Substrate Rigidity And TGF-?1 Affect Cell Function Of Human Normal Liver Cells Based On RNA-sequencing

Objective: Based on high-throughput sequencing technologies, to explore the change of substrate rigidity and TGF-?1 on cell function of normal liver cells, as well asto study of liver cell biology behavior and the relationship between the liver fibrosis and provides new perspectives for the treatment of liver tissue pathological changes.Methods:Usingpolyvinyl alcohol gel as cell culture substrate and the rigidity of substrate implied in this study were 4.6 kPa, 19 kPa and 37 kPa, in accordance with normal liver tissue, early and advanced stage of liver fibrosis respectively. Cultivation of L02 cells on different hardness of PVA substrate,flow cytometry to detect cell apoptosis ratio. High-throughput sequencing technology was used to select differentially expressed genes under the coordinating role of substrate rigidity and TGF-?1. Immunofluorescence technique was applied to test cell morphology and the change of skeleton conformation. Using RT-PCR and western blot to detect gene and protein expression level of E-cadherin,Bate-catenin,Alpha-SMA,Vinculin,Integrin?1 and Collagen-1.Result:we established a stiffness controllable in vitro cell culture model by using polyvinyl hydrogel, which mimic the same physical niche as fibrotic liver. L02 cells cultured on PVA substrate present the fusion growth after 48 hours, and cell apoptosis rates were lower than 10% on three differest hardness of PVA, with the increase of hardness, apoptosis rates were decreased, proved that PVA substrate as a cell culture basementhas good biological compatibility. Cells deform factorincreased with the substrate turns stiffer. A significant increase in cell skeleton development was observed with increase in substrate stiffness, which gradually and markedly showed extended morphology with more precisely arranged microfilaments. There was on obvious change of skeletonconformation between TGF-?1 and the control group, but TGF-?1 promote the development of skeleton on the stiffer substrate. In addition, the expression level of cell-substrate(Integrin-?1 and vinculin) and cell-cell(E-cadherin and Bate-catenin) adhesive system related cytoskeleton proteins showed an increase in the former while decrease in the later on stiffer substrates, and TGF-?1 promote this trend. There were more abundant expressions of Collagen-1 and Alpha-SMA on stiffer substrate in TGF-?1 group, which indicated that TGF-?1 promot the process of Epithelial-Mesenchymal Transition and extracellular matrix collagen deposition play a important role in the liver fibrosis.Conclusion: Based on results, we conclude thatsubstrate-rigidity can induce hepatocyte phenotype transformation and promote the impact of TGF-?1on hepatocyte metabolic behavior.Our findings give a clue that further studies are desirable to explore in depth mechanism of human liver fibrosis.