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研究生:陳長親
研究生(外文):Chang-Chin Chen
論文名稱:分析紅血球生成基因表現
論文名稱(外文):Characterization of gene expression profile in erythropoiesis.
指導教授:孫德珊
指導教授(外文):De r - S h a n S u n
口試委員:朱崧肇陳紀雄
口試委員(外文):Sung-Chao ChuJi-Hshiung Chen
口試日期:2014-07-29
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物暨人類遺傳學系碩士班
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:2014
畢業學年度:102
語文別:中文
論文頁數:97
中文關鍵詞:紅血球生成骨髓分化不良症候群CD235aCD71CD36
外文關鍵詞:Erythropoiesismyelodysplastic syndromeCD235aCD71CD36
相關次數:
  • 被引用被引用:1
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  • 下載下載:38
  • 收藏至我的研究室書目清單書目收藏:0
骨髓分化不良症候群(Myelodysplastic syndrome)一直以來都是臨床上的一大難題,其成因複雜又難以治癒。然而,該病往往發生不典型病
徵,其中以難治性貧血為少數的共同特徵。流式細胞儀發展至今已被廣泛使用於白血病的細胞標記檢測。然而,目前對於紅血球系列每個階段尚無專一性的細胞標記。本研究係挑選無貧血症狀之白血病患者常規骨髓,同時利用三種紅血球細胞標記(CD235a、CD71、CD45)以流式細胞儀進行檢測後,並且嘗詴最佳化儀器設定。以CD71 (Y 軸)及CD235a (X 軸),建立四區正常骨髓紅血球分化圖形:第一區主要為紅血球芽母細胞及嗜鹼性紅血球芽母細胞(CD45dim/low/CD71dim/bright/ CD235a–),佔四區2 to 5%;第二區主要為多色性紅血球芽母細胞及正色性紅血球芽母細胞(CD45-/CD71bright/CD235a+),佔四區比例最高(80-90%);第三區主要為網狀紅血球(CD45-/CD71dim/CD235a+),佔四區比例最低(小於1%);第四區主要為成熟紅血球(CD45-/CD71-/CD235a+),佔四區比例0~20%。經分析骨髓分化不良症候群患者骨髓後,發現第二區細胞有CD71 表現低下之情形。為了找出更多相關的標記,來分析骨髓分化不良症候群患者紅血球生成的障礙,本研究利用細胞分選儀,將正常人類及小鼠紅血球分化第一區與第二區細胞分選出來,然後進行微陣列及基因功能分析,找出紅血球細胞膜相關基因。再透過MetaCore 軟體資料庫,找出CD36 等多個與CD71 有關之具有潛力應用於骨髓分化不良症候群的診斷、預後與治療追蹤的細胞表面標記。此外,這些細胞標記同樣也將對於以流式細胞儀區分紅血球生成各階段有所幫助。
A complex and incurable leukemia disease, myelodysplastic syndrome (MDS), is always a course in clinical. The patients who suffered from MDS may be developing refractory anemia finally. Nowadays, flow cytometry is developed for diagnosis and prognosis of lymphoproliferative and myeloproliferative disorders; however, the difficulty of standardizing flow cytometry diagnosis has increased because of insufficient specific clusters of differentiation (CD) markers of every erythroid stage for dyserythropoiesis disorders. In this study, 3 known CD markers (CD45, CD71, and CD235a) were used to gate 4 normal bone marrow erythroid differentiation groups. The first group is mainly the erythroblast and the basophilic normoblast (CD45dim/low/CD71dim/bright/CD235a–) (about 2 to 5% of all erythroid populations). The second group comprises mostly the polychromatophilic normoblast and the orthochromatophilic normoblast (CD45-/CD71bright/CD235a+), which occupies the highest cell proportion of all erythroid populations (80 to 90%). The third group is mainly the reticulocyte (CD45-/CD71dim/CD235a+), which occupies the lowest cell proportion (less than 1%). The fourth group is mainly the mature erythrocytes (CD45-/CD71-/CD235a+) (about 0 to 20%). After analyzing the samples from MDS patients, our results showed that the expression of CD71 was reduced in the second group. In order to find out more novel erythroid differentiation markers to delineate the erythropoiesis defect in 4 MDS patients, the first and second group erythroid cells of humans and mice were sorted for microarray and gene ontology analysis. Then, erythroid cell membrane related candidate genes were selected, and the MetaCore software was also used to identify novel CD71-related CD markers. The results showed CD36 and many other cell surface markers will be used to help the clinical diagnosis for MDS patients. Furthermore, these markers also will be helpful for distinguishing erythropoiesis stages.
中文摘要 ........................................................................................................ 2
英文摘要 ........................................................................................................ 3
1 緒 論 ...................................................................................................... 5
1.1 骨髓分化不良症候群(Myelodysplastic syndrome,MDS) ........ 5
1.2 骨髓分化不良症候群診斷 .......................................................... 7
1.3 紅血球生成及相關細胞表面抗原表現 ...................................... 9
1.4 實驗目的與動機......................................................................... 11
2 實驗材料與方法 .................................................................................... 13
2.1 病患骨髓紅血球系列流式細胞儀分析 .................................... 13
2.2 人類骨髓紅血球生成基因表現分析 ........................................ 18
2.3 小鼠骨髓紅血球生成基因表現分析 ........................................ 20
2.4 人類紅血球基因功能分析策略 ................................................ 22
2.5 CD71 關聯紅血球細胞膜蛋白分析策略 .................................. 23
3 結果 ........................................................................................................ 25
3.1 病患骨髓紅血球系列流式細胞儀分析結果 ............................ 25
3.2 人類與小鼠骨髓紅血球基因表現分析結果 ............................ 29
3.3 人類紅血球基因功能分析結果 ................................................ 30
3.4 CD71 關聯紅血球細胞膜蛋白分析結果 .................................. 31
4 討 論 ...................................................................................................... 35
5 圖表 ........................................................................................................ 46
6 參考文獻 ................................................................................................ 83
7 附圖 ........................................................................................................ 88
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