【作者】 李英博；

【导师】 王莎莉；

【作者基本信息】 重庆医科大学 ， 组织工程与细胞工程， 2010， 博士

【摘要】 目的神经干细胞因具有自我更新和多向分化的生物学特征,已成为组织细胞工程研究的基础模型和移植治疗神经系统多种疾病的种子细胞,也为以往认为不可能治愈的神经系统退行性疾病的治疗带来了新的希望。神经干细胞的研究已成为当今生物学领域最热门、最前沿、最具活力的课题之一。各国政府都在投入更大的人力、物力和财力,希望在此领域中获得突破,掌握先机,为全球社会的经济发展和人民的健康做出巨大贡献。迄今为止,增殖和定向诱导分化仍然是神经干细胞研究的核心内容之一。而国内外的研究大多集中于细胞因子对NSCs的影响上,利用传统中药诱导NSCs的增殖分化研究甚少。人参是我国传统名贵中药,具有“补气生血”的强大功效,人参皂苷Rgl是人参的主要药效成分。药理学研究表明,人参皂苷Rgl具有抗衰老、减少神经细胞损伤、促进脑功能恢复、促进脑内蛋白质的合成、增加突触数目、增强记忆等功效,但对NSCs诱导分化的研究较少,特别是利用基因芯片技术筛选出人参皂苷Rg1促进NSCs分化的主要分子靶点,通过分化细胞的电生理特性,研究其功能分化水平,更是未见报道。我们研究NSCs就是利用它结构与功能的可塑性去治疗神经系统损伤及疾病。新生儿缺氧缺血性脑病患病率高达4‰,是导致脑永久性损伤,脑性瘫痪的重要原因,是临床上难治性疾病。我们将人参皂苷Rg1诱导的NSCs应用在缺血缺氧性新生模型鼠中,以进一步从体内实验证明人参皂苷Rg1诱导的NSCs功能分化的效果以及应用潜力。人参皂苷Rb1是人参另一重要活性成分。现已证实Rb1在心脑血管系统的疾病治疗中有重要的作用。例如阻滞心肌细胞的钙超载,减轻缺血和/或缺氧诱导的神经细胞死亡,增加神经元的可塑性等。蛛网膜下腔出血是一种老年人常见疾病,常伴有脑组织广泛严重损伤和高死亡率、高致残率,虽然全世界进行了广泛的研发,以寻找新的药物和治疗手段,但疗效仍然远远不如人意。因此,我们也探讨了人参皂苷Rb1对出血性脑损伤的治疗作用,期望在更大程度上挖掘人参这一“百草之王”的重大药用价值,为临床治疗新生儿缺氧缺血性脑病和蛛网膜下腔出血提供新的理论与实验资料。方法:1.从7～12w流产人胚胎大脑皮层中分离NSCs,采用悬浮细胞培养方法反复传代培养,纯化NSCs;经光学显微镜观察形态和免疫细胞化学对其鉴定;并观察不同浓度的Rgl对NSCs增殖、分化的影响。2.通过基因芯片技术,观察Rg1诱导人NSCs向神经元分化7d时靶基因表达情况,通过图形比对分析、Pathway分析、数据演算最终筛选出Rg1促进NSCs分化的最主要的目的基因和信号转导途径,再采用western blot和免疫组化的方法对筛选出的ERK靶基因进行验证。3.采用全细胞膜片钳技术分析人参皂苷Rg1诱导人胚胎神经干细胞分化7d时,神经元样细胞的膜特性以及钠、钾离子通道的功能表达,通过电生理特性证实检测细胞的功能成熟水平,以证实人参皂苷Rg1能促进NSCs功能分化成熟。4.将人参皂苷Rg1诱导的神经干细胞移植入缺血缺氧的新生鼠模型侧脑室,采用TTC染色和行为学观察对模型进行评价。通过水迷宫、体感诱发电位观测其脑功能的恢复情况,免疫组化检测移植的神经干细胞生长、分化状况。5.将人参皂苷GRb1应用到蛛网膜下腔出血的大鼠模型中,通过统计死亡率,检测脑水含量、大脑基底动脉的血管壁厚度和管腔面积,血脑屏障通透性,电镜检测血管壁的损伤情况,以及神经功能学评分来客观评价Rb1的治疗效果。通过检测脑神经元凋亡情况和p53、caspase-3、Bax以及Bcl-2等关键性凋亡通路相关蛋白的表达来探讨GRb1作用的可能机理。结果:1.体外一定浓度的Rg1有明显的促进NSCs增殖和分化的功效。Rg1浓度为120μg/ml时,MTT的OD值最大,促增殖效果最好。Rg1浓度为10μg/ml时,NSE、GFAP和Gal-c的细胞阳性率开始升高,20μg/ml时,分化的细胞达到高峰,并比IL-1组效果略好。但继续再增加Rg1的浓度对提高分化细胞的阳性率没有帮助。2.基因芯片检测和数据分析可以筛选出人参皂苷Rg1促进NSCs增殖分化的主要靶基因和信号转导通路。基因芯片共检测到Rg1诱导NSCs分化过程中差异表达基因675个,其中显著上调的基因有255个,显著下调的基因有420个。基因种类主要有与细胞生物合成、细胞代谢、转录正向调控、中枢神经系统发育、细胞分化、离子通道活性等相关基因。分别占差异基因总数的21.6%、11.7%、6.4%、5.1%、3.4%和1.5%。其中与NSCs分化相关最主要的上调基因有:syntaxin 1基因,α-tubulin基因,下调基因有Wnt抑制因子-1。与调控离子通道相关的基因有:编码受体门控性阳离子通道(ROC)蛋白的基因,编码4型内向整流K+通道的基因和编码TRPm2通道蛋白的基因。通过Pathway分析发现, MAPK(丝裂原活化蛋白激酶)通路中的ERK(细胞外信号调节蛋白激酶)在Rg1诱导NSCs分化过程中扮演重要角色。而CAMP(环磷酸腺苷)-PKA(蛋白激酶A)和PI3K(磷脂酰肌醇-3激酶)-AKT信号传导通路在Rg1促增殖过程中发挥重要作用。经western blot和免疫组化检测,Rg1诱导NSCs分化过程中,ERK1/2蛋白明显上调,并且其磷酸化可以被Rg1激活,30min时达到高峰,60min时消失。在使用ERK阻断剂PD98059后,NSCs的分化率明显下降。得到与基因芯片相同的结果。3.全细胞膜片钳检测证实,人参皂苷Rg1能促进NSCs电生理特性的表达和功能分化成熟。人参皂苷Rg1(20μg/ml)诱导NSCs分化7天时神经元样细胞的膜静息电位:45.70±2.63 mv;膜电容:26.89±1.91 pf;膜输入阻抗:877.51±20.44 M?;均较对照组有明显差异(P<0.05)。显示了更为成熟的神经元细胞膜特性。记录到电压依赖性的快速激活、快速失活的内向Na+电流,并可被TTX阻断,其平均峰值为711.48±158.03 pA,检出率50%,对照组267.24±71.15 pA,检出率22%,均有统计学意义。记录到电压依赖性的外向K +电流(经鉴定为快速激活的瞬时外向型K+电流和延迟整流型的外向K +电流),平均峰值1070.42±177.18 pA,对照组:798.11±100.02 pA,(P<0.05)。4.移植Rg1诱导后的NSCs,可以在治疗新生大鼠缺血缺氧性脑损伤中发挥较好的作用。移植Rg1诱导的人NSCs到的缺血缺氧模型大鼠体内1月后:水迷宫实验潜伏期:60.38±13.5 s,游泳路程:686.52±142.75 cm,较对照组明显缩短;目标象限探索时间:40.72±6.14 s,较对照组明显延长(P<0.05)。体感诱发电位实验潜伏期18.42±1.79 ms,较对照组明显缩短;振幅:227.28±19.38μv,较对照组明显增大(P<0.05)。抗人NSE抗体免疫组化染色显示:移植组的脑切片中,分化的神经元样细胞在皮层呈散在表达,而在海马区域呈集中表达并围绕缺血损伤区域生长。5.