Beta-catenin信号在低氧条件下新生小鼠海马神经干细胞增殖中的作用及机制

发布时间：2018-01-04 15:50

  本文关键词：Beta-catenin信号在低氧条件下新生小鼠海马神经干细胞增殖中的作用及机制　出处：《第三军医大学》2007年博士论文　论文类型：学位论文

  更多相关文章： 海马 神经干细胞 低氧 增殖 β-catenin

【摘要】： 多种疾病,包括脑外伤、脑中风、癫痫等,均可导致脑组织缺氧,是引起神经元不可逆死亡的共同病理机制之一。神经元死亡后常导致靶器官功能障碍,导致严重残疾。因此,采取有效措施来补充或替代缺失的神经细胞,恢复对靶器官的支配,促进缺氧性脑损伤后功能恢复,具有重要作用。 研究发现哺乳动物中枢神经系统终身存在神经干细胞( neural stem cells , NSCs),主要集中在侧脑室下区(subventricular zone,SVZ)和海马齿状回颗粒下区(subgranular zone,SGZ)。在一定条件下(主要是脑缺氧损伤)这两处的NSCs均表现出增殖、迁移和多方向分化的能力。新生成的神经元可替代丢失的神经细胞,参与损伤修复。显然,利用这种内源性NSCs进行治疗具有无创、无伦理学问题、无免疫排斥等优点,是治疗缺氧性脑损伤的理想途径。 然而,虽然脑缺氧损伤可以激活NSCs原位增殖,但自发条件下NSCs增殖能力有限,更为重要的是,目前对内源性NSCs在缺氧性脑损伤后的增殖机制尚不清楚,这是影响我们通过调控这种内源性NSCs来修复中枢神经损伤的关键障碍。研究表明,Wnt/β-catenin信号通路在胚胎神经发育和肿瘤发生过程中均发挥了重要的作用。胚胎发育、肿瘤发生过程存在着共性,即处于低氧环境。新近研究发现,Wnt/β-catenin信号通路参与哺乳动物成体神经再生。综上所述,本课题首次提出β-catenin信号通路可能在低氧引发的NSCs增殖过程中具有重要的作用,为此建立了新生绿色荧光蛋白(GFP)转基因小鼠海马NSCs和星形胶质细胞体外低氧模型,利用它探讨Wnt/β-catenin信号在低氧条件下NSCs增殖过程中的作用及其机制。 本课题研究包括以下三个部分: 第一部分低氧对新生绿色荧光蛋白(GFP)转基因小鼠海马神经干细胞(NSCs)增殖和分化的影响 本实验通过械分离和无血清培养法获取新生GFP转基因小鼠海马NSCs。体外培养的NSCs呈悬浮生长,传代后可再次形成神经球,目前已稳定传代至15代。神经球经免疫荧光化学染色和免疫细胞组织化学染色鉴定呈Nestin抗原和Musashi1抗原阳性,神经球内绝大部分细胞BrdU表达阳性。神经球可被10%胎牛血清诱导分化成神经元(NSE阳性)、星形胶质细胞(GFAP阳性)和少突胶质细胞(MBP阳性)。神经球在增殖和诱导分化过程中GFP稳定表达不丢失。综上表明成功从新生24 h内的GFP转基因小鼠海马组织获取具有自我更新和多向分化潜能的NSCs。 利用新生GFP转基因小鼠海马NSCs建立体外低氧(5%O2)模型,探讨低氧对新生小鼠海马NSCs增殖和分化的影响。结果发现,低氧条件下,NSCs克隆形成率,BrdU阳性细胞率和MTT值均高于常氧条件(P0.05)。NSCs诱导分化为神经元和星形胶质细胞的数量与常氧条件相比,分比增加了31.06%和19.79%(P0.05)。表明低于传统培养的低氧条件,可以促进新生小鼠海马NSCs的体外增殖,增加分化细胞尤其是神经元的数量。 第二部分Beta-catenin信号在低氧条件下海马NSCs增殖中的作用 本实验首先通过RT-PCR法检测到体外培养的海马NSCs表达Wnt/β-catenin信号通路的主要分子,包括Wnt膜受体(Frz1),β-catenin,Axin1,GSK-3β和LEF1,表明海马NSCs具备对Wnt信号反应的能力。荧光素酶活性检测法检测发现低氧条件,海马NSCs内荧光素酶活性明显提高,间接表明低氧增加细胞内β-catenin的含量。通过Western Blotting法进一步检测发现,低氧培养NSCs 12h和24h后,胞浆和胞核中β-catenin均有所增加,尤以胞核增加明显(P0.05)。结果还发现,Wnt/β-catenin的靶基因cyclinD1表达也增高(P0.05)。 依据上述结果,我们推断β-catenin信号可能参与低氧条件下海马NSCs增殖。因此采用电穿孔的方法分别增加和抑制NSCs内β-catenin的表达,通过MTT法检测NSCs增殖情况,Western Blotting法检测β-catenin下游的靶基因cyclinD1的表达情况。结果发现,增加NSCs内β-catenin的表达,可以进一步促进低氧条件下NSCs的增殖和cyclinD1的表达(P0.05);抑制NSCs内β-catenin的表达,降低低氧条件下NSCs的增殖和cyclinD1的表达(P0.05),但仍高于常氧组(P0.05)。表明β-catenin信号通过调节cyclinD1的表达参与低氧引起的NSCs的增殖过程。 第三部分低氧增加海马NSCs内β-catenin表达的机制 低氧增加NSCs内β-catenin含量的机制尚不明确。研究表明,Wnt3和Wnt3a蛋白对胚胎海马发育和成体海马神经再生具有重要的作用。星形胶质细胞不仅是脑内主要的支持细胞,而且还通过分泌一些因子(如Wnt3)参与其他细胞的调控。因此本实验首先探讨低氧培养是否具备上调海马星形胶质细胞内Wnt3,Wnt3α的作用。通过RT-PCR法检测低氧条件下新生小鼠海马星形胶质细胞内Wnt3,Wnt3α的表达变化,结果发现,常氧培养的海马星形胶质细胞内存在Wnt3的表达,低氧下调Wnt3的表达(P0.05);而常氧和低氧条件培养的海马星形胶质细胞内均未不表达Wnt3a。研究表明,丝氨酸/苏氨酸(Ser/Thr)蛋白激酶AKT激活后,通过磷酸化糖原合成酶激酶-3β(GSK-3β)的Ser 9残基抑制其活性,进而增加β-catenin积聚。本研究通过Western Blotting检测发现,低氧上调海马NSCs内Akt蛋白和GSK-3β蛋白的磷酸化水平(P0.05)。结果提示低氧增加β-catenin的含量与与海马星形胶质细胞分泌的Wnt3,Wnt3a无关,与低氧促进海马NSCs内Akt蛋白和GSK-3β蛋白的磷酸化有关。 综上所述,低氧促进体外新生GFP转基因小鼠海马NSCs增殖和分化,β-catenin信号通过调节cyclinD1的表达参与低氧条件下海马NSCs的增殖过程,β-catenin的作用发挥与海马星形胶质细胞分泌的Wnt3,Wnt3a无关,与低氧促进NSCs内Akt蛋白和GSK-3β蛋白的磷酸化有关。本研究对于通过调控内源性NSCs增殖来促进中枢神经损伤后的修复具有重要的意义。
[Abstract]:A variety of diseases, including brain trauma, stroke, epilepsy, which can lead to brain hypoxia, which is one of the common pathological irreversible neuronal death mechanisms. Target organ dysfunction often leads to neuronal death, lead to severe disability. Therefore, to take effective measures to supplement or replace the loss of nerve cells, reinnervation of target organ. Promote the hypoxic brain function recovery after injury, plays an important role.
The study found that the mammalian central nervous system lifetime of neural stem cells (neural stem cells, NSCs), mainly concentrated in the subventricular zone (subventricular zone, SVZ) and the hippocampal dentate gyrus (subgranular, zone, SGZ). Under certain conditions (mainly brain injury) of these two NSCs show the ability of proliferation, migration and differentiation of newly generated neurons can replace lost nerve cells involved in wound healing. Obviously, were treated with noninvasive using the endogenous NSCs, no ethical problem, the advantages of no rejection, is an ideal way for the treatment of hypoxic brain injury.
