RNA-Seq experiment of primary hepatic stellate cells (HSCs) and LX-2 cell line treated with TGFb. RNA-Seq experiment of primary hepatic stellate cells (HSCs) and LX-2 cell line treated with TGFb

Liver fibrosis stands as the most prominent predictor of overall mortality in non-alcoholic steatohepatitis (NASH). The fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from "activated" hepatic stellate cells (HSCs). Whereas targeting HSC in fibrosis therapeutics shows promise, the current identification of human genetic regulators driving HSC activation is far from complete. This knowledge gap largely emanates from the limited understanding of the vast array of long non-coding RNAs (lncRNAs). Analyzing differentially regulated human lncRNAs under various regulatory patterns often provides the most practical means to inform their function. To get human lncRNA regulators in driving HSC activation, we cultured both primary human stellate cells and LX-2 cells. Then, we treated them with TGFb to mimic the activation process in vitro. Differentially expressed lncRNAs can be obtained from RNAseq results, which may guide the characterization of lncRNAs relevant to HSC activation. Overall design: Human primary HSCa were thawed in Human Stellate Cell Growth Media (Lonza) and seeded at a density of 4,000-5,000 cells/cm2 on a Collagen I coated plate using the growth media above. The medium is changed every other day. Cryopreserved, immortalized LX-2 cells were obtained from Millipore. The standard procedure was followed by the manufacturer's instructions for thawing, splitting, and freezing of the cell line. For TGFβ treatment, 10ng/ml of reconstituted TGFβ was added to the media. Cells were treated for 24 hours before collecting for RNA isolation.

肝纤维化是非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）患者总体死亡率的最显著预测因子。纤维化肝脏的特征为细胞外基质（extracellular matrix, ECM）过度沉积，而该基质主要由“激活态”肝星状细胞（hepatic stellate cells, HSCs）产生。尽管以HSC为靶点的纤维化治疗方案已展现出应用潜力，但目前我们对驱动HSC激活的人类遗传调控因子的认知仍存在显著缺口。这一认知空白很大程度上源于我们对种类繁多的长链非编码RNA（long non-coding RNAs, lncRNAs）的理解不足。分析不同调控模式下的差异调控人源lncRNAs，往往是揭示其功能的最实用途径。为筛选驱动HSC激活的人源lncRNA调控因子，我们分别培养了原代人肝星状细胞与LX-2细胞，并通过添加转化生长因子β（TGFβ）以体外模拟HSC激活过程。从RNA测序（RNAseq）结果中可获取差异表达的lncRNAs，这将为表征与HSC激活相关的lncRNAs提供指导。整体实验设计：将冻存的原代人肝星状细胞置于人星状细胞生长培养基（Lonza）中复苏，以4000~5000个细胞/cm²的密度接种于Ⅰ型胶原蛋白包被的培养板中，使用上述生长培养基进行培养，每2天更换一次培养基。我们从Millipore公司获取了冻存的永生化LX-2细胞系，并严格遵循制造商说明书完成该细胞系的复苏、传代与冻存操作。在TGFβ处理实验中，向培养基中添加终浓度为10ng/ml的重组TGFβ，处理细胞24小时后收集细胞并进行RNA提取。