不同培养体系对猪胚胎多能样干细胞分离培养的影响

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摘要为深入研究猪 (Sus scrofa)胚胎干细胞(porcine embryonic stem cells, pESCs)分离培养自我更新和分化潜能，本研究利用不同培养条件，从孤雌胚胎(parthenogenetic embryos, PA)、细胞核移植胚胎(somatic cell nuclear transfer, SCNT)和体内受精胚胎(in vivo-produced, IVP)中分离pESC样细胞。对PA和SCNT胚胎的培养条件和关键多能基因进行检测，比较培养液中添加白血病抑制因子(leukemia inhibitory factor, LIF)、人碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)、糖原合成酶激酶3β抑制剂(inhibitors of glycogen synthase kinase 3β, CHIR99021)和有丝分裂原激活蛋白激酶1(mitogen-activated protein kinase 1, PD0325901)对分离pESCs的影响。结果显示，猪受精卵培养基3(porcine zygote medium 3, PMZ3)适合PA胚胎的发育，NCSU23培养基适合SCNT胚胎的发育。在PA和SCNT囊胚胎中，多能基因八聚体结合转录因子4 (octamer-binding transcription factor 4, OCT4)的表达量比提尔纳瑙伊 (Tir na n-Og, NANOG)和性别决定区Y-box 2(sex determining region Y-box 2, SOX2)显著高；碱性成纤维细胞生长因子受体(basic fibroblast growth factor receptor, bFGFR)基因的表达显著上调，表明从PA和SCNT囊胚胎中分离pESCs需要同时添加bFGF。另外，通过添加bFGF，提高了从体内胚胎分离pESCs克隆的效率。细胞形态呈外胚层样干细胞，免疫荧光检测有NANOG和SOX2的表达，碱性磷酸酶检测阳性。本研究为分离pESCs提供了参考数据和方法。

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	王宁
	俞晓丽
	刘晓鹏
	马洋洋
	成德
	王华岩

关键词 ：猪, 孤雌胚胎(PA), 八聚体结合转录因子4(OCT4), 多能干细胞, 分离培养

Abstract：Although the isolation of porcine (Sus scrofa) embryonic stem cells (pESCs) has been reported, the self-renewal and differentiation potential of pESCs were limit. In this study, the isolation of pESCs from parthenogenetic embryos (PA), somatic cell nuclear transfer embryos (SCNT) and in vivo-produced embryos (IVP) based on the different culture media were researched. The culture condition and key pluripotent genes in PA and SCNT embryos were analyzed by the qRT-PCR, and the effects of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), inhibitors of glycogen synthase kinase 3β (CHIR99021), and mitogen-activated protein kinase 1 (PD0325901) on pESCs isolation were investigated. The results showed that PZM3 and NCSU23 media were suitable for PA and SCNT embryos development in vitro, respectively. The 4 octamer-binding transcription factor 4(OCT4) expression was significantly increased in PA and SCNT embryos compared with NANOG and SOX2 in PA and SCNT embryos. Basic fibroblast growth factor receptor(bFGFR) gene expression was also significantly increased, suggesting that growth factor bFGF was needed for pESCs isolation from PA and SCNT embryos. Furthermore, addition of bFGF in medium could elevate the efficiency of pESCs isolation and cloning from in vivo-produced embryos, which showed morphology of the epiblast-like stem cells (EpiSCs), expressed NANOG and SOX2 proteins by immunochemistry, and had alkaline phosphatase(AP) activity. The results may contribute to a new method to isolate pESCs.

Key words： Porcine Parthenogenetic embryos (PA) Octamer-binding transcription factor 4(OCT4) Pluripotent stem cell Isolate culture

收稿日期: 2016-04-05 出版日期: 2016-07-22

基金资助:国家自然科学基金;国家自然科学基金

通讯作者: 王华岩 E-mail: hhwang101@163.com

引用本文:

王宁俞晓丽刘晓鹏马洋洋成德王华岩. 不同培养体系对猪胚胎多能样干细胞分离培养的影响[J]. , 2016, 24(9): 1429-1438.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I9/1429

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