臺灣博碩士論文加值系統

當細胞在進行DNA複製及基因重組時偶而會有錯誤配對的情形產生，此時DNA配對錯誤修補系統會進行錯誤配對的修補以維持細胞遺傳基因的穩定性。MutS homolog 6蛋白質，簡稱MSH6，為真核生物之DNA錯誤配對辨識蛋白，其可針對單一鹼基配對錯誤及嵌入或刪除之未配對寡核酸環進行結合並與其他蛋白質形成複合體進行修補工作。目前於斑馬魚中已經選殖出MSH2及MSH6之基因，原位雜合實驗結果發現MSH2基因於受精後12小時至36小時會集中表現在眼睛及周圍腦室區域，並且60小時後其mRNA的表現明顯減弱，顯示MSH蛋白質在胚胎發育時期除參與配對錯誤辨識外也可能與神經發育有關。
本篇論文利用全覆式mRNA定位雜合實驗觀察MSH6於斑馬於胚胎發育早期之基因表現位置情形以及北方轉漬法mRNA表現定量分析，發現MSH6基因於受精後尚未進行細胞分裂之胚胎 ( one cell stage )即可偵測到其表現，而在6小時後卻無表現直至9小時後才又再度表現，是屬於母系之基因，並且MSH6 mRNA在授精後24小時明顯地出現在眼睛及周圍腦室的神經管區域，36及48小時則只集中表現在midbrain- hindbrain boundary ( MHB )位置，往後到了60小時基因表現的情形卻明顯的減弱；而在西方轉漬法的實驗結果發現 MSH6蛋白質在不同時期的斑馬魚胚胎當中含量穩定，並未隨mRNA變動而有起伏的情形，而MSH6 mRNA之表現與MSH2 mRNA表現相似，集中在眼睛、周圍腦室之神經管及MHB的位置。
爾後利用顯微注射的方式將MSH6 morphlino反意寡核酸以12ng的劑量送入斑馬魚one –cell stage 胚胎當中，發現MSH6蛋白質的產生明顯受抑制，觀察其神經發育相關基因之活性，結果發現在48小時之胚胎NeuroD表現開始出現異常增多的情形，而施打劑量達到20ng時，36小時之胚胎，NeuroD開始有表現異常的情形產生；而48小時之胚胎，其NeuroD在頭部的表現情形有明顯異常增強的情形產生，表現增強的區域集中在視網膜以及後方的神經節區域，由此顯示MSH6 可能直接或間接的抑制NeuroD的基因表現，但是屬於NeuroD所調控的pax6b基因卻沒有受到影響；另外在抑制MSH6蛋白質的表現後，與NeuroD 調控機制功能相似參與肌肉細胞分化的MyoD基因並未受到影響，顯示MSH6在參與基因調控的工作上具有其專一性，然而MSH6參與NeuroD詳細的調控機制仍然有待進一步證實與深入探討。

As replication or homologous recombination errors generated during cell division of living organisms, the mismatch repair system should be efficiently corrected to maintain the integrity of genetic materials. MutS homolog 6 (MSH6) is an eukaryotic DNA mismatch recognition protein binds simple mispairs and small insertion-deletion loops and associated with other mismatch repair proteins to repair DNA damage. The msh2 and msh6 genes in zebrafish have previously been cloned and recombinant MSH2 or MSH6 was shown to bind preferentially to G-T mispair.
In this study, we used whole-mount in situ hybridization to detect the msh6 gene expression during development in early zebrafish embryos and preformed Northern blot assays to quantify the msh6 mRNA transcription. msh6 mRNA was observed between one cell stage and 3.7 hpf. and disappeared at 6 hpf. Following 9 hpf, msh6 mRNA expression was again observed and by 24 hpf. , msh6 expression was expressed strongly in eye and brain. The expression region of msh6 in 36 hours embryos was mainly at midbrain-hindbrain boundary. It suggested that the expression of msh6 mRNA was maternally derived mRNA and maybe had an another function besides the mismatch repair.
The MSH6 expression in embryos was significantly inhibited by microinjection of 12 ng MSH6 morphlino antisense oligonucleotide. The expressions of neural growth related genes were also monitored. The dosages of MSH6 morphlino was increased to 20 ng, NeuroD started to express abnormally in 36 hours embryos. The ectopic expression of NeuroD at head was observed in 48 hours embryos. The ectopic expression region was restricted on retina and its backward ganglions. As the result, NeuroD gene expression could be directly or indirectly inhibited my MSH6. However, pax6b ‚The NeuroD down-stream regulatory gene , was not affected by msh6 knock down. After the inhibition of MSH6, the gene expression of MyoD, which is an important regulatory protein of muscle differentiation and its mechanism is similar to NeuroD, was not affected. It revealed that MSH6 maybe has a specificity to NeuroD gene expression. The detailed NeuroD regulation mechanism regarding MSH6 was still waiting for proof and further investigation.

中文摘要 …………………………………………………………………1
英文摘要 …………………………………………………………………3

壹、前言 …………………………………………………………………5
貳、實驗材料與方法……………………………………………………11
一、材料…………………………………………………………………11
二、方法…………………………………………………………………23
參、結果…………………………………………………………………45
一、斑馬魚早期胚胎發育msh6 基因表現位置分佈 …………………45
二、msh6 mRNA表現及蛋白質表現的定量分析 ………………………46
三、利用msh6 morpholino抑制MSH6蛋白質表現 ……………………47
四、Msh6 knock down對於pax6b表現的影響…………………………48
五、Msh6 knock down對於NeuroD表現的影響 ………………………49
六、Msh6 knock down對於myoD表現的影響 …………………………50
肆、討論…………………………………………………………………52
伍、參考文獻……………………………………………………………57
陸、實驗圖表及附圖……………………………………………………64

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