臺灣博碩士論文加值系統

始基生殖細胞（primordial germ cell, PGC）係生殖細胞分化成為雌性或雄性配子（gamete）過程中最初始之細胞。PGC無法於一般體外培養條件下存活超過一周。增添白血病抑制因子 (leukemia inhibitory factor, LIF)、幹細胞因子（stem cell factor, SCF）及FGF2 (basic fibroblast growth factor)於培養液並培養於飼養層細胞（feeder layer）上，則可使具單一分化能力之PGC轉變為具分化多能性之胚生殖細胞（embryonic germ cell, EGC）。本論文主要目的為探討小鼠EGC細胞株建立之培養條件並分析細胞株之特性。試驗一旨在測試PGC轉變為EGC之培養條件；分離自11.5 dpc （days post coitum）之小鼠PGC，以1,500 cells/cm2之密度培養於含有新鮮STO飼養層細胞上，並供給不同濃度之LIF、 forskolin（FK）及FGF2。結果顯示小鼠PGC於培養7日後，類EGC之產率為0.1-0.28%。經免疫細胞染色結果顯示此等EGC不僅具備表現鹼性磷酸酶 （alkaline phosphatase, AP），與具備能力表現多能性細胞標記蛋白，諸如：Oct4、Nanog及SSEA1等。然在此等培養系統下其產生之類EGC僅能被培養至第3代。試驗二除探討GSK3（glycogen-synthase-kinase-3）與MEK抑制劑（inhibitor）之添加，對於EGC產率之影響外，並嘗試於抑制劑系統下建立EGC細胞株。抑制劑處理組之PGC，經培養於含有LIF、FK與FGF2環境下48 h後，更換為含有LIF、FK、MEK inhibitor與GSK3 inhibitor（2i）之培養液後培養至第7天。篩選後發現對照組僅得到一株類EGC，而試驗組（2i組）則得到12株；兩者經繼代培養後且分別皆可維持至第6代。經細胞特性分析結果顯示，2i組之類EGC具AP表現；進一步且RT-PCR結果且證明類EGC不僅能順利表現oct4、sox2、stat3、c-myc及klf4等多能性基因，且能表現mvh與stella等生殖細胞標記基因。進一步藉由免疫細胞染色分析結果，且證明彼等細胞液充分具備表現包括Oct4、Sox2、Nanog, Mvh及Stella等蛋白質。此外，於2i系統下所建立之EGC，於適當之環境下更具備表現形成具三胚層之類胚體（embryoid body, EB）之能力。綜上所述，小鼠PGC可於LIF、FK、FGF2與2i培養系統下可被順利誘導形成具備EGC特徵之類細胞株；惟欲證明此等細胞株確為EGC，則將之誘導形成畸胎瘤（teratoma）與透過嵌合（chimera）測試以證明其確有產生嵌合體小鼠（chimera mouse）之能力之試驗有待進行。

Primordial germ cells (PGCs) are the primitive forms of germ cells, which ultimately differentiate into male and female gametes. PGCs cannot survive under in vitro culture conditions for more than one week. However, they would transform intopluripotent embryonic germ cells (EGCs) in vitro if they are cocultured with feeder cells supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF), basic fibroblast growth factor (FGF2). The primary goal of this study was to establish mouse embryonic germ cell lines under various conditions and characterizethem. In Experiment 1, to testthe culture conditions capable of convertingPGCs into EGCs, mouse PGCs derived from 11.5 dpc (days post coitum) mouse fetus were seeded by 1,500 cells/cm2 on STO feeders in medium supplemented with various concentrations of LIF, forskolin (FK) and FGF2. EG-like colonies were observed 4 days post the initial culture and after 7 days of culture the formation rate of EG-like colonies reached0.1-0.28%. Those EG-like cells expressed pluripotency markers alkaline phosphatase (AP) and Oct4, Nanog and SSEA1 on protein levelsand were maintained to passage 3 only. In Experiment 2, GSK3 and MEK inhibitors were added into the culture medium to examine the efficiency of EG-like colony formation. Mouse PGCs were cultured in medium supplemented with LIF, FK and FGF2 for 48 h, and then in the medium containing LIF, FK, MEK inhibitor and GSK3 inhibitor (2i) for additional five days. The EG-like cells which reprogram completely had been selected by trypsin. Twelve EG-like colonies were selected by the2i culture system as opposed to one colony by the untreated control system. All the EG-like cells can be maintained over 6 passages. The EG-like cells derived from 2i treatments expressed AP, pluripotency and germ cell marker genes oct4, sox2, stat3, c-myc, klf4, mvh and stella on RNA levels ; Oct4, Sox2, Nanog, Mvh and Stella on protein levels. Furthermore, the tested clone, N2, showed the capability of forming embryoid bodies (EBs) as evident by the expression of three germ layer marker genes. Taken together, several EG-like cell lines had been established from mouse PGCs with the presence of LIF, FK, FGF2, and GSK3 and MEK inhibitors.The full potency of these EGC-line clones awaits further confirmation by testing their capability in teratoma formation and in chimera formation via germline transmission.

目 次
頁次
摘要
文獻檢討…………………………………………………………………………1
前言…………………………………………………………………………………2
始基生殖細胞之發生………………………………………………………………2
胚胎生殖細胞之背景及其特性介紹………………………………………… 4
一 EGC之建立歷史及現況………………………………………………4
二 PGC與 EGC之異同………………………………………………7
三 促進PGC轉變為EGC之因子…………………………………………7
四 纖維母細胞生長因子2促進細胞自我更新……………………………9
五 Forskolin提升細胞內cAMP之濃度…………………………………10
六 Blimp1調控細胞之基因外修飾……………………………………….10
七 Trichostatin A可協助FGF2協助PGC轉變為EGC……………………10
八 訊息傳導路逕抑制劑之添加促進EGC之產生…………………………11
研究目的…………………………………………………………………………11
研究試驗…………………………………………………………………………14
試驗一 小鼠PGC之分離與體外培養條件之測試………………………………15
前言……………………………………………………………………15
材料與方法…………………………………………………………………16
結果……………………………………………………………………21
試驗二 添加GSK與MEK抑制劑以改善小鼠類EGC之培養模式與建立類
EGC細胞株………………………………………………………………27
前言…………………………………………………………………………27
試驗設計………………………………………………………………………28
材料與方法…………………………………………………………………28
結果……………………………………………………………………40
討論……………………………………………………………………56
結論……………………………………………………………………58
附錄一…………………………………………………………………………59
參考文獻…………………………………………………………………………65

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