Diferential gene expression in Trophoblast cell cultures

The syncytiotrophoblast develops as a result of the fusion of placental villous cytotrophoblast cells. These multinuclear cells are essential for fetal-maternal exchange and production of pregnancy-related hormones. Placental deficiency is considered a major risk factor associated with intrauterine growth restriction and preeclampsia. Better understanding of placental patho-physiology requires knowledge on gene expression regulation involved in trophoblast differentiation. Using the well-established in vitro trophoblast differentiation model, we have performed a microarray analysis on mRNA expression in trophoblast and syncytiotrophoblast cell cultures. Dramatic changes in gene expression patterns were detected during trophoblast differentiation. As many as 3524 novel and known genes are found to be up- or down regulated for more than 2-fold. Real-time PCR analysis of a group of genes confirmed the reliability of the microarray data. Overall, this study provided a global view on the molecular events underlying trophoblast differentiation. Subsequent characterization on regulation and function of the identified genes may lead to discovery of new pathways important for placental differentiation and function. Keywords: Trophoblast culture cells Affymetrix GeneChipTM Human Genome U133 Plus 2.0 microarrays were used for mRNA profiling. RNA samples were subject to Agilent analysis for quality controls. cDNA was prepared from 10 µg of RNA, quantified by spectrometry, and used as a template for the synthesis of biotinylated cRNA using RNA transcript labeling reagent. The quality of the cRNA probe was verified by gel electrophoresis as well as a pilot hybridization using the Test-3 arrays. Hybridization solution containing fragmented cRNA probes and control cRNA was supplemented with herring sperm DNA and bovine serum albumin. The probe solution was heated at 99° C for 5 min followed by incubation at 45° C for 5 min before use. Hybridization was carried out at 45° C for 16 hr with constant rotation at 60 rpm. The arrays were washed and stained with streptavidin-phycoerythrin. The GeneChip 5.0 (Affymetrix) program was used to scan and quantitatively document the hybridization signals. Compilation of candidate gene and calculation of changes were performed on SpotFire and Microsoft Excel programs. To minimize the false-positive conclusion, only genes satisfying the following two criteria were considered candidate regulation targets for further analysis: 1) The gene’s hybridization signal reached an absolute level that was significantly higher than that of the background (P<0.05); 2) The changes is more than 2-fold when compared to control group.合体滋养层细胞（syncytiotrophoblast）由胎盘绒毛细胞滋养层细胞融合发育而来。这类多核细胞对于胎儿-母体间物质交换以及妊娠相关激素的合成分泌至关重要。 胎盘功能不全被认为是宫内生长受限与子痫前期的主要危险因素之一。深入解析胎盘病理生理学机制，有赖于对滋养层细胞分化过程中基因表达调控机制的认知。 本研究借助已成熟建立的体外滋养层细胞分化模型，对滋养层细胞与合体滋养层细胞培养物中的mRNA表达水平开展了芯片分析。在滋养层细胞分化过程中，可检测到基因表达谱发生显著改变：共计3524个新发现基因与已知基因的表达量出现了2倍以上的上调或下调。针对一组基因的实时荧光定量PCR（real-time PCR）分析验证了芯片数据的可靠性。 综上，本研究全面揭示了滋养层细胞分化过程背后的分子事件。后续针对已筛选基因的调控机制与生物学功能进行的解析，有望发现胎盘分化与功能维持相关的全新信号通路。 关键词：滋养层细胞培养；采用Affymetrix GeneChip™ 人类基因组U133 Plus 2.0芯片进行mRNA表达谱分析。 RNA样品通过安捷伦（Agilent）分析进行质量控制。以10 μg总RNA为起始材料合成cDNA，经光谱定量后，以其为模板，利用RNA转录标记试剂合成生物素标记的cRNA。通过凝胶电泳以及采用Test-3芯片进行的预杂交实验，验证cRNA探针的质量。向包含片段化cRNA探针与对照cRNA的杂交液中补加鲑鱼精DNA与牛血清白蛋白。探针溶液使用前需先于99℃加热5分钟，随后在45℃孵育5分钟。杂交反应于45℃下进行16小时，同时以60转/分钟的转速持续旋转。芯片经洗涤后，采用链霉亲和素-藻红蛋白进行染色。采用Affymetrix GeneChip 5.0软件扫描芯片并定量记录杂交信号。候选基因的汇总与表达变化倍数的计算均通过SpotFire与微软Excel（Microsoft Excel）软件完成。 为最大限度降低假阳性结论的出现，仅满足以下两项标准的基因才被纳入后续分析的候选调控靶标：1）该基因的杂交信号显著高于背景信号（P<0.05）；2）与对照组相比，其表达变化幅度达到2倍以上。