發(fā)布時(shí)間：2018-01-04 15:50

  本文關(guān)鍵詞：Beta-catenin信號(hào)在低氧條件下新生小鼠海馬神經(jīng)干細(xì)胞增殖中的作用及機(jī)制　出處：《第三軍醫(yī)大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 海馬 神經(jīng)干細(xì)胞 低氧 增殖 β-catenin

【摘要】： 多種疾病,包括腦外傷、腦中風(fēng)、癲癇等,均可導(dǎo)致腦組織缺氧,是引起神經(jīng)元不可逆死亡的共同病理機(jī)制之一。神經(jīng)元死亡后常導(dǎo)致靶器官功能障礙,導(dǎo)致嚴(yán)重殘疾。因此,采取有效措施來補(bǔ)充或替代缺失的神經(jīng)細(xì)胞,恢復(fù)對(duì)靶器官的支配,促進(jìn)缺氧性腦損傷后功能恢復(fù),具有重要作用。 研究發(fā)現(xiàn)哺乳動(dòng)物中樞神經(jīng)系統(tǒng)終身存在神經(jīng)干細(xì)胞( neural stem cells , NSCs),主要集中在側(cè)腦室下區(qū)(subventricular zone,SVZ)和海馬齒狀回顆粒下區(qū)(subgranular zone,SGZ)。在一定條件下(主要是腦缺氧損傷)這兩處的NSCs均表現(xiàn)出增殖、遷移和多方向分化的能力。新生成的神經(jīng)元可替代丟失的神經(jīng)細(xì)胞,參與損傷修復(fù)。顯然,利用這種內(nèi)源性NSCs進(jìn)行治療具有無創(chuàng)、無倫理學(xué)問題、無免疫排斥等優(yōu)點(diǎn),是治療缺氧性腦損傷的理想途徑。 然而,雖然腦缺氧損傷可以激活NSCs原位增殖,但自發(fā)條件下NSCs增殖能力有限,更為重要的是,目前對(duì)內(nèi)源性NSCs在缺氧性腦損傷后的增殖機(jī)制尚不清楚,這是影響我們通過調(diào)控這種內(nèi)源性NSCs來修復(fù)中樞神經(jīng)損傷的關(guān)鍵障礙。研究表明,Wnt/β-catenin信號(hào)通路在胚胎神經(jīng)發(fā)育和腫瘤發(fā)生過程中均發(fā)揮了重要的作用。胚胎發(fā)育、腫瘤發(fā)生過程存在著共性,即處于低氧環(huán)境。新近研究發(fā)現(xiàn),Wnt/β-catenin信號(hào)通路參與哺乳動(dòng)物成體神經(jīng)再生。綜上所述,本課題首次提出β-catenin信號(hào)通路可能在低氧引發(fā)的NSCs增殖過程中具有重要的作用,為此建立了新生綠色熒光蛋白(GFP)轉(zhuǎn)基因小鼠海馬NSCs和星形膠質(zhì)細(xì)胞體外低氧模型,利用它探討Wnt/β-catenin信號(hào)在低氧條件下NSCs增殖過程中的作用及其機(jī)制。 本課題研究包括以下三個(gè)部分: 第一部分低氧對(duì)新生綠色熒光蛋白(GFP)轉(zhuǎn)基因小鼠海馬神經(jīng)干細(xì)胞(NSCs)增殖和分化的影響 本實(shí)驗(yàn)通過械分離和無血清培養(yǎng)法獲取新生GFP轉(zhuǎn)基因小鼠海馬NSCs。體外培養(yǎng)的NSCs呈懸浮生長,傳代后可再次形成神經(jīng)球,目前已穩(wěn)定傳代至15代。神經(jīng)球經(jīng)免疫熒光化學(xué)染色和免疫細(xì)胞組織化學(xué)染色鑒定呈Nestin抗原和Musashi1抗原陽性,神經(jīng)球內(nèi)絕大部分細(xì)胞BrdU表達(dá)陽性。神經(jīng)球可被10%胎牛血清誘導(dǎo)分化成神經(jīng)元(NSE陽性)、星形膠質(zhì)細(xì)胞(GFAP陽性)和少突膠質(zhì)細(xì)胞(MBP陽性)。神經(jīng)球在增殖和誘導(dǎo)分化過程中GFP穩(wěn)定表達(dá)不丟失。綜上表明成功從新生24 h內(nèi)的GFP轉(zhuǎn)基因小鼠海馬組織獲取具有自我更新和多向分化潛能的NSCs。 利用新生GFP轉(zhuǎn)基因小鼠海馬NSCs建立體外低氧(5%O2)模型,探討低氧對(duì)新生小鼠海馬NSCs增殖和分化的影響。結(jié)果發(fā)現(xiàn),低氧條件下,NSCs克隆形成率,BrdU陽性細(xì)胞率和MTT值均高于常氧條件(P0.05)。NSCs誘導(dǎo)分化為神經(jīng)元和星形膠質(zhì)細(xì)胞的數(shù)量與常氧條件相比,分比增加了31.06%和19.79%(P0.05)。表明低于傳統(tǒng)培養(yǎng)的低氧條件,可以促進(jìn)新生小鼠海馬NSCs的體外增殖,增加分化細(xì)胞尤其是神經(jīng)元的數(shù)量。 