Normal human osteoblasts and MDA-MB-231 mono and coculture

As a model for investigating changes in gene expression in response to epithelial-osteoblast interactions in bone metastases of breast carcinomas, cells that represented malignant epithelial cell compartments (MDA-MB-231) and cells that represented skeletal compartments (Normal Human (NH) Osteoblasts) were examined in an in vitro mixed co-culture setting. These two types of cells were co-cultivated for 48 h in a low-serum medium [0.2% fetal bovine serum (FBS)] to allow reciprocal signal exchange with minimal background from undefined molecular signals that are inherent in fetal bovine serum. We examined the effects of co-cultivation on each cell pairing in two independent biological replicates. The gene expression profiles of the co-cultures were compared to the expression profiles of the corresponding cells that were kept in monoculture using HEEBO microarrays. After mono or co-culturing, total RNA was extracted and amplified using a modified Eberwine procedure. The amplified RNA was labeled with the fluorescent dye Cy5 and pooled with Cy3 labeled reference RNA, and then the pooled RNA was hybridized onto HEEBO microarrays. After hybridization and washing, arrays were scanned on a fluorescent microscope scanner. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

本数据集以探究乳腺癌骨转移中上皮细胞-成骨细胞相互作用诱导的基因表达变化为研究模型。实验在体外混合共培养体系中开展，分别选取代表恶性上皮细胞组分的MDA-MB-231细胞，以及代表骨骼微环境组分的正常人成骨细胞（Normal Human (NH) Osteoblasts）作为研究对象。将两类细胞置于含0.2%胎牛血清（fetal bovine serum, FBS）的低血清培养基中共同培养48小时，以保障细胞间的双向信号交流，同时最大程度降低胎牛血清中固有未明确分子信号所带来的背景干扰。本实验设置两组独立生物学重复，分析共培养对该细胞配对的影响。采用HEEBO微阵列（HEEBO microarrays），将共培养细胞的基因表达谱与对应单培养细胞的基因表达谱进行对比分析。单培养或共培养结束后，采用改良Eberwine法（modified Eberwine procedure）对总RNA进行提取与扩增。将扩增得到的RNA用荧光染料Cy5标记，并与经Cy3标记的参照RNA混合，随后将混合后的RNA样本杂交至HEEBO微阵列上。杂交与洗涤完成后，使用荧光显微镜扫描仪对微阵列进行扫描。本实验采用全配对实验设计方案，即所有标记后的提取物之间均进行两两比较。