人参皂苷Rb1对蛛网膜下腔出血(SAH)的脑损伤有积极的治疗和保护作用。20mg/kg Rb1治疗SAH模型大鼠后:死亡率15.15%;假手术组6.67%,生理盐水组24.32%。自发活动评分提高;脑水肿明显降低;血脑屏障通透降低;血管内皮细胞电镜及光镜组织病理学明显改善;大脑基底动脉管腔面积明显增大而管壁厚度明显降低;神经元凋亡指数明显减少。促凋亡蛋白p53,以及由p53激活的Bax蛋白和caspase-3蛋白的表达明显下调,抗凋亡蛋白Bcl-2明显上调。结论:1.一定浓度的Rg1有明显的促进NSCs增殖和分化的功效。2.通过基因芯片检测可以筛选出Rg1促NSCs增殖、分化过程中的重要靶基因。其中与分化相关的主要基因是:syntaxin 1基因,α-tubulin基因,WIF-1基因等;与离子通道调控相关的靶基因主要是:ROC基因,TRPm2基因,4型内向整流K+通道基因等。而数据分析得出人参皂苷Rg1促进NSCs增殖、分化的机制与CAMP-PKA、PI3K-AKT、ERK信号通路的激活有关。3.人参皂苷Rg1能促进NSCs电生理特性的表达和功能分化成熟。4.移植Rg1诱导后的NSCs,可以在治疗新生大鼠缺血缺氧性脑损伤中发挥较好的作用。5.人参皂苷Rb1对蛛网膜下腔出血(SAH)诱发的脑损伤有积极的治疗和保护作用。更多还原

【Abstract】 ObjectiveNeural stem cells has become the basis model in the research of tissue engineering and seed cells in transplantation treatment for a variety of diseases nervous system because of self renewal and differentiation of biological characteristics, but also bring a new hope for the neurodegenerative diseases that can not be cured in the past. Neural stem cells biology research has become one of the most popular, cutting-edge and active issue. Governments are putting more human, material and financial resources, hoping to get a breakthrough in this field, to master the initiative, and make a significant contribution for the economic development of the global community and the health of the people.So far, induction of proliferation and directed differentiation is still one of the cores in neural stem cell research. Domestic and foreign research focused largely on the impact of cytokines on the NSCs, but the use of traditional Chinese medicine induced proliferation and differentiation of NSCs is very little. Ginseng is a traditional precious medicine in China with the powerful effect of“Supplement Qi and Engendering Blood”, Ginsenoside Rgl is the major components of ginseng. Pharmacology studies have shown that ginsenoside Rgl has the effect of anti-aging, reducing nerve cell injury, promoting recovery of brain function and brain protein synthesis, increasing the number of synapses, enhancing memory and other effects. But the research on induced differentiation of NSCs is very less. Especially, using gene chip technology to select major molecular target of ginsenoside Rg1-induced NSCs differentiation, and investigate the level of functional differentiation by the electrophysiological characteristics of differentiated cells are not reported.Our research on induced NSCs is to use its structure and function of plasticity solving very difficult nervous system injury and degenerative disease in clinical. Currently, the prevalence of neonatal hypoxic ischemic encephalopathy is 4‰, and the disease will cause permanent brain damage. It’s an important factor leading to cerebral palsy. In response to this disease, we will apply the Rg1-induced NSCs to make exploratory study.In addition, Ginsenoside Rb1 is one of the main active ingredients in the ginseng, and the research for its pharmacological effects has been studied for many years. In the past decade, Rb1 has been demonstrated in heart and brain systems played an important role, such as blocking calcium overload in myocardial cells, reducing ischemia and/or hypoxia-induced neurons death, increasing the neuronal plasticity and so on. In recent years, the research of subarachnoid hemorrhage (SAH) is in full swing. The professional association for this study is extensively seeking drugs and advanced treatment methods for easing the serious consequences caused by bleeding around the world, but progress is very limited. In view of broad and strong pharmacological effect in the central nervous system, we will focus on examining the role of Rb1 in the treatment of hemorrhagic brain injury(SAH), and look forward to digging significant medicinal value of "King of Herbs" in greater extent. We will provide new theoretical and experimental data for the treatment of neonatal hypoxic ischemic encephalopathy and subarachnoid hemorrhage.Methods1. We isolated NSCs from 7~12w human embryo cortex, and purified NSCs by using the suspensory cell culture. We observed the shape of NSCs by optical microscopy, identified by immunocytochemistry. And we investigated the effects of different concentrations of Rgl on NSCs proliferation and differentiation.2. By gene chip technology to observe the target gene expression in Rg1 induced NSCs differentiate into neurons at 7d. Through the graphical comparative analysis, Pathway analysis and data calculations, the main purpose genes and signal transduction pathway were selested in differentiationed NSCs. Finally, using western blot and immunohistochemical methods the ERK target genes were verified.3. Using whole cell patch clamp, differentiated neuron-like cells were assayed for the membrane properties as well as sodium, potassium channel functional expression at 7d. Through the electrophysiological properties to confirm the cell function level of maturity and confirm that ginsenoside Rg1 can promote the differentiation and maturation of NSCs function.4. Transplanted ginsenoside Rg1-induced NSCs into the neonatal rat model of hypoxic ischemic by lateral ventricle injection, and evaluated the model by using TTC staining and behavioral observation. Through the water maze and somatosensory evoked potential observed recovery of brain function and through the immunohistochemical detected growth and differentiation status about transplanted NSCs.5. Ginsenoside GRb1 was applied to rat model of subarachnoid hemorrhage. Through the statistics of mortality, measurement of brain water content, basilar artery wall thickness and luminal area, blood-brain barrier permeability, electron microscopy detection of vascular wall damage, and nerve function score the therapeutic effect of Rb1 was evaluated, And then, the possible mechanisms of GRb1 were explored by detecting the neuronal apoptosis and the key proteins of apoptosis pathway related such as p53, caspase - 3, Bax and Bcl - 2 .Results1. Rg1 significantly promoted NSCs proliferation and differentiation in vitro.