An Experimental Study Of The Proliferation And Oriented Differentiation Of Rat Oval Cell Into Hepatocytes Regulated By Bone Morphogenetic Protein-4 (BMP-4)

Background & ObjectiveHepatic oval cell (HOC) is the major source of pluripotent stem cells in adult liver. The proliferation and differentiation of HOC, which is regulated by a series of cell growth factors, is one of the most important approaches and mechanisms of liver regeneration and tissue recovery following liver injury. Bone morphogenetic protein-4 (BMP-4) is a critical regulator involved in the treatment of different acute and chronic organs injury using several kinds of inherent and transplanted stem cells. WB-F344 cell, which was an adult rat HOC cell line, had been proved to be directionality differentiated into hepatic phenotype in our previous studies. However, the effects and the cellular signaling transduction mechanisms of BMP-4 on the proliferation and differentiation of the primary rat HOC remain unclear. The aims of the present stydy are to investigate the regulative effects and the intracellular signaling moleculars of BMP-4 on rat primary HOC proliferation and orientational differentiation into hepatocytes. The results will help us to have a better understanding for the biological functions and the molecular mechanisms of BMP-4 regualtion on HOC proliferation and orientational differentiation into hepatocytes, so as to provide the theoretical principle and clinical application basis for the treatment of liver injury by taking the advantages of BMP-4 and its cellular signaling transduction to promote liver regeneration and tissue revovery.MethodsThe animal models with proliferated HOC was set up by the methods of N-2-fluorenyl-acetamide gavage in combined with 2/3 hepatectomy in adult male Sprague-Dawley rats. Rat HOC was isolated and purified by in situ liver perfusion with Collagenase IV/ Pronase, which was followed by cellular gradient centrifugation using three density gradients of Percoll. The isolated primary rat HOC was cultured and sub-cultured to be amplified in vitro. The effects of BMP-4 on the proliferation of primary rat HOC was detected by MTT assay. RT-PCR was employed to determine mRNA expression of TypeⅠand TypeⅡBMP-4 receptors on rat HOC cytomembrane. To understand the intracellular signaling moleculars and transduction pathways of BMP-4 in rat HOC, the techniques of Real-time PCR, Western blot and immunofluorescent staining were used to explore mRNA and protein expression, as well as the phosphorylated protein expression and its accumulation in HOC nucleus of Smad1 and extracellular regulated protein kinases 1/2 (ERK 1/2), which were two major transcription factors of BMP-4. To approach the induction of BMP-4 on HOC orientational differentiation into hepatocytes, the cell markers of rat HOC, mature hepatocytes and biliary epithelia cell (BEC) were measured by RT-PCR for their mRNA expression. The concentrations of urea and albumin secreted by differentiated HOC into HOC culture medium were tested by automated biochemistry examination and ELISA assay. The HOC ultrastructures and cell-cell junctions were checked on a transmission electron microscope (TEM). Rat BMP-4 and BMP4 shRNA recombinant adenovirus were constructed applying gene recombination. The HOC infected by either BMP-4 or BMP4 shRNA recombinant adenovirus was applied in allograft models of rat spleen transplantation to observe the effects of BMP-4-carrying HOC transplantation on the recovery of liver tissue and functions following acute liver injury by carbon tetrachloride (CCl4) gavage.ResultsAbout 5.63±0.56 x 106 HOC with 90.32%±3.26% living cells were isolated from each Sprague-Dawley rat successfully established HOC proliferation in vivo. Recombinant BMP-4 protein could boost the proliferation of HOC in vitro in both dose-dependent and time-dependent manners. Comparing with either lower or higher BMP-4 concentrations. 50 ng/ml of BMP-4 was the optimal concentration on HOC proliferation promotion, which reached its peak at the sixth day of BMP-4 treatment at the concentration of 50 ng/ml. The existence and mRNA expression of BMPR-1A, BMPR-1B and BMPR-II on HOC cytomembrane were proved by RT-PCR. BMP-4 treatment at the concentration of 50 ng/ml could not influence mRNA and protein expression of both Smadl and ERK 1/2. However, BMP-4 intervention significantly enhanced phosphorylated Smad1 and ERK 1/2 proteins and its accumulation in HOC nucleus. Following 12-day treatment of BMP-4 at 50 ng/ml, the stem cell marker of c-kit and BEC markers of CK19 andβ4-integrin significantly faded or vanished, but the mRNA markers of matured hepatocytes including TAT-1,Alb and G6Pase were significantly increased in rat HOC. Meanwhile, the urea concentrations secreted by HOC into the cultured medium in BMP-4 treatment group (1.32±0.32 mmol/L) exhibited 2.16-fold and 2.24-fold of that in Noggin treament group (0.61±0.09 mmol/L) and the blank control (0.59±0.13 mmol/L), and the Alb concentrations in HOC cultured medium in BMP-4 group (68.256±13.256 ng/ml) were 4.63-fold and 4.29-fold of that in Noggin group (14.735±4.012 ng/ml) and the blank control (15.897±4.283 ng/ml), respectively (p< 0.05, respectively). The HOC ultra-structures and intercellular junctions observed under TEM indicated that BMP-4 induced rat primary HOC to differentiate into epithelium-like cells in vitro. Noggin, which acted as BMP-4 antagonist, could specifically block the regulative effects of BMP-4 on rat HOC proliferation and its differentiation into hepatocytes. The rat HOC infected with the constructed BMP-4 and BMP4 shRNA adenovirus were applied in rat spleen allografts. On the ninth day after intraspleenic HOC transplantation into homogenous rats receiving CCl4 gavage, the fixation of hepatocytes differentiated from transplanted HOC and the specific "hepaticized spleen", obviously recovered liver tissue, significanltly improved general conditions of rats'mortality, hair, spirit, movement and appetite, as well as the liver functional indices including total bilirubin (TBIL), albumin, ALT and AST were observed in spleen transplantation of the HOC infected with BMP-4 adenovirus, as compared with that of the other five experimental control groups including BMP4 shRNA adenovirus-infected HOC transplantation, etc.ConclusionsFollowing the binding to TypeⅠandⅡreceptors on rat HOC cytomembrane, BMP-4 transducts its intracellular signal through its transcription factors of Smadl and ERK-1/2 to induce HOC proliferation and orientational differentiation into hepatocytes with protein synthesis and secretion functions, as well as the ultrastructures and intercellular junctions of mature hepatocytes. Rat HOC spleen allografts regulated by BMP-4 will promote the liver tissue regeneration and liver function recovery in the rats with acute liver injury.