The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.
The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.
复制标题
使用 ROCK 抑制剂 Y-27632 从猪体外受精和单性生殖囊胚中有效衍生滋养层细胞并进行培养
DOI：
10.1371/journal.pone.0142442
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Hou D;Su M;Li X;Li Z;Yun T;Zhao Y;Zhang M;Zhao L;Li R;Yu H;Li X
滋养细胞是胎盘的特化细胞，在胚胎着床过程中起着重要作用。滋养细胞的体外培养为研究其着床机制提供了重要手段。本研究以猪体外受精（IVF）和孤性生殖激活（PA）囊胚为材料，在STO饲喂蛋鸡上添加敲除血清替代（KOSR）和碱性成纤维细胞生长因子（bFGF）的培养基中培养猪滋养细胞，并检测ROCK （rho相关线圈蛋白激酶）抑制剂Y-27632对细胞培养的影响。5株PA胚泡衍生细胞系和2株IVF胚泡衍生细胞系培养20余代；1株PA细胞株传代达110代，形态无明显改变。衍生的滋养细胞形态呈上皮样，富含脂滴，与饲养细胞有明显的边界。细胞组织化学染色碱性磷酸酶阳性。采用免疫荧光染色、反转录PCR和实时荧光定量PCR检测细胞中CDX2、KRT7、KRT18、TEAD4、ELF5、HAND1等TR系标记、IGF2、PEG1、PEG10等印迹基因和端粒酶活性相关基因TERC、TERF2的表达。PA和IVF胚泡衍生的滋养层细胞在体外均具有向成熟滋养层细胞分化的能力。Y-27632的添加促进了PA和IVF囊胚衍生细胞系的生长，并增加了滋养层基因的表达。本研究为IVF和PA胚胎着床期和胎盘发育的研究提供了另一种高效的培养滋养细胞的方法。
Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos.