Genome-wide transcript profiling for native porcine valvular interstitial cells and those cultured on TCPS and treated with TGF-β1

Fibrotic diseases have significant health impact and have been associated with differentiation of the resident fibroblasts into myofibroblasts. In particular, stiffened extracellular matrix and TGF-β1 in fibrotic lesions have been shown to promote pathogenic myofibroblast activation and progression of fibrosis in various tissues. To better understand the roles of mechanical and chemical cues on myofibroblast differentiation and how they may crosstalk, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves and studied how traditional TCPS culture, which presents a non-physiologically stiff environment, and TGF-β1 affect native VIC phenotypes. We carried out gene expression profiling using porcine genome microarrays from Affymetrix and found that traditional TCPS culture induces major changes in gene expression of native VICs, rendering these cells more activated and similar to cells treated with TGF-β1. We also monitored time-dependent effects induced by TGF-β1 by examining gene expression changes induced by TGF-β1 at 8 hours and 24 hours. Porcine aortic VICs were isolated and cultured with or without TGF-β1 treatment for RNA extraction and hybridization on Affymetrix microarrays. We included 3 biological replicates for each condition. P0 VICs were freshly isolated cells which had not been cultured. P2 VICs were cells that had been passaged 2 times and cultured on plastic plates in low serum media. Some of the P2 VICs were treated with TGF-β1 at 5ng/ml for 8 hours or 24 hours. All the control and TGF-β1-treated conditions were collected at the same time on day 3 of culture.

纤维化疾病对健康具有显著危害，且与驻留成纤维细胞向肌成纤维细胞的分化密切相关。具体而言，纤维化病灶中硬化的细胞外基质与转化生长因子-β1（TGF-β1）已被证实可促进多种组织内致病性肌成纤维细胞的活化及纤维化进展。为深入探究机械信号与化学信号对肌成纤维细胞分化的调控作用及其潜在串扰机制，本研究培养了从猪主动脉瓣膜中分离得到的原代瓣膜间质细胞（valvular interstitial cells, VICs），并考察了传统组织培养聚苯乙烯（TCPS）培养体系——其提供的基质硬度不符合生理状态——与TGF-β1对天然VIC表型的影响。本研究使用Affymetrix公司的猪基因组微阵列开展基因表达谱分析，发现传统TCPS培养可显著改变天然VIC的基因表达模式，使这些细胞的活化程度升高，表型更接近TGF-β1处理组细胞。此外，本研究通过检测TGF-β1分别处理8小时与24小时后引发的基因表达变化，分析了TGF-β1诱导的时间依赖性效应。研究人员从猪主动脉瓣膜中分离VICs，设置是否经TGF-β1处理的实验组与对照组，收集样本用于RNA提取并在Affymetrix微阵列上进行杂交。每个实验条件均设置3次生物学重复。P0代VICs为未经过体外培养的新鲜分离细胞；P2代VICs为经2次传代、于低血清培养基中在塑料培养板上培养的细胞。部分P2代VICs经5ng/ml的TGF-β1分别处理8小时或24小时；所有对照组与TGF-β1处理组样本均在培养第3天同一时间点收集。