第二部分Beta-catenin信號(hào)在低氧條件下海馬NSCs增殖中的作用 本實(shí)驗(yàn)首先通過RT-PCR法檢測到體外培養(yǎng)的海馬NSCs表達(dá)Wnt/β-catenin信號(hào)通路的主要分子,包括Wnt膜受體(Frz1),β-catenin,Axin1,GSK-3β和LEF1,表明海馬NSCs具備對(duì)Wnt信號(hào)反應(yīng)的能力。熒光素酶活性檢測法檢測發(fā)現(xiàn)低氧條件,海馬NSCs內(nèi)熒光素酶活性明顯提高,間接表明低氧增加細(xì)胞內(nèi)β-catenin的含量。通過Western Blotting法進(jìn)一步檢測發(fā)現(xiàn),低氧培養(yǎng)NSCs 12h和24h后,胞漿和胞核中β-catenin均有所增加,尤以胞核增加明顯(P0.05)。結(jié)果還發(fā)現(xiàn),Wnt/β-catenin的靶基因cyclinD1表達(dá)也增高(P0.05)。 依據(jù)上述結(jié)果,我們推斷β-catenin信號(hào)可能參與低氧條件下海馬NSCs增殖。因此采用電穿孔的方法分別增加和抑制NSCs內(nèi)β-catenin的表達(dá),通過MTT法檢測NSCs增殖情況,Western Blotting法檢測β-catenin下游的靶基因cyclinD1的表達(dá)情況。結(jié)果發(fā)現(xiàn),增加NSCs內(nèi)β-catenin的表達(dá),可以進(jìn)一步促進(jìn)低氧條件下NSCs的增殖和cyclinD1的表達(dá)(P0.05);抑制NSCs內(nèi)β-catenin的表達(dá),降低低氧條件下NSCs的增殖和cyclinD1的表達(dá)(P0.05),但仍高于常氧組(P0.05)。表明β-catenin信號(hào)通過調(diào)節(jié)cyclinD1的表達(dá)參與低氧引起的NSCs的增殖過程。 第三部分低氧增加海馬NSCs內(nèi)β-catenin表達(dá)的機(jī)制 低氧增加NSCs內(nèi)β-catenin含量的機(jī)制尚不明確。研究表明,Wnt3和Wnt3a蛋白對(duì)胚胎海馬發(fā)育和成體海馬神經(jīng)再生具有重要的作用。星形膠質(zhì)細(xì)胞不僅是腦內(nèi)主要的支持細(xì)胞,而且還通過分泌一些因子(如Wnt3)參與其他細(xì)胞的調(diào)控。因此本實(shí)驗(yàn)首先探討低氧培養(yǎng)是否具備上調(diào)海馬星形膠質(zhì)細(xì)胞內(nèi)Wnt3,Wnt3α的作用。通過RT-PCR法檢測低氧條件下新生小鼠海馬星形膠質(zhì)細(xì)胞內(nèi)Wnt3,Wnt3α的表達(dá)變化,結(jié)果發(fā)現(xiàn),常氧培養(yǎng)的海馬星形膠質(zhì)細(xì)胞內(nèi)存在Wnt3的表達(dá),低氧下調(diào)Wnt3的表達(dá)(P0.05);而常氧和低氧條件培養(yǎng)的海馬星形膠質(zhì)細(xì)胞內(nèi)均未不表達(dá)Wnt3a。研究表明,絲氨酸/蘇氨酸(Ser/Thr)蛋白激酶AKT激活后,通過磷酸化糖原合成酶激酶-3β(GSK-3β)的Ser 9殘基抑制其活性,進(jìn)而增加β-catenin積聚。本研究通過Western Blotting檢測發(fā)現(xiàn),低氧上調(diào)海馬NSCs內(nèi)Akt蛋白和GSK-3β蛋白的磷酸化水平(P0.05)。結(jié)果提示低氧增加β-catenin的含量與與海馬星形膠質(zhì)細(xì)胞分泌的Wnt3,Wnt3a無關(guān),與低氧促進(jìn)海馬NSCs內(nèi)Akt蛋白和GSK-3β蛋白的磷酸化有關(guān)。 綜上所述,低氧促進(jìn)體外新生GFP轉(zhuǎn)基因小鼠海馬NSCs增殖和分化,β-catenin信號(hào)通過調(diào)節(jié)cyclinD1的表達(dá)參與低氧條件下海馬NSCs的增殖過程,β-catenin的作用發(fā)揮與海馬星形膠質(zhì)細(xì)胞分泌的Wnt3,Wnt3a無關(guān),與低氧促進(jìn)NSCs內(nèi)Akt蛋白和GSK-3β蛋白的磷酸化有關(guān)。本研究對(duì)于通過調(diào)控內(nèi)源性NSCs增殖來促進(jìn)中樞神經(jīng)損傷后的修復(fù)具有重要的意義。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 莊述娟;陳小玉;劉慶山;;神經(jīng)干細(xì)胞增殖的分子機(jī)制及中藥干預(yù)研究進(jìn)展[J];中國實(shí)驗(yàn)方劑學(xué)雜志;2013年03期

，

本文編號(hào)：1379070