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研究生:林文音
研究生(外文):Wen-Yin Lin
論文名稱:MMP-2基因多型性與miRNA對子宮內膜異位症之影響
論文名稱(外文):MMP-2 Polymorphism and miRNA effects associated with endometriosis
指導教授:卓夙航
指導教授(外文):Suh-Hang Hank Juo
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學遺傳學研究所
學門:生命科學學門
學類:生物科技學類
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:93
中文關鍵詞:子宮內膜異位症基因多型性微核醣核酸
外文關鍵詞:endometriosispolymorphismmicroRNA
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背景:子宮內膜異位症是婦科常見的疾病,此疾病名稱是指子宮內膜組織生長在子宮以外的地方而稱之。越來越多的證據顯示基質金屬蛋白酵素(Matrix metalloproteinases,MMPs)在子宮內膜異位症的致病機轉中扮演著很重要的角色。因此,本研究的目的之一就是為了探索基質金屬蛋白酵素-2(MMP-2)與子宮內膜異位症相關性;另一個目的則是鑑定MMP-2上3’-UTR 的基因多型性與microRNA的關係。
方法:本研究鑑定了192位只患有子宮內膜異位症,這些病例組都經病理切片確診。此外,對照組有400位婦女,其中醫院來源的有162 位、社區來源的則有238 位。本研究所選的都是tSNPs,而且病例組和對照組都符合哈溫平衡。最後用多變量迴歸分析來評估基因作用及校正共變項來分析。利用GFP reporter assay來了解MMP-2 3’-UTR的基因多型性對microRNA的影響,將兩種基因型分別建構在pEGFP的質體上,在經由transfection將兩個建構成功的質體送入H1299細胞株內,也分別加了has-miR-520 precursor 與anti- has-miR-520到細胞中,並觀察螢光量是否有差異。
結果:根據實驗的結果,我們發現在位於MMP-2 3’-UTR位置上的tSNP 有達到統計上的意義,與AA基因型相比,帶AC基因型校正後的危險對比值是1.92 (P=0.005)。帶有C allele的質體所產生的螢光量比帶有A allele的質體高,及C allele所產生的mRNA比A allele的量高。並且帶有A allele的質體,所產生的GFP會受到has-miR-520 precursor 與anti- has-miR-520的影響而有不同,而 C allele不會。
結論:根據基因型分析與螢光的結果,miRSNP會影響miRNA的作用,相對也會在MMP-2的基因上調控其蛋白質的產生,預防子宮內膜異位症的形成。
Background:Endometriosis is a common gynecological disorder in which endometrial tissue is found at locations outside the uterine cavity. There is accumulating evidence suggesting that MMP-2 might play a important role in the formation of endometriosis. One the aim of this study was to investigate the relationship between MMP-2 and endometriosis; the other was to identify the SNP located at MMP-2 3''-UTR .
Method:We genotyped 192 cases with endometriosis. The disease
status was confirmed by pathology. 400 disease-free controls including 162 enrolled at the hospital and 238 enrolled from the community were compared with either disease. We selected all the tagging single nucleotide polymorphisms (tSNPs) for MMP-2. Hardy-Weinberg equilibrium was checked in both cases and controls. We used logistic regression to estimate the genetic effect and adjust the covariates. We used the EGFP reporter gene and constructed with 3’-UTR of MMP-2 two different types. Then co-transfected plsmid into H1299 cell line with hsa-miR-520 precursor and anti- hsa-miR-520. Detect fluorescence by fluorescence reader.
Result(s):We found significant association between one MMP-2 tSNP at the 3’-UTR with an adjusted odds ratio of 1.92 (P=0.005) for the AC type carriers. The fluorescence of type C plasmid is higher than type A plasmid. The has-miR-520 precursor and anti- has-miR-520 can not affect the fluorescence of type C.
Conclusion:According to result of SNP and fluorescence, we suggest that miRSNP will influence function of miRNA. Relatively, in vivo, protein expression is regulated by the same mechanisms. MMP-2 may overexpression if the woman has type C allele.
