【摘要】：干細胞一直吸引著人們的研究,是因為它可以在維持自身未分化狀態(tài)(自我更新)的同時產(chǎn)生多種子代細胞。神經(jīng)干細胞(NSC)是神經(jīng)系統(tǒng)發(fā)育的基礎,它們?yōu)閿?shù)不多卻具有高度的可塑性,能夠增殖分裂、獲得位置信息、繼而分化出不同的子代細胞(神經(jīng)元和膠質細胞),最終發(fā)育成為整個神經(jīng)系統(tǒng)。NSC的這些基本特征使其成為治療多種神經(jīng)退行性疾患和中樞神經(jīng)損傷的最有前途的侯選方法,然而,在充分理解NSC的增殖和分化調控機理之前,將NSC應用于臨床治療是不可能的。因此研究NSC的增殖和分化機理不僅有助于神經(jīng)發(fā)育的理論探討,更是將NSC應用于臨床的前提。 目前已知NSC的增殖分化受到多種細胞自主性和細胞非自主性因素的調節(jié),其中以細胞膜受體Notch為基礎的信號途徑在神經(jīng)干細胞增殖分化的調控中發(fā)揮重要作用。Notch途徑對于神經(jīng)發(fā)生的各個方面均有重要的調控作用,如維持NSC的未分化狀態(tài),調控NSC分化過程中的命運選擇,以及調控子代細胞的最終成熟。但是關于Notch途徑發(fā)揮調控作用的具體機制,即它是如何調控NSC和神經(jīng)祖細胞(INP)的增殖和分化,尤其是體內的狀況,還有待于進一步的研究。 在本課題中,我們擬構建中樞神經(jīng)系統(tǒng)(CNS)特異性阻斷Notch途徑的轉基因小鼠,并在此基礎上在體觀察Notch途徑阻斷后NSC的各種異常表型,包括NSC自身的增殖分化的異常、Notch阻斷的NSC分化出的子代細胞是否有異常等。最終,我們希望能夠在體闡明Notch途徑在CNS發(fā)育的各個過程中所發(fā)揮的不同作用,并初步探討其作用的機制。 本課題所取得的成果分述如下: 1、首先,我們成功構建了NesCre轉基因小鼠,并鑒定出其Cre重組酶的表達范圍主要包括基底前腦和中腦腹側等。再通過NesCre小鼠和先期構建成功的RBP-J-floxed小鼠交配,獲得在CNS特異性剔除Notch途徑中的關鍵轉錄因子RBP-J的條件性基因剔除小鼠。 2、其次,在剔除小鼠中,我們首先觀察到NSC分化的異常。在胚胎發(fā)育的早期(即E11.5時)RBP-J剔除區(qū)域的組織體外培養(yǎng)神經(jīng)球的數(shù)目有所增多。但是,這些增多的神經(jīng)球卻以神經(jīng)元祖細胞(INP)為主,提示RBP-J剔除后存在NSC向INP的提前分化。在胚胎發(fā)育的晚期(E17.5和新生時期),由于NSC池的提前消耗,RBP-J剔除區(qū)域的體外培養(yǎng)神經(jīng)球數(shù)目降低,在體觀察NSC數(shù)目也減少,同時伴隨有神經(jīng)元和膠質細胞等子代細胞的分化增加。除此以外,在E11.5時我們觀察到神經(jīng)元的發(fā)生卻有所降低。這些結果提示在正常胚胎發(fā)育過程中RBP-J介導的Notch途徑可以抑制NSC向INP的分化,并有可能對某些早期生成的神經(jīng)元的發(fā)生具有重要的調控作用。 3、再次,由RBP-J剔除的NSC分化而來的神經(jīng)元的形態(tài)也有所改變。NSC中的Notch途徑阻斷后,不僅向神經(jīng)元的分化增加,而且分化出來的神經(jīng)元的突起分支數(shù)也增多。 4、最后,我們進行了深入的調控機制研究。芯片結果提示一些miRNA分子在NSC增殖分化過程中可能發(fā)揮重要的調控作用。而在神經(jīng)元形態(tài)發(fā)育方面,我們發(fā)現(xiàn)microRNA342-5p可以促進神經(jīng)元的突起分支長出,而Notch途徑可以抑制microRNA342-5p的表達,從而實現(xiàn)對神經(jīng)元形態(tài)發(fā)育的調控作用。 綜上所述,我們的研究闡明了經(jīng)典Notch途徑在NSC向INP分化過程中的作用,提示microRNA分子可能參與其中;Notch途徑在神經(jīng)元的終末成熟過程中也發(fā)揮著重要的作用,microRNA342-5p參與其中,受到Notch途徑的調控。這些研究為我們進一步理解CNS發(fā)育提供了詳實的實驗數(shù)據(jù),尤其是Notch途徑與microRNA分子的相互作用研究,為Notch途徑調控CNS發(fā)育的機制研究提供了新的理論知識。這些研究也為將來Notch途徑作為臨床治療潛在靶點提供了堅實的理論基礎。
[Abstract]:Stem cells have attracted much attention because they can produce a variety of offspring while maintaining their undifferentiated state (self-renewal). Neural stem cells (NSCs) are the basis for the development of the nervous system. They are small but highly plastic, capable of proliferating and dividing, obtaining location information, and then differentiating into different molecules. These basic characteristics of NSC make it the most promising candidate for treatment of many neurodegenerative diseases and central nervous system injury. However, it is impossible to apply NSC to clinical treatment until the mechanism of regulation of proliferation and differentiation of NSC is fully understood. Therefore, the study of the proliferation and differentiation mechanism of NSC is not only conducive to the theoretical study of neural development, but also a prerequisite for the clinical application of NSC.
It is known that the proliferation and differentiation of NSC are regulated by a variety of cellular autonomy and non-autonomic factors. Notch-based signaling pathway plays an important role in the regulation of neural stem cell proliferation and differentiation. Notch pathway plays an important role in all aspects of neurogenesis, such as maintaining the absence of NSC. Differentiation status, fate selection during differentiation of NSC and final maturation of progeny cells are regulated. However, the specific mechanism of Notch pathway, that is, how it regulates the proliferation and differentiation of NSC and neural progenitor cells (INP), especially in vivo, remains to be further studied.
