NUDT9在月經週期不同時期中子宮內膜腺體細胞之表現

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 博碩士論文 > Item 310902500/624

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题名:	NUDT9在月經週期不同時期中子宮內膜腺體細胞之表現 Expression of NUDT9 in human endometrial gland cells during different phases of menstrual cycle
作者:	溫保英 Pao-Ying Wen
贡献者:	中山醫學大學：生化暨生物科技研究所;陳凌雲
关键词:	子宮腺體細胞增殖期;分泌期;子宮內膜癌 NUDT9(Nucleoside diphosphate-linked moiety X motif 9)
日期:	2005/07/20
上传时间:	2010-03-12T02:37:20Z (UTC)
摘要:	子宮內膜腺體在月經週期的不同時期會呈現不同的形態，形態的差異可能與某些荷爾蒙或基因的調控有關。在本研究中，首先承接先前實驗DDRT-PCR去找出子宮腺體細胞增殖期及分泌期具表現差異的基因，再與NCBI BLAST網站搜尋比對，發現數個值得繼續分析的基因。本篇研究針對NUDT9蛋白的功能及表現做進一步探討。目前已知人類NUDT9是Nudix hydrolase family的成員之一，它具有ADP-ribose pyrophosphatase的活性。本論文首先將NUDT9 cDNA部份片斷選殖出來進行定序，接著以 Ni-NTA純化得到pET32a (Thioredoxin)/NUDT9融合蛋白並以此蛋白去免疫兔子得到抗體，再利用免疫組織化學染色對處於月經週期之不同時期的子宮內膜組織細胞做分析，結果發現NUDT9蛋白在增殖期表現量較分泌期表現量高。另外，取子宮內膜癌(細胞過度增生)觀察NUDT9染色的結果，腫瘤組織與正常組織相比呈現明顯的染色。再者，利用免疫螢光染色法對三株細胞株(A549、Huh7、HUVEC)染色以共軛焦顯微鏡觀察得知，此蛋白除表現在細胞質上，另外也可明顯地看到蛋白在細胞核的表現。綜合本研究之實驗結果可知NUDT9確實在細胞增生時明顯表現，至於細胞核內確切的作用機轉仍待進一步研究。 The glandular epithelial cells of the endometrium manifest distinct and different morphology during the different phases of the menstrual cycle. The differences may involve many hormones and are regulated by many genes. In our laboratory, we have identified the specific gene, NUDT9 (Nucleoside diphosphate-linked moiety X motif 9), expressed in the early proliferative phase or in the secretory phase. In this study we focused on the function and expression of NUDT9 protein. NUDT9 gene functions as a highly specific ADP-ribose (ADPR) pyrophosphatase. The human NUDT9 gene has been mapped to 4q22 and shown to give rise to two alternatively spliced mRNAs, the NUDT9α and NUDT9β, encoding a member of the Nudix hydrolase family. First we performed sequencing on the NUDT9 gene we identified, purified this 33 kDa protein with Ni-NTA, and then used this protein to immunize the rabbit to obtain the polyclonal antibody. We found that NUDT9 protein is differentially expressed in the endometrial gland cell by immunohistochemical stain. Besides, NUDT9 was found to express much more prominently in the endometrial carcinoma cells when compared with the surrounding non-cancer cells. According to the immunofluorescence stain of the three cell lines (A549、Huh7、HUVEC) with the assistance of confocal microscopy, we further demonstrated this protein is expressed in both the cytoplasm and nucleus, suggests that NUDT9 is expressed in cell cycle progression(S phase of DNA synthesis), and subsequently promotes the cellular proliferation. However, the exact mechanism was still largely unknown, and awaits future study.
URI:	http://140.128.138.153:8080/handle/310902500/624
显示于类别:	[生化微生物免疫研究所] 博碩士論文

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