目 次
致謝……………...….. ………………….…………………. ..……………Ⅰ
中文摘要………………. …………………. …………………... ……...…Ⅲ
Abstract……..…………………………………………………….......…….Ⅴ
第一篇 子宮內膜異位症與MMP2基因多型性之相關性………...……..1
 第一章 研究背景…………………………………………………..……2
  第一節 子宮內膜異位症形成學說理論及其分類………………..…2
    1.1 植入學說………………….. ………………………………..2
    1.2 淋巴與血流轉移學說………………….. …………………..3
    1.3. 胚胎化生學說………………….. ……………………….....3
    1.4. 直接擴散學說………………….. ………………………….4
    1.5. 遺傳因素………………….. ……………………………….4
    1.6. 免疫防禦功能缺陷………………….. …………………….4
  第二節 基因對子宮內膜異位症之影響……………………………..6
    2.1 基因與子宮內膜異位症…………………………………….6
    2.2 家族聚集與雙胞胎研究……….…..…... ……...…………...6
  第三節 子宮內膜異位症與基質金屬蛋白酵素(MMPs)之關係……8
    3.1 MMPs在子宮內膜異位症的角色…………………………..8
    3.2 MMP2的重要性………………….. ………………………...8
    3.3 MMPs分類與特性背景………………….. ………………...9
      3.3.1 MMPs分類………………….. ………………………9
      3.3.2 MMPs基質金屬蛋白酵素家族有幾項特性……….11
      3.3.3 MMPs在生理狀況可受兩種不同的方式調控…….11
  第四節 研究動機與目的………………….. ……………………….13
 第二章 研究材料與方法………………………………………………14
  第一節 研究對象及資料收集………………………………………14
    1.1 病例組…………………………………………...…………14
    1.2. 對照組……………………………………………………..14
  第二節 DNA 萃取………………………………………………......16
    2.1 DNA萃取實驗步驟……………………………………......16
    2.2 溶液配置…………………………………………………...17
  第三節 基因挑選及基因型鑑定……………………………………18
    3.1 基因挑選…………………………………………………...18
    3.2 基因型鑑定原理…………………………………………...18
    3.3 基因型鑑定實驗步驟……………………………………...19
  第四節 統計分析……………………………………………………21
    4.1 哈溫平衡…………………………………………………...21
    4.2 多變量迴歸分析…………………………………………...21
 第三章 研究結果………………………………………………………22
    3.1 基本人口學特徵…………………………………………...22
    3.2 疾病與基因多型性分析…………………………………...22
第二篇 microRNA結合在MMP2的基因多型性 …………….……...24
 第一章 小分子RNA …………………………………………………25
  第一節 小分子RNA干擾現象………………………………..……26
  第二節 siRNA ………………………………………….………...….27
    2.1 siRNA背景……………………………………………….....27
    2.2 siRNA分子機制…………………………………………….27
  第三節 微核醣核酸(microRNA) …………………………………...28
    3.1 microRNA背景……………………………………………..28
    3.2 microRNA分子機制………………………………………..29
  第四節 siRNA和microRNA差異性的比較……………………….31
    4.1 來源的差異…………………………………………………31
    4.2 路徑的差異…………………………………………………31
  第五節 microRNA與SNP ………………………………………….33
    5.1 後轉錄調節的關鍵…………………………………………33
    5.2 結合位上的多型性…………………………………………33
  第六節 研究目的…………………………………………………….34
 第二章 研究材料與方法……………………………………………….35
  第一節 研究方法流程……………………………………………….35
    1.1 microRNA 與microRNA 結合位預測…………………….35
    1.2 表現載體之構築……………………………………………35
    1.3 表現載體與細胞株轉染作用………………………………36
    1.4 帶有表現載體之細胞株與外源性miRNA進行反應…….36
  第二節 細胞培養…………………………………………………….37
    2.1細胞來源與培養基………………………………………….37
    2.2細胞的培養程序…………………………………………….37
