臺灣博碩士論文加值系統

類鐸受體(Toll-like receptor, TLR) 是先天性免疫系統(innate immune system)內諸多種樣式辨識受體(pattern recognition receptor)當中被研究最為透徹的一種，它們能辨識外來病原體上的病原相關分子樣式(pathogen-associated molecular pattern)及由損傷組織所釋放出來的損傷相關分子樣式(damage-associated molecular pattern)，進而引起發炎反應去清除外來的病原菌或是調控組織修復機制。TLR除了在免疫系統當中扮演重要的角色之外，近期亦有許多研究指出TLR也會被表現於神經細胞(neuron)當中，並且參與在神經發育的調控機制當中。我們實驗室過去的研究也明確地指出，TLR7會表現於神經細胞當中，且對於神經突起(neurites)的生長具有負向調節(negative regulation)的功能。若在初級神經細胞培養(primary neuronal culture)中加入TLR7的促效劑(agonist)，則可以清楚地觀察到神經元的樹突生成(dendritic arborization)會受到負向影響，整個樹突(dendrite)會變得較不複雜且總長度較短。另一方面，我們亦在Tlr7基因剔除小鼠(knockout mice)的海馬體CA1區(hippocampal CA1 area)當中，觀察到神經細胞的樹突生長變得更好更為複雜。至此，雖然我們已經非常確定TLR7在神經細胞的形態生成(neuronal morphogenesis)中扮演至關重要的角色，但神經元本身的TLR7 (neuronal TLR7)在這過程中所扮演的角色依然未知。為了解決這個難題，在這個研究當中，我們利用Cre-loxP系統建立二個神經專一性(neuron-specific) Tlr7條件式基因剔除(conditional knockout, cKO)小鼠模式系統(mouse model)，包括Syn1-Cre-mediated Tlr7 cKO (Tlr7Syn1KO)小鼠以及NEX-Cre-mediated Tlr7 cKO (Tlr7NEXKO)小鼠，接著使用高基氏染色法(Golgi staining)去觀察與分析該基因型小鼠內的海馬體CA1區的神經細胞的樹突生成狀況。我們的神經形態分析(neuronal morphometry)結果顯示，Tlr7Syn1KO小鼠的CA1區內的神經元的樹突總長度確實有顯著地比同窩(littermate)野生型(wild-type)小鼠的樹突還要長，而且在樹突複雜度上亦可觀察到相同的趨勢。然而，我們卻發現Tlr7NEXKO小鼠的CA1區中的神經元樹突生長比起同窩的NEXCre/+小鼠還要簡單，且樹突總長度明顯較短。似乎從我們那二個小鼠模式系統得到的結果是完全相反的。由於我們的Tlr7NEXKO小鼠其NEX-Cre等位基因(allele)是嵌入型(knockin)，設計上就已經破壞了原本的NEX基因編碼序列(coding sequence)，這也表示該等位基因實際上已經是一個無效等位基因(null allele)，可能因此影響了神經發育。此外，我們亦有發現NEXCre/+小鼠在行為實驗當中有表現顯著較多與焦慮(anxiety)相關的行為（此實驗不包含於本研究中）。相較之下，由於Tlr7Syn1KO小鼠的Syn1-Cre等位基因是一個轉殖基因(transgene)，所以該小鼠基因體(genome)中所有基因的編碼序列並不受影響。因此我們認為，在那二個相互牴觸的結果當中，從Tlr7NEXKO小鼠得到的結果可能並不能代表神經細胞內TLR7的作用。於是，我們得以推論神經元本身的TLR7確實在其形態生成的過程中扮演著關鍵而且是負向調控的角色。

Toll-like receptors (TLRs), the most well-studied pattern recognition receptors in innate immune system, are able to recognize both pathogen-associated molecular patterns of foreign pathogens and damage-associated molecular patterns derived from damaged tissues, then triggering inflammatory responses to clear invading pathogens and mediate tissue repair, respectively. In addition to their critical role in innate immunity, Toll-like receptors, including TLR3, TLR4, TLR7, and TLR8, have been shown to be expressed in neurons and involved in neurodevelopment. For TLR7, our previous studies have demonstrated that activation of TLR7 with agonists in primary neuronal cultures restricts the dendrite outgrowth. Conversely, facilitation of dendritic arborization is observed in both Tlr7 knockout cultured neurons and hippocampal CA1 neurons of P7 Tlr7 knockout mice. Nevertheless, the genuine role of neuronal TLR7 in neural morphogenesis and brain function remains elusive. In this thesis, neuron-specific Tlr7 conditional knockout (cKO) mouse models, including Syn1-Cre-mediated Tlr7 cKO (Tlr7Syn1KO) mice and NEX-Cre-mediated Tlr7 cKO (Tlr7NEXKO) mice, and Golgi staining were employed to investigate the role of neuronal TLR7 in dendritic arborization of hippocampal CA1 neurons. The results of neuronal morphometry indicate that in comparison with their wild-type (WT) littermates, total dendrite length of CA1 neurons in Tlr7Syn1KO mice is indeed significantly longer. Though there is no statistically significant difference, total dendritic tip number of Tlr7Syn1KO neurons is also obviously more than WT neurons. However, dendritic growth of CA1 neurons in Tlr7NEXKO mice seems to be repressed compared with their NEXCre/+ littermates, and this result is totally opposite to our expectation. Owning to the fact that NEX-Cre allele in Tlr7NEXKO mice is a knock-in allele with disruption of original coding sequence of NEX gene, therefore the function of that NEX allele, the development of neurons may be impeded in those mice. Furthermore, abnormal behaviors related to anxiety are also observed in NEXCre/+ mice (data not shown). In contrast, Syn1-Cre allele in Tlr7Syn1KO mice is a transgenic allele without disruption of any coding sequence in the genome. Thus, it is possible that the quantification results of Tlr7NEXKO mice cannot reflect the real function of neuronal TLR7. Taken them all together, the results conclude that neuronal TLR7 exactly plays a key role and acts as a negative regulator in neuronal morphogenesis.

Acknowledgement i
Chinese Abstract iii
English Abstract v
Contents vi
I. Introduction 1
II. Materials & Methods 8
III. Results 16
IV. Discussion 23
V. Bibliography 28
VI. Figures & Tables 32
VII. Supplementary Information 43

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