Establishment of hepatic stellate cell line with stable expression of HBx protein and its preliminary characterization

Objective To establish a LX-2 cell line with stable expression of HBV X protein (HBx) and preliminary study the effect of HBx to hepatic stellate cell (HSC) proliferation and collagen Ⅰ expression.Methods A recombinant lentivirus vector LV5-X and a vehicle control plasmid were constructed,both of which subsequently transfected into LX-2 cells.Then a stable HBx-expressing LX-2-X cell line and a control LX-2-C cell line were established after puromysin selection.The transfection rate was determined by fluorescene microscope and HBx expression was detected by western blot.For the preliminary characterization of the LX-2 cell line,the cell proliferation was detected by CCK-8,the mRNA expression of collagen Ⅰ was qualified by Real-time PCR and the collagen Ⅰ secretion was measured by ELISA.Results The recombinant lentivirus LVS-X was successfully constructed and then infected LX-2 cell to establish the cell lines named LX-2-X and LX-2-C.After selection of puromysin for 3 d,approximately 100％ of the LX-2-X cells expressed green fluorescent protein(GFP) and western blot showed that LX-2-X cell line expressed HBx stably.LX-2-X cells showed the higher proliferation compared to LX-2 and LX-2-C cells after 72 h and 96 h (P＜0.05).The collagen Ⅰ expression of LX-2-X cells at both mRNA and protein level were higher than LX-2 and LX-2-C cells (P＜0.05).Conclusions The LX-2 cell line named LX-2-X which can express HBx stably is successfully established.HBx could promote the proliferation of HSC and increase the expression of collagen Ⅰ. Key words: Hepatitis B virus ;  HBx protein;  Recombinant lentivirus vector;  LX-2 cell line;  Hepatic fibrosis