家兔始基生殖細胞體外再程式化與分化能力之研究

外文標題: 
In Vitro Reprogramming and Differentiation Potential of Rabbit Primordial Germ Cells
校院系所: 
東海大學畜產與生物科技學系
指導教授: 
羅能文
出版年份: 
2015年
主題類別: 
摘要: 

始基生殖細胞(primordial germ cells, PGC)是生殖前軀細胞。在體外,PGC能夠再程式化為胚源生殖細胞(embryonic germ cells, EGCs)。本研究為了瞭解家兔PGCs在早期發育階段胚胎體內的遷移過程,首先(試驗一)觀察不同發育天數之家兔胚胎(fetuses)生殖脊與性腺相關構造之形態,作為後續試驗與相關研究之基礎。結果指出,在9.5 dpc前之胚胎生殖脊(genital ridges)其形態無法清晰被分辨與分離,反之,同一發育時期小鼠胚胎生殖脊形態已具基本的輪廓。11.5 dpc是可辨識家兔胚胎生殖脊與中腎(mesonephros)聯合組織發育的最早時間點,而13.5 dpc的兔生殖脊發育形態則與11.5 dpc的小鼠生殖脊形態相似。為期建立體外之EGCs,試驗二乃自12.5 dpc之兔胚胎分離PGC培養於兔胚幹細胞(rabbit embryonic stem cells, rESCs)培養液、添加MEK與GSK3 抑制劑之mouse EGC培養液、mTeSR™培養液或rabbit EGC培養液等處理組;同時也測試最佳之抑制劑與生長因子的添加濃度。結果指出,家兔EGC只有與小鼠飼養層細胞(mouse embryonic fibroblast, MEF)共培養以及添加LIF (1,000 U/mL)、 bFGF (20 ng/mL)、 forskolin (10 µM) 與 ROCK inhibitor Y-27632 (10 ng/mL)之rabbit EGC培養液中才能被繼代與表現多能性標記蛋白Oct-4與生殖細胞專一標記蛋白Vasa。為探求PGC分化為生殖細胞的潛能,試驗三以12.5 dpc之PGC與生殖脊體細胞共培養於含有BMP4 (500 ng/mL)、BMP8a (500 ng/mL)、SCF (100 ng/mL)、EGF (50 ng/mL) 與 10% 之豬濾泡液(porcine follicle fluid, pFF)之誘導環境中,結果發現PGC與生殖脊體細胞能夠形成直徑小於100 μm的初級細胞聚集體(cell aggregates, CA)並表現Oct-4與 VASA。持續培養14天後CA可成長至直徑超過100 μm且能夠表現減數分裂專一蛋白標記Scp3,並在培養至30天時表現卵子專一標記蛋白Gdf9,此時期的Gdf9螢光表現模式與兔濾泡之Gdf9螢光表現相似。本研究證明自12.5 dpc兔胚胎中所分離的PGC可形成類EGC並保持多能性標記的表現。此外,當12.5 dpc之PGC與生殖脊體細胞共培養時,可被誘導朝向雌性生殖細胞分化並進入減數分裂。

外文摘要: 

Primordial germ cells (PGCs) starting as germline precursor cells can give rise to mature germ cells in vivo via a cascade of differentiation processes. They can also be reprogrammed in vitro into pluripotent stem cells, i.e., embryonic germ cells (EGCs). To understand the rabbit PGC migration process in early developmental stages, we anatomized New Zealand White rabbit fetuses at different days of development. Before 9.5 dpc, PGCs in the rabbit fetus were hardly isolatable due to the undistinguishable structure of genital ridge, whereas at the same time point the mouse fetus already has a basic exterior shape of a fetus. The first distinguishable rabbit mesonephros-associated genital ridges appeared at 11.5 dpc. The morphology of rabbit genital ridges at 13.5 dpc resembles that of the mouse at 11.5 dpc. These observations are likely to provide a distinct timeline of rabbit germ cell migration helpful for the understanding of embryonic gonad physiology and technical separation of rabbit PGCs or genital ridges. We therefore isolated rabbit PGCs from the 12.5 dpc fetus and compared several culture media including rESC medium, mouse EGC medium with MEK and GSK3 inhibitors, mTeSR™ medium and rabbit EGC medium. Different concentrations of inhibitors or growth factors were also tested. The rabbit EGCs can be long-term co-cultured with mouse embryonic fibroblast (MEF) cells for several passages only in rabbit EGC medium with the presence of LIF (1,000 U/mL), bFGF (20 ng/mL), forskolin (10 µM) and ROCK inhibitor Y-27632 (10 ng/mL). These EG-like cells expressed pluripotency marker Oct-4, and germ cell marker Vasa as shown by the immunocytochemical (ICC) staining at an undifferentiated state. The in vitro differentiation abilities of rabbit PGC were also confirmed. We collected 12.5 dpc PGCs and co-cultured them with somatic cells from genital ridges in medium with BMP4 (500 ng/mL), BMP8a (500 ng/mL), SCF (100 ng/mL), EGF (50 ng/mL) and 10% porcine follicle fluid (pFF). The results showed these two kinds of cells could coexist and form primary cell aggregates (CA) which were less than 100 μm in diameter and expressed Oct-4 and VASA by ICC staining. After being cultured for 14 days, the CA could be grown over 100 μm in diameter and expressed the meiosis specific marker Scp3 and oocyte specific marker Gdf9 at Day 30. The Gdf9 expression pattern was similar to that of rabbit follicles. This study demonstrates that rabbit PGCs isolated from 12.5 dpc fetuses can be reprogrammed into EG-like cells and remain pluripotent as in mouse PGCs. Furthermore, when 12.5 dpc PGCs were co-cultured with genital ridge somatic cells, they could form a CA structure with presumed meiotic activity inside.