRNA of NSCs in vitro respectively. HBO alone increased the level of Ngn1 protein and decreased BMP4 protein of ncNSCs;HIBD and normal brain extract cultures increased the level of Ngn1 protein and decreased BMP4 protein of ncNSCs,and the effectus of HIBD brain extract cultures is superior to the latter; it is more significant for HIBD accompany HBO to increased the level of Ngnl protein; transfection ofβ-catenin siRNA could down-regulate the expression of Ngnl protein and up-regulate BMP4 protein of NSCs in vitro respectively. Conclusions (1) HBO alone increased the level of Ngn1 gene and decreased BMP4 gene of ncNSCs, (2) transfection ofβ-catenin siRNA could down-regulate the expression of Ngnl and up-regulate the expression of BMP4 of NSCs in vitro (3) there is potential cooperative actions of BMP4 and Ngn1 on differentiating rat neural stem cell in cerebral ischemic brain. The ability of Ngnl to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor.The main contents and conclusions of the research are summarized as following:1. The shRNA eukaryotic expression plasmid targeting atβ-catenin gene is constructed and selected successfully.2. HBO could promote NSCs cultured with HIBD brain extract cultures differentiate into neuronal or Oligodendrocyte, and inhibited them differentiate into astrocytes.3. HBO therapy promotes the proliferation of NSCs in vitro, an effect that is correlated withβ-catenin protein.4. HBO therapy could promote neurogenesis byβ-catenin-induced activated Ngn1, could repress astrocytogenesis byβ-catenin-induced down regulated BMP4.更多还原