Differences between fibroblasts derived from normal and IPF lungs. Differences between fibroblasts derived from normal and IPF lungs

Objective: To evaluate the characteristics of IPF lungs in fibroblasts, we performed RNA-Sequencing of fibroblasts derived from normal and IPF lungs Method: NHLF（Normal human lung fibroblast） and DHLF-IPF (DIPF; Diseased human lung fibroblasts derived from idiopathic pulmonary fibrosis) were purchased from Lonza (Walkersville, MD, USA) .Total RNA was extracted from fibroblasts (NHLF and DIPF) using an RNeasy® Mini Kit (#74106; Qiagen, Valencia, CA, USA). Preparation of a next-generation sequencing library was performed using a SMARTer® Stranded Total RNA Sample Prep Kit–Pico Input Mammalian (TaKaRa, Shiga, Japan). Sequencing was performed on an Illumina HiSeq 2500 platform in 75-base single-end mode with Illumina Casava 1.8.2 software for base calling. Sequenced reads were mapped to the human reference genome sequence (hg19) using TopHat v.2.0.13 software, in combination with Bowtie 2 v.2.2.3 and SAMtools v.0.1.19. Result: Of the 26,257 genes analyzed, for NHLF and DHLF-IPF (DIPF), FPKM was required to be greater than or equal to 0.3. Under these conditions, 764 genes were up regulated inDHLF-IPF (DIPF) and 691 genes were down regulated. Overall design: 2 cell lines; NHLF (Normal human lung fibroblast) and DHLF-IPF (DIPF; Diseased human lung fibroblasts derived from idiopathic pulmonary fibrosis)

研究目的：为评估特发性肺纤维化（Idiopathic Pulmonary Fibrosis, IPF）患者肺成纤维细胞的相关特征，我们对正常肺组织及IPF患者肺组织来源的成纤维细胞开展了RNA测序分析。
实验方法：正常成人肺成纤维细胞（Normal Human Lung Fibroblast, NHLF）与特发性肺纤维化来源病变肺成纤维细胞（DHLF-IPF，即DIPF，Diseased Human Lung Fibroblasts Derived from Idiopathic Pulmonary Fibrosis）购自Lonza公司（美国马里兰州沃克斯维尔市）。使用RNeasy® Mini Kit（货号#74106；凯杰Qiagen公司，美国加利福尼亚州巴伦西亚市）从上述两种成纤维细胞中提取总RNA。采用SMARTer® Stranded Total RNA Sample Prep Kit–Pico Input Mammalian试剂盒（日本滋贺县TaKaRa公司）制备下一代测序文库。测序工作在Illumina HiSeq 2500平台上以75碱基单端测序模式完成，并使用Illumina Casava 1.8.2软件进行碱基识别。将测序读数通过TopHat v.2.0.13软件，结合Bowtie 2 v.2.2.3与SAMtools v.0.1.19软件，比对至人类参考基因组序列（hg19）。
实验结果：本次共分析26257个基因，筛选条件为NHLF与DHLF-IPF（即DIPF）的FPKM值不低于0.3。满足该筛选条件的基因中，DHLF-IPF（DIPF）细胞内共有764个基因呈上调表达，691个基因呈下调表达。
整体实验设计：本实验设置两组细胞系，分别为正常成人肺成纤维细胞（Normal Human Lung Fibroblast, NHLF）与特发性肺纤维化来源病变肺成纤维细胞（DHLF-IPF，即DIPF，Diseased Human Lung Fibroblasts Derived from Idiopathic Pulmonary Fibrosis）。