【摘要】：目的 :建立豬naive-like誘導(dǎo)性多能干(induced pluripotent stem,i PS)細(xì)胞系,并對(duì)其進(jìn)行紅色熒光標(biāo)記,為通過(guò)示蹤豬naive i PS細(xì)胞發(fā)育和分化的相關(guān)研究奠定基礎(chǔ)。方法:首先利用核轉(zhuǎn)染技術(shù)向巴馬小型豬胎兒成纖維細(xì)胞(porcine embryonic fibroblast,PEF)中轉(zhuǎn)入鼠源OCT4、SOX2、KLF4和c-MYC轉(zhuǎn)錄因子表達(dá)載體Tet O-FUW-OSKM,并同時(shí)轉(zhuǎn)入激活表達(dá)載體FUW-M2rt TA,采用白血病抑制因子(leukemia inhibitory factor,LIF)結(jié)合堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,b FGF)的培養(yǎng)體系進(jìn)行培養(yǎng),通過(guò)在培養(yǎng)液中添加鹽酸多西環(huán)素(doxycycline hyclate,DOX)進(jìn)行誘導(dǎo),建立起豬i PS細(xì)胞系,并對(duì)細(xì)胞系的多能性進(jìn)行鑒定。在此基礎(chǔ)上,向i PS細(xì)胞系轉(zhuǎn)入紅色熒光蛋白表達(dá)載體,對(duì)其進(jìn)行標(biāo)記,并鑒定被紅色熒光標(biāo)記后的細(xì)胞是否仍然具有多能性。結(jié)果:所建立的i PS細(xì)胞系克隆呈三維立體生長(zhǎng),可以進(jìn)行單細(xì)胞傳代培養(yǎng)至30代以上,核型正常,堿性磷酸酶染色呈陽(yáng)性,表達(dá)多種干細(xì)胞多能因子,利用LIF/STAT3信號(hào)通路維持其增殖,體外可分化形成表達(dá)三胚層相關(guān)基因的類(lèi)胚體,為豬naive-like i PS細(xì)胞系。成功對(duì)所建立的i PS細(xì)胞系進(jìn)行紅色熒光標(biāo)記,堿性磷酸酶染色和免疫熒光染色結(jié)果顯示被紅色熒光標(biāo)記的i PS細(xì)胞的多能性依然存在。結(jié)論:成功建立了穩(wěn)定表達(dá)紅色熒光蛋白的豬naive-like i PS細(xì)胞系。
[Abstract]:Aim: to establish a pig naive-like induced pluripotent (induced pluripotent stem,i PS cell line and label it with red fluorescence, so as to lay a foundation for the study of the development and differentiation of pig naive i PS cells. Methods: firstly, mouse OCT4,SOX2,KLF4 and c-MYC transcription factor expression vector Tet O 鈮，

本文編號(hào)：2487195