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研究生:姚俊年
研究生(外文):Chuan-Nien
論文名稱:小鼠卵細胞特異性表現同源箱基因Cphx的功能分析
論文名稱(外文):Functional characterization of an oocyte specific homeobox Gene, Cphx
指導教授:王淑紅
指導教授(外文):Sue-Hong Wang
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:74
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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。在過去的研究中,我們發現一個在卵細胞及胚胎幹細胞中大量表現的基因—Cphx(Cytoplasmic Polyadenylation Homeobox gene)。Cphx是一個專一性表達在卵細胞的母源性(maternal effect)基因,從胚胎第13.5天開始到成熟的卵細胞都有Cphx的表現,本研究論文以實驗室產製的Cphx多株抗體分析Cphx蛋白質的表現,結果顯示Cphx蛋白質在卵巢組織的表現量由三週開始逐漸增加,且僅表現於卵巢組織中次級濾泡的卵細胞中。本論文進一步以RNAi的策略來探討Cphx基因的功能,利用Zp3(Zona pellucida glycoprotein 3)promoter(專一性在卵細胞中啟動)驅動Cphx序列,使其能轉錄出雙股RNA干擾Cphx基因的表現,將此Cphx-RNAi construct注射入小鼠受精卵中,製作出基因轉殖鼠。在我們所得到五株不同copy number數的基因轉殖鼠中,轉殖基因套數較多M50D轉殖鼠(高:8 copy;其他:3 copy以下),發現有明顯的生育能力下降的狀況(正常:約8隻/胎;轉殖鼠:約4隻/胎),而其他株在生育能力方面,則沒有較明顯的變化。我們亦以real-time PCR進行了RNA表現的分析,在M31、M47、M48、M50及M50D這四株十週大轉殖基因鼠的卵巢組織中,均發現Cphx mRNA表現量下降的情況。我們更進一步分析基因轉殖鼠M50D與衛兵鼠卵巢中卵細胞的數目,發現M50D卵細胞數目比衛兵鼠減少16%;而另一方面,我們也找到Cphx可能的上游基因Figlα,從近來的文獻中指出,Figlα的基因剃除鼠中,也同時偵測到Cphx基因的表現量降低,於是我們構築了Figlα蛋白質表現系統,另外將Cphx promoter區域接上reporter gene,共轉殖入細胞中,觀察到Figlα能促進Cphx promoter後的reporter gene表現量上升,我們推測Cphx可能直接受到Figlα的調控,另外轉殖鼠中Cphx 表現下降的同時,Gdf9的表現也明顯的減少,因此推測Gdf9可能是受Cphx調控的下游基因。

The homeobox gene products act as transcription factors during animal developmental process. From previous results, we identified a novel homeobox-containing gene, Cphx (Cytoplasmic Polyadenylation Homeobox gene), which is preferentially expressed in embryonic stem cells and oocytes. De novo expression of Cphx started at 13.5 days postcoitum in the ovaries, which coincides with the initiation of oogenesis. Since oocyte maturation is a complex process and the mechanism of gene regulation during oogenesis is unclear, studies of Cphx functions in vivo may have a great help to decipher them. Here, we used anti-Cphx polyclonal antibodies to analyze Cphx protein expression pattern. The results of Western blotting and immunohistochemistry indicated that Cphx proteins were increasingly expressed from 3-week ovaries and only in secondary follicles. We further used RNA interference technique to explore the in vivo functions of the Cphx gene. We generated transgenic mice expressing a double-stranded interfering Cphx RNA, drived by an oocyte-specific Zp3 promoter, to interfere in vivo Cphx gene expression. Among five transgenic lines, only the line M50D with the highest copy of transgenes (eight copies) showed significant decrease in female fertility than control littermates. Results of real-time PCR also showed decrease in Cphx gene expression in adult ovaries of these four transgenic lines M31, M47, M48, M50 and M50D. Compared the oocyte numbers of M50D transgenic mice with control littermate, there is only 16% decrease in M50D line. We also found Figlα might directly regulate Cphx expression. The significant decrease of Cphx expression in Figlα knockout mice was recently identified. Figlα is a gene which only expresses in germline cell and can regulate the development of oocytes and follicles. Thus, we constructed a Figlα expression vector and cotransfect with reporter genes drived with different lengths of Cphx promoter. Reporter genes with Cphx promoter were upregulated by Figlα expression. Therefore, we presumed that Figlα directly regulates Cphx expression. Since the expression of Cphx and Gdf9 was decreased at the same time in Cphx RNAi transgenic mice, we suggest that Gdf9 expression was regulated by Cphx.

頁次

一、 摘要---------------------------------------------1
二、 前言---------------------------------------------5
三、 材料與方法--------------------------------------15
四、 結果--------------------------------------------27
五、 討論--------------------------------------------42
六、 圖表--------------------------------------------51
七、 參考文獻----------------------------------------61
八、 附錄--------------------------------------------64
九、 附圖--------------------------------------------69




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