Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin
Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin
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DOI：
10.1016/j.preghy.2014.09.003
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发表时间：
2014-10-01
影响因子：
2.2
通讯作者：
Hannan, Natalie J.
中科院分区：
文献类型：
--
作者：
Kaitu'u-Lino, Tu'uhevaha J.;Tong, Stephen;Hannan, Natalie J.
目的：可溶性膜样酪氨酸激酶-1 （sFlt-1）和可溶性内啡肽（sEng）是子痫前期研究最多的分子。然而，大多数滋养细胞系不分泌这两种因子。因此，我们开始对原代滋养细胞中sFlt-1和sEng的功能进行研究。研究设计：采用percoll梯度法从足月胎盘中分离原代滋养细胞，然后用CD9抗体进行阴性选择。通过细胞角蛋白7免疫染色检测纯度。我们首先研究了CD9阴性选择对sFlt-1、sEng和hCG分泌的影响，以及福斯克林增强合胞的能力。然后，我们检查了缺氧对sFlt-1产生的影响，并使用siRNA评估了基因敲除。结果：CD9阴性选择产生纯初代滋养细胞群体。当cd9阳性细胞被去除时，sEng的分泌降低了5倍，sFlt1不变，hCG显著升高。纯化人群的hCG分析显示自发合胞，福斯克林没有增强。Forskolin同样没有改变sFlt-1的分泌。缺氧如预期的那样显著增加sFlt-1的分泌。重要的是，高基因沉默效率很容易实现。结论：总之，我们提出了一种方案，产生高纯度的初级滋养细胞，产生丰富的sFlt-1和低但可检测水平的sEng。此外，这些细胞很容易被sirna沉默，因此适合功能研究。(C) 2014年国际妊娠高血压研究协会出版，Elsevier B.V.版权所有。
Objectives: Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast.Study design: Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA.Results: CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved.Conclusion: In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies. (C) 2014 International Society for the Study of Hypertension in Pregnancy Published by Elsevier B.V. All rights reserved.