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系統識別號 U0007-1704200714562863
論文名稱(中文) 精原幹細胞的培養與鑑定研究
論文名稱(英文) Cultivation and Characterization of Mouse Germ Line Stem Cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 92
學期 2
出版年 93
研究生(中文) 吳采蓉
研究生(英文) Tsai-Jung Wu
學號 M102091023
學位類別 碩士
語文別 中文
口試日期
論文頁數 81頁
口試委員 指導教授-黃彥華
中文關鍵字 幹原細胞  精原幹細胞  胚胎幹細胞 
英文關鍵字 stem cell  germ line  Oct-4 
學科別分類
中文摘要 幹原細胞精原幹細胞(germ line stem cells)為一群具多功能(pluripotent)分化特性的早期幹原細胞,其可以自體新生(self-renew),若分化為精細胞,攜帶遺傳相關的基因,產生子代,進行世代交替。在小鼠系統中精原幹細胞包括三個部份:原生殖細胞 (primordial germ cells),生殖細胞 (gonocytes),及未分化的A型精原母細胞 (type A spermatogonia / spermatogonial stem cells)。 我們利用特別的培養系統,將新生小鼠體內的睪丸取出,讓其內部的細胞釋出,進行體外培養實驗,數天後觀察細胞形成群落(clones),其型態類似早期培養胚胎幹細胞時會出現的胚體 (embryoid body; EB),利用染色鑑定得知此群細胞呈現很強的鹼性磷酸酶 (alkaline phosphatase; AP) 活性表現。從RT-PCR與細胞免疫染色的結果得知其表現Oct-4的基因與蛋白質,其細胞表面抗原的表現則為SSEA1+,α6-integerin+,β1-integerin+,c-Kit-,CD31-,CD34-。除此之外檢測在培養皿中的細胞,則有Oct-4+ Mvh+ Stella+ Fragilis+ Daz+ Piwi+ Tex14+ Gcnf+ 的基因表現。 將此群落的細胞打散,培養在含condition medium的Methylcellulose培養基,可以看到細胞 recolonization的現象。我們進一步利用添加維他命A酸 (retinoic acid; RA),來刺激精原幹細胞進行增生。初步的結果顯示在0.5-2 μM的RA處理下可促進細胞增生最高至175 %。
英文摘要 Germ line stem cells are unique in stem cell biology and transmit genetics to next generation. Mouse germ line stem cells includes primordial germ cells (PGCs), gonocytes, and spermatogonial stem cells (SSCs). SSCs are responsible for maintaining spermatogenesis throughout life in the male by continuous production of daughter cells that differentiate into spermatozoa. We successfully got some germ cell clones from culture neonatal ICR mice testis cells at specific condition in vitro. These clones look like embryoid body in morphology and show strong alkaline phosphatase activity, suggesting its primordial cells potential. In this report, we identify this clone by detection several genes by RT-PCR including Oct-4pos Mvhpos Stellapos Fragilispos Dazpos Piwipos Tex14pos Gcnfneg . In addition, we characterized the Oct-4 protein and other cell surface antigen expression by immunocytochemistry. This clone shows Oct-4pos, SSEA1pos, α6-integerinpos, β1-integerinpos, c-Kitlow, CD31neg, and CD34neg , like PGC or SSC origin. As retinoic acid (RA) acts to stimulate proliferation of PGCs, we also test the cell proliferation ability of these clones induced by RA in vitro. Our preliminary result shows that these clones could be recolonized and the cell number increased to 175% at 2 μM RA concentration in condition - methylcellulose culture medium.
論文目次 第一章 緒論 1.1 幹原細胞 1.1.1 胚胎幹細胞(embryonic stem cell;ESC) 1.1.2 成體幹細胞(adult stem cell) 1.2 精原幹細胞 1.2.1原生殖細胞 (Primordial Germ Cell;PGC) 1.2.2 前精原幹細胞 (Gonocyte) 1.2.3 精原幹細胞 (Spermatogonial Stem Cell; SSC) 1.3 精子的生成與發育 1.3.1 精原幹細胞期 / 精原細胞期 (Spermatogonia) 1.3.2 精母細胞期 (Spermatocyte) 1.3.3 精細胞期 (Spermatid) 1.4 多功能幹細胞的研究 1.5 精原幹細胞的標記物 1.5.1 鹼性磷酸酶(Alkaine phosphatase; AP) 1.5.2 轉錄因子Oct-4 1.5.3 其他標記物 1.6 精原幹細胞功能上的鑑定與應用 1.7 本論文研究重點 第二章 材料與實驗方法 2.1動物與細胞培養 2.2細胞製備 2.2.1 睪丸組織細胞初級培養 2.2.2 TM4細胞株培養 2.3 反轉錄聚合酶連鎖反應RT-PCR 2.3.1 RNA isolation from the clones 2.3.2 First-Strand cDNA Synthesis Reaction 2.3.3 聚合酶連鎖反應PCR 2.3.4 洋菜膠體電泳 2.4 鹼性磷酸酶活性測定 2.4.1 BCIP / NBT 2.4.2 AP試劑套組 2.5 細胞免疫染色 2.6 維他命A酸處理細胞 2.6.2 Methylcellulose 2.6.1 Poly-lysine coating 第三章 實驗結果 3.1 不同時期睪丸組織切片H&E染色 3.2 組織免液染色:Oct-4 3.3 精原幹細胞培養 3.4 鹼性磷酸酶(Alkaline Phosphatase) 3.5 反轉錄-聚合酶連鎖反應 (RT-PCR) 3.6 細胞免疫染色 3.7細胞增生(Reclonization) 第四章 討論 4.1 組織學鑑定 4.2 細胞形態上變化 4.3 細胞特性分析 4.3.1 鹼性磷酸酶 4.3.2 轉錄因子Oct-4與其他標記 4.4 精原幹細胞的增生 4.5 精原幹細胞株的建立 第五章 結論 圖次 參考文獻
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