  第三節 建構pEGFP-C/A……………………………………………39
    3.1 DNA檢體來源……………………………………………...39
    3.2 載體………………………………………………………....39
    3.3 酵素…………………………………………………………39
    3.4 套組試劑……………………………………………………40
    3.5建構pEGFP-A/C載體程序………………………………...40
      3.5.1擴增MMP2 3’-UTR基因片段……………………...41
      3.5.2 DNA純化……………………………………………42
      3.5.3 以限制酶剪切DNA片段與載體…………………..42
      3.5.4 DNA片段與載體的接合作用………………………43
      3.5.5 Transformation…………………………………….....43
      3.5.6萃取小量質體DNA………………………………....44
      3.5.7 DNA sequence……………………………………….44
      3.5.8 萃取大量質體DNA………………………………...45
  第四節 Transfetion…………………………………………………...47
    4.1預測microRNA 與microRNA 結合位…………………....47
    4.2 套組試劑…………………………………………………....47
    4.3 plasmid transfection………………………………………....47
    4.4 microRNA transfection……………………………………...48
    4.5 測定螢光數值……………………………………………....49
  第五節 quantitative gene expression………………………………...50
    5.1 套組試劑……………………………………………………50
    5.2 萃取microRNA…………………………………………….50
    5.3 Reverse Transcription ……………………………………….52
    5.4 Real-time polymerase chain reaction………………………...53
 第三章 研究結果………………………………………………………54
    3.1預測microRNA與microRNA結合位……………………54
    3.2質體DNA定序…………………………………………….54
    3.3pEGFP-A與pEGFP-C plasmid在H1299細胞株中綠螢光蛋
     白的表現…………………………………………………… 54
    3.4外原性microRNA與anti-microRNA對pEGFP-A plasmid
     在細胞中綠螢光蛋白的影響………………………………55
    3.5外源性microRNA與anti-microRNA對pEGFP-C plasmid
     在細胞中綠螢光蛋白的影響………………………………55
    3.6測定不同細胞株內hsa-miR520g的含量………………..56
    3.7不同細胞株對於pEGFP-A與pEGFP-C plasmid在細胞中綠
     螢光蛋白的表現……………………………………………56
討論………………………………………………………………………58
結論與未來展望…………………………………………………………64
參考文獻…………………………………………………………………65


表 目 錄
表一、17個tSNP位置表………………………………………………...76
表二、基本人口學…………...…………………………………………...77
表三、MMP-2 tSNP基因分型…………………………………………...77
表四、MMP-2 rs7201基因分型………………………………………….80


圖 目 錄
圖一、MMP2的基因序列,t-SNP位置圖……………………………...81
圖二、pEGFP_C1 載體 Map與Multiple Cloning Site(MCS) ………...82
圖三、pDsRed_C1 載體 Map與Multiple Cloning Site(MCS) ………..83
圖四、hsa-miR-520g和hsa-miR-520h與MMP-2 3’-UTR相對應結合序
列比對圖…………………………………………………………..84
圖五、hsa-miR-520h與hsa-miR-520g的結構………………………….85
圖六、所建構之兩種不同基因型(A/C)plasmid定序結果……………...86
圖七、表現載體於共同轉染24小時之後細胞株生長情形……………87
圖八、不同A/C基因表現載體之綠螢光蛋白表現差異……………….88
圖九、正反向外原性miRNA對pEGFP-A之綠螢光蛋白GFP表現影
響…………………………………………………………………..89
圖十、正反向外原性miRNA對pEGFP-C之綠螢光蛋白GFP表現影
響…………………………………………………………………..90
圖十一、不同細胞株內has-miR520g相對含量之比較………………..91
圖十二、分別轉染pEGFP-C與pEGFP-A對不同細胞株其綠螢光蛋白表
現之影響…………………………………………………………..92
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