Transcriptome analysis of human umbilical cord fibroblasts from babies whose mother experienced preeclampsia. Homo sapiens

In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. Overall design: RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).

子痫前期（preeclampsia, PE）妊娠中，已有研究证据表明胎儿-胎盘单位处于氧化应激状态。本研究针对正常妊娠产妇与早发型子痫前期产妇分娩的脐带（umbilical cords, UC）所建立的原代细胞系展开分析，旨在明确二者是否存在可区分的表型。尽管所有脐带组织在4%氧浓度环境下建立培养时均可长出细胞集落，但子痫前期组脐带在常氧（20%氧浓度）条件下的培养成功率显著更低（P < 0.05）。此外，已建立的子痫前期组细胞系的增殖速率，在4%氧浓度下与对照组无明显差异，但在20%氧浓度下则显著低于对照组。在4%氧浓度下培养的子痫前期组细胞系，同样比对照组对促氧化剂马来酸二乙酯更为敏感，且与对照组不同，其细胞存活无法被谷胱甘肽所保护。转录组分析显示，在4%氧浓度条件下，子痫前期组与对照组细胞系间仅有少量差异表达基因，但证实了子痫前期组细胞系在20%氧浓度下处于更为严重的应激状态。本研究结论认为，从子痫前期产妇分娩的脐带中建立的原代细胞系，对氧化应激的易感性在多次细胞传代后仍保持稳定，但这种易感性的基础是遗传还是表观遗传调控，目前仍不明确。

实验设计：在4%氧浓度条件下，当脐带成纤维细胞系（子痫前期组15株，对照组（control, CTL）9株，均培养于T25培养瓶）传代早期（<第5代）汇合度达到约90%时，提取其总RNA。为收集20%氧浓度条件下细胞的总RNA，将传代第4、5或6代的细胞系在汇合度约为20%时，从4%氧浓度环境转移至20%氧浓度环境中培养。当细胞汇合度达到约90%时（4%氧浓度下通常需2天，20%氧浓度下需3-4天），弃去培养基，立即向每个T25培养瓶中加入1 ml RNA STAT60试剂（Tel-Test公司，弗里德伍德，得克萨斯州），并按照产品说明书进行总RNA提取。将RNA样品提交至得克萨斯大学西南医学中心微阵列核心实验室（https://microarray.swmed.edu/），使用Illumina HumanHT-12 v4表达微珠芯片进行微阵列分析。原始强度数据使用BeadStudio软件进行背景校正，并依据高级工作流程方案（百分位移法及标记过滤（检出）），通过GeneSpring 12.6软件（安捷伦科技公司，圣克拉拉，加利福尼亚州）进行后续分析。