Phillippe Mark, Adeli Sharareh
Vincent Center for Reproductive Biology, Department of Obstetrics & Gynecology, Massachusetts General Hospital, Boston, MA.
PLoS One. 2017 Jun 16;12(6):e0178845. doi: 10.1371/journal.pone.0178845. eCollection 2017.
Although suggested that "fetal" cell-free DNA (cfDNA) is derived from trophoblast cells, the exact origin is unclear. The studies in this report sought to demonstrate that placental tissue releases cfDNA in parallel with cell death, that the size range of cfDNA is similar to that found in maternal plasma, and that the cfDNA fragments are able to stimulate a proinflammatory cytokine response. Placentas were harvested from near term pregnant CD-1 mice and cultured in DMEM/Ham's F12/FBS media in 8% or 21% O2. After centrifugation to remove cells and cellular debris, the cfDNA was extracted from the media and quantified by DNA spectrophotometry. The cfDNA fragments were sized using a 1.5% TAE gel. Cell death was quantified by lactate dehydrogenase assay; and tissue homogenates were used to quantify caspase activity and BAX expression. Cultured RAW-264.7 macrophage cells were used to determine IL6 stimulation by cfDNA. The cfDNA levels released in 8% O2 (placental normoxia) were not significantly different from explants cultured in 21% O2 (placental hyperoxia). The cfDNA fragments ranged in size from < 100 -< 400 bp. The cfDNA release increased when cultured with LPS, whereas it decreased with trolox (vitamin E analog). Explant release of cfDNA increased in parallel with cell death. The cfDNA release and cell death of trophoblast appears to involve components of the apoptosis signaling pathway as suggested by LPS enhancement of placental caspase activity, suppression of cfDNA release by a pan-caspase inhibitor and the trend toward increased Bax protein expression. Studies with cultured macrophage cells confirmed the ability of cfDNA to stimulate an IL6 response. In summary, these studies have confirmed the ability of placental tissue to release significant amounts of cfDNA, a phenomenon that appears to be mediated, at least in part, by apoptosis; and that the cfDNA released by the placental explants is able to stimulate a significant proinflammatory response. Thus, these studies provide support for the hypothesis that cell-free fetal DNA released by placental tissue potentially plays a mechanistically important role during the events leading to the onset of parturition.
尽管有人提出“胎儿”游离DNA（cfDNA）源自滋养层细胞，但其确切来源尚不清楚。本报告中的研究旨在证明胎盘组织在细胞死亡的同时释放cfDNA，cfDNA的大小范围与母体血浆中的相似，并且cfDNA片段能够刺激促炎细胞因子反应。从接近足月妊娠的CD-1小鼠获取胎盘，并在含8%或21%氧气的DMEM/Ham's F12/FBS培养基中培养。离心去除细胞和细胞碎片后，从培养基中提取cfDNA并通过DNA分光光度法定量。使用1.5%的TAE凝胶对cfDNA片段进行大小测定。通过乳酸脱氢酶测定法定量细胞死亡；并使用组织匀浆定量半胱天冬酶活性和BAX表达。使用培养的RAW-264.7巨噬细胞来确定cfDNA对IL6的刺激作用。在8%氧气（胎盘正常氧含量）条件下释放的cfDNA水平与在21%氧气（胎盘高氧）条件下培养的外植体无显著差异。cfDNA片段大小范围为<100 -<400 bp。与脂多糖（LPS）一起培养时cfDNA释放增加，而与生育三烯酚（维生素E类似物）一起培养时则减少。外植体cfDNA释放与细胞死亡同时增加。胎盘滋养层细胞的cfDNA释放和细胞死亡似乎涉及凋亡信号通路的成分，这表现为LPS增强胎盘半胱天冬酶活性、泛半胱天冬酶抑制剂抑制cfDNA释放以及Bax蛋白表达增加的趋势。对培养的巨噬细胞的研究证实了cfDNA刺激IL6反应的能力。总之，这些研究证实了胎盘组织释放大量cfDNA的能力，这一现象似乎至少部分由凋亡介导；并且胎盘外植体释放的cfDNA能够刺激显著的促炎反应。因此，这些研究为胎盘组织释放的游离胎儿DNA在导致分娩开始的事件中可能发挥重要机制作用这一假说提供了支持。