●Rg1(120μg/ml) had the maximum MTT OD value and the best effect for NSCs proliferation.●When Rg1 concentration was 10μg/ml, the positive rate of NSE, GFAP, and Gal-c began to increase. When the concentration reached 20μg/ml, the differentiation of cells reached a peak. And the effect was slightly better than IL-1 group. But continued to increase the concentration of Rg1 did not help to promote the positive rate of differentiated cells.2. Gene chip detection and data analysis could select main purpose genes and signal transduction pathway in Rg1 induced NSCs proliferation and differentiation.●Gene chips detected that the differentially expressed genes number are the 675 in Rg1 induced NSCs differentiation. The significantly increased genes were 255, and significantly reduced genes were 420.●Gene types are mainly associated with cell biology synthesis, cell metabolism, positive regulation of transcription, central nervous system development, cell differentiation, ion channel activity, respectively, 21.6%, 11.7%, 6.4%, 5.1%, 3.4% and 1.5%. The most related upregulation genes in NSCs differentiation were syntaxin 1 gene,α-tubulin gene, and the downregulation gene was Wnt inhibitory factor -1. Associated with the regulation of ion channel genes were encoding receptor-gated cation channel (ROC) protein gene and TRPm2 channel proteins gene.●Through Pathway analysis, the ERK (extracellular signal-regulated protein kinase) /MAPK(mitogen-activated protein kinase) pathway played an important role in Rg1 induced NSCs differentiation. However the CAMP (cyclic adenosine monophosphate)-PKA (protein kinase A) and PI3K (phosphatidylinositol-3 kinase)-AKT signaling pathway played an important role in Rg1 induced NSCs proliferation.●Through the western blot and immunohistochemistry analysis, ERK1/2 protein was significantly increased during Rg1 induced NSCs differentiation, and its phosphorylation could be activated by Rg1, reached its peak in 30min, disappeared in 60min. After using the PD98059 of ERK inhibitor, NSCs differentiation rate decreased significantly. So we got the same results with the gene chip.3. The whole cell patch clamp test confirmed that ginsenoside Rg1 can promote the expression of NSC electrophysiological properties and functional differentiation.Ginsenoside Rg1 (20μg/ml) induced differentiation of NSCs at 7 days●The resting membrane potential of neuron-like cells: 45.70±2.63 mv; membrane capacitance: 26.89±1.91 pf, membrane input impedance;877.51±20.44 M?; these data were significantly different compared with the control group (P <0.05), and shows a more mature neurons membrane properties.●Recorded voltage dependence inward Na+ currents of fast activation, fast inactivation, and could be blocked by TTX, the average peak value of 711.48±158.03 pA , the detection rate of 50%. However in the control group, the average peak value was 267.24±71.15 pA and the detection rate was 22%. There were significant differences compared with the control group(P <0.05).●Voltage-dependent outward K+ current (identified as rapidly activating transient outward K+ current and delayed rectifier outward K+ current) was recorded. The average peak value was 1070.42±177.18 pA and the value was 798.11±100.02 pA in control group ( P <0.05).4. Transplantation of NSCs induced by Rg1 could play a better role in the treatment of HIE neonatal rats with brain injury.Transplanted NSCs induced by Rg1 into the rats of ischemia and hypoxia after 1 month:●Water maze test: The latency was 60.38±13.5 s, and the swimming distance was 686.52±142.75 cm, significantly shorter than the control group. Target quadrant exploration time was 40.72±6.14 s, significantly longer than the control group (P <0.05).●Somatosensory evoked potentials: The latency was 18.42±1.79 ms, significantly shorter than the control group. The amplitude was 227.28±19.38μv, significantly increased compared with the control group (P <0.05).●Anti-human NSE antibody staining showed that: neuron-like cells were scattered in the cortex, but the expression was concentrated in the hippocampus and grew around the ischemic injury area in transplantation group.5. Ginsenoside Rb1 had active therapeutic and protective effect on subarachnoid hemorrhage (SAH) induced brain injury. 20mg/kg Rb1 treatmented rats after SAH:●15.15% Mortality in 20mg/kg Rb1 group; 6.67% in sham operation group, 24.32% in vehicle group;●Spontaneous activity score was increased; ●Brain edema and blood-brain barrier permeability were significantly reduced;●Vascular endothelial cells histopathology were significantly improved by electron microscopy and light microscopy; Cerebral basilar artery lumen was increased and the wall thickness was decreased significantly;●Neuronal apoptosis was significantly reduced.●The expression of pro-apoptotic protein p53, the Bax and caspase-3 protein activated by p53 were significantly reduced, however anti-apoptotic protein Bcl-2 were increased.Conclusion1. A certain concentration of Rg1 significantly promoted NSCs proliferation and differentiation in vitro.2. Gene chip detection could select main purpose genes. The most related upregulation genes in NSCs differentiation were syntaxin 1 gene,α-tubulin gene, and the downregulation gene was Wnt inhibitory factor -1. Associated with the regulation of ion channel genes were encoding receptor-gated cation channel (ROC) protein gene and TRPm2 channel proteins gene. Through Pathway analysis, the ERK /MAPK pathway played an important role in Rg1-induced NSCs differentiation. However the CAMP-PKA and PI3K-AKT signaling pathway played an important role in Rg1-induced NSCs proliferation.3. Ginsenoside Rg1 can promote the expression of NSC electrophysiological properties and functional differentiation.4. Transplantation of NSCs induced by Rg1 could play a better role in the treatment of HIE neonatal rats with brain injury.5. Ginsenoside Rb1 had a active therapeutic and protective effect on subarachnoid hemorrhage (SAH) induced brain injury.更多还原