Prevention Of Liver Fibrosis By Intrasplenic Injection Of High Density Cultured Bone Marrow Cells In A Rat Model

Objective: The purpose of this study was to establish a stable rat chronic liver fibrosis model first and subsequently using this model to evaluate the anti-fibrogenic potential of high density cultured bone marrow cells through intrasplenic injection. In addition, the possible mechanism involved in the anti-fibrosis process was investigated.Methods: 1. Chronic liver fibrosis model in Wistar rat was established by intra-peritoneal injection of CCl4. 2. The rats were divided into three groups: high density(HD) group, regular density(RD) group and phosphate puffer polution(PBS) group. Two million of CM-Di L labeled HD cells or RD cells were suspended in 200 ul PBS and injected intrasplenicly. Rats received same volume of PBS were served as controls. 3. Blood samples of all experimental rats were tested to analyze liver function. Hematoxylin & Eosin and Picric acid-Sirius Red(PSR) staining of the liver tissues were performed to reveal the extracellular collagen deposition. Type I collagen and α- smooth muscle actin(SMA) expressions were detected by immunohistochemical(IHC) staining. Furthermore, gene expression of α2-procollagen, α- SMA, fibronectin(FN) and transforming growth factor(TGF-β) in the liver tissue were detected by quantitative real-time polymerase chain reaction(q RT-PCR) analysis. 4. After immