- 學位論文
小鼠雙同源箱基因(mDux)表現分佈與功能分析
Expression profiles and functional analyses of the mouse double homeobox gene : mDux
摘要
同源箱基因是一群具有轉錄調節能力的基因，此基因群可藉由轉 譯出具有同源箱區的蛋白質（homeodomain proteins）來調控胚胎發 育過程的細胞分化及決定細胞的命運。之前實驗室根據同源箱區的高度保留性氨基酸序列來設計退化性核酸引子（degenerated oligonucleotide primers），以polymerase chain reaction（PCR）的方式篩選到一個新的同源箱基因，且可同時轉譯出兩個同源箱區，因此將它並名為mDux （murine double homeobox gene）， mDux基因可能是人類DUX4基因在小鼠的同源基因，研究指出，DUX4基因可能是造成顏面及肩胛肱肌肉失養症（FSHD：Facioscapulohumeral Muscular Dystrophy）的候選基因。mDux基因具有兩種轉錄物，mDux與mDux-s。以RT-PCR分析mDux基因在成鼠組織之表現，顯示mDux基因主要表現於小鼠性腺、眼球和大腦。以切片原位雜交技術和免疫組織化學染色分析卵巢、睪丸和眼睛組織，顯示mDux表現於卵母細胞、精原細胞和神經節細胞。進一步以全胚胎原位雜交分析胚胎發育時期mDux基因的表現，顯示mDux在早期胚胎主要表現在四肢和尾巴，進一步以免疫組織化學染色分析不同天數胚胎，顯示mDux蛋白表現在四肢的纖維狀已分化肌肉細胞，與MyoD染到的單核肌纖維母細胞明顯的不同，以RT-PCR分析比較mDux與MyoD基因在四肢發育過程中的表現趨勢，發現mDux與MyoD一樣在四肢肌肉發育過程持續的表現，但成熟肌肉組織測不到mDux與MyoD基因的表現，由於mDux基因的表現由胚胎12.5到15.5有明顯的增加，推測mDux與MyoD可能表現在不同分化時期的四肢肌肉細胞，另外我們也證實了mDux蛋白表現於體腔骨骼肌，包括epaxial及hypaxial 中的肌纖維細胞。以RT-PCR分析不同培養分化時期之肌纖維母細胞細胞萃取RNA發現，mDux的表現趨勢和myogenin的表現趨勢相近。同時以細胞免疫螢光染色技術分析顯示，mDux表現在肌管（myotube）肌肉細胞的細胞核中。以RNAi抑制肌纖維母細胞內mDux基因的表現，造成分化中肌纖維母細胞死亡，對於mDux在肌肉發育的影響還需進一步的研究。我們也將建立肌肉專一性表達mDux基因的基因轉殖鼠，分析當轉殖鼠成體肌肉過度表達mDux基因時，是否出現類似FSHD病人的肌肉萎縮現象。
關鍵字
同源箱基因並列摘要
The homeobox gene products act as transcription factors that regulate the cell differentiation and decide the cell fates during animal developmental process. We performed a degenerated oligonucleotide polymerase chain reaction （PCR） to screen and isolate a novel homeobox gene, murine double homeobox gene （mDux）, which encodes two homeodomains. mDux gene is the ortholog of human DUX4 gene, which is one of candidate genes of causing facioscapulohumeral muscular dystrophy （FSHD）. The mDux has two different transcripts froms: mDux and mDux-s. mDux was predominantly expressed in gonad, eye and brain by reverse transcription （RT）-PCR analysis. Moreover, mDux expressed in oocyte, spermatogonia and ganglion cell was confirmed by in situ hybridization and immunohistochemical staining. Further, whole mount in situ hybridization results show that mDux transcripts expressed in limbs and tail beginning at E9.5 and maintained to E12. MDux signals were localized in fiber-like muscle cells but not in the mononuclear cells that were labeled with MyoD （myoblast marker gene） signals by section in situ hybridization and immuno-histochemistry analyses. During limb myogenesis, mDux expressions, similar to MyoD, were continuously in postnatal limbs （3d） but slightly detectable in adult muscles by RT-PCR analysis. In addition, mDux also expressed in the trunk muscle, including epaxial and hypaxial myofiber. During trunk myogenesis, mDux expression was beginning at E12.5 embryo and increased in E13.5 embryo, myotube formation stage. We also examined the mDux expression during in vitro differentiation of C2C12 （myoblast） cell line. The mDux expression was slightly detected in confluent myocyte, but largely increased in differentiated cells following 2days in differentiation medium by RT-PCR analysis. The expression pattern of mDux is more similar to myogenin and both gene expression increase following myotube formation by RT-PCR analysis. Meanwhile, cellular immunofluorescence analysis also detected the mDux expression in nuclear of myotube. Furthermore, inhibition the expression of mDux in differentiated myoblast by mDux small interference RNA caused the cell death of differentiated myoblast. The influence of mDux in myogenesis must be clarified further. In the future, we will establish the muscle specific overexpression of mDux transgenic mice system to understand whether overexpression of mDux results in the present of FSHD phenotype.