In this study, we intend to construct transgenic mice that specifically block Notch pathway by the central nervous system (CNS), and then observe the abnormal phenotypes of NSC in vivo after Notch pathway blockade, including the abnormal proliferation and differentiation of NSC itself, and whether the offspring cells differentiated from NSC blocked by Notch are abnormal or not. To elucidate the different roles of Notch pathway in the development of CNS in vivo and to explore its mechanism.
The results of this research are as follows:
1. First, we successfully constructed NesCre transgenic mice and identified the expression range of Cre recombinase mainly in the basal forebrain and ventral midbrain. Mice.
2. Secondly, we observed the abnormal differentiation of NSC in the rejected mice. In the early embryonic development (E11.5), the number of cultured neurospheres in the rejected RBP-J region increased in vitro. However, these increased neurospheres were mainly neuron progenitor cells (INP), suggesting that there was an early differentiation of NSC into INP after RBP-J rejection. In the late stages of fetal development (E17.5 and neonatal period), the number of cultured neurons in the RBP-J exclusion region decreased due to the early consumption of NSC pools, and the number of NSCs decreased in vivo, accompanied by an increase in the differentiation of neurons and glial cells. These results suggest that the Notch pathway mediated by RBP-J may inhibit the differentiation of NSC into INP during normal embryonic development, and may play an important role in the regulation of neurogenesis in early stage.
3. Thirdly, the morphology of neurons differentiated from NSC excluded by RBP-J also changed. After the blockade of Notch pathway in NSC, not only the differentiation of neurons increased, but also the number of neurite branches of differentiated neurons increased.
4. Finally, we conducted in-depth study of the regulatory mechanisms. The results of the microarray suggest that some of the microRNAs may play an important role in the proliferation and differentiation of NSC. Thus, it can regulate the morphological development of neurons.
In summary, our study elucidates the role of the classical Notch pathway in the differentiation of NSC into INP, suggesting that microRNA molecules may be involved; Notch pathway also plays an important role in the terminal maturation of neurons, in which microRNA342-5p is involved and regulated by Notch pathway. Development provides detailed experimental data, especially the interaction between Notch pathway and microRNA molecules, and provides new theoretical knowledge for the mechanism of Notch pathway regulating CNS development.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R33

【引證文獻】

相關碩士學位論文 前1條

1 王俊偉;ADAM10在大腦皮層神經(jīng)細胞發(fā)育過程中調節(jié)作用的研究[D];浙江大學;2012年

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