However, although hypoxia can activate NSCs in situ proliferation, but spontaneous proliferation of NSCs under the condition of limited, more importantly, the proliferation mechanism of endogenous NSCs in hypoxic brain injury is not clear, this is our impact through the regulation of this endogenous NSCs to fix the key barriers to research shows that damage to the central nervous system. Wnt/, beta -catenin signaling pathway in the process of embryonic development and tumorigenesis have played an important role. Embryonic development and tumorigenesis have common features, namely in the hypoxic environment. Recent studies showed that Wnt/ beta -catenin signal pathway in mammalian somatic nerve regeneration. In conclusion, this paper puts forward for the first time plays an important role in NSCs the proliferation of beta -catenin signaling pathway in hypoxia caused, for the establishment of new green fluorescent protein (GFP) transgenic mice hippocampus NSCs and astrocytes The model of hypoxic cells in vitro was used to explore the role and mechanism of Wnt/ beta -catenin signal in the proliferation of NSCs under hypoxia.
This research includes the following three parts:
The first part of hypoxia effect on the proliferation and differentiation of hippocampal neural stem cells (NSCs) in GFP transgenic mice
Through the experiments of mechanical isolation and serum-free culture method to obtain the newborn GFP transgenic mice hippocampus NSCs. NSCs in vitro was suspended growth, after the passage of neurospheres formed again, has stable passage to the 15 generation. The neurospheres by immunofluorescence staining and immunohistochemical staining showed Nestin antigen and Musashi1 antigen positive. Neurospheres most cells expressed BrdU. The neurospheres can be differentiate into neurons induced by 10% fetal bovine serum (NSE positive), astrocytes (GFAP positive) and oligodendrocytes (MBP positive). Neurospheres in proliferation and differentiation of GFP stable expression is not lost. These results show that the successful acquisition of self-renewal and multilineage differentiation potential of NSCs. within 24 h from newborn GFP transgenic mice hippocampus
In vitro hypoxia using newborn GFP transgenic mice hippocampus NSCs (5%O2) model, to investigate the effect of hypoxia on the proliferation and differentiation of newborn mouse hippocampus NSCs. The results showed that under hypoxic conditions, NSCs clone formation rate, the ratio of BrdU positive cells and MTT value were higher than normoxia (P0.05) induce.NSCs differentiation into neurons and glial number cells compared with normoxia, ratio increased by 31.06% and 19.79% (P0.05). Hypoxia condition suggests that less than traditional culture, can promote the proliferation of NSCs in hippocampus of newborn mice in vitro, differentiated cells increase especially the number of neurons.
The role of the second part of Beta-catenin signal in the proliferation of hippocampal NSCs under hypoxic condition
The main molecular experiment first detected by RT-PCR in cultured hippocampal NSCs Wnt/ expression of -catenin signaling pathway, including Wnt receptor (Frz1), beta -catenin, Axin1, GSK-3 and LEF1 show that the hippocampus NSCs beta, have the ability to Wnt signal response. Luciferase activity detection detection of hypoxia, NSCs in hippocampus luciferase activity was significantly increased, suggesting that hypoxia increased the intracellular concentration of beta -catenin detected by Western Blotting. Further, NSCs 12h and 24h after hypoxia, the cytoplasm and nucleus of beta -catenin were increased, especially in the nucleus increased significantly (P0.05). The results also show that the target gene cyclinD1 Wnt/ beta the expression of -catenin also increased (P0.05).
According to the above results, we concluded that the beta -catenin signal may participate in hypoxic conditions. The expression of NSCs in proliferation of electroporation were increased and inhibition of NSCs beta -catenin, by detecting the proliferation of NSCs MTT method, the expression of downstream target genes of cyclinD1 beta -catenin Western Blotting detection method. The results showed that increased expression of NSCs beta -catenin, can further promote the proliferation and expression of cyclinD1 NSCs under hypoxic condition (P0.05); inhibit the expression of NSCs beta in -catenin, reduce the proliferation and expression of cyclinD1 NSCs under hypoxic condition (P0.05), but still higher than the normal oxygen group (P0.05). That involved in hypoxia induced NSCs proliferation of beta -catenin signal by regulating the expression of cyclinD1.
The third part of hypoxia increases the mechanism of the expression of beta -catenin in the hippocampal NSCs
Hypoxia increased -catenin content in the mechanism of beta NSCs is not clear. The study shows that the Wnt3 and Wnt3a protein plays an important role in the development of embryonic hippocampus and hippocampal neurogenesis. Astrocytes not only support cells mainly in the brain, but also through the secretion of some factors (such as Wnt3) is involved in regulation of other cells. Therefore in this study, to investigate the effect of hypoxia training have increased hippocampal astrocytes in Wnt3, Wnt3 alpha. By RT-PCR method under hypoxia condition detection of neonatal mouse hippocampal astrocytes in Wnt3, expression of Wnt3 alpha, it was found that normoxic cultured hippocampal astrocytes in the expression of Wnt3, down regulated expression of hypoxia Wnt3 (P0.05); and the cultivation of normal and hypoxic conditions of hippocampal astrocytes were not within the expression of Wnt3a. study showed that serine / threonine protein kinase (Ser/Thr) activation of AKT, pass After phosphorylation of glycogen synthase kinase -3 beta (GSK-3 beta) Ser 9 residues inhibit its activity, and increased beta -catenin accumulation. In this study, Blotting detected by Western, NSCs Akt in hippocampus hypoxia up-regulated protein and GSK-3 phosphorylation level (P0.05). The results suggest that hypoxia increased content of beta -catenin Wnt3. With the secretion of astrocytes in hippocampus of Wnt3a has nothing to do with hypoxia in hippocampal NSCs of Akt protein and GSK-3 protein phosphorylation.
To sum up, promote the in vitro hypoxic newborn GFP transgenic mice hippocampus NSCs proliferation and differentiation, beta -catenin signaling by regulating the expression of cyclinD1 in hippocampus NSCs proliferation under hypoxic conditions, beta -catenin secretion and play the role of astrocytes in hippocampus of Wnt3, Wnt3a, NSCs and Akt in hypoxia promotes protein and GSK-3 phosphate related. This study has important significance for promoting the CNS injury repair by regulating the proliferation of endogenous NSCs.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

【引证文献】

相关期刊论文 前1条

1 庄述娟;陈小玉;刘庆山;;神经干细胞增殖的分子机制及中药干预研究进展[J];中国实验方剂学杂志;2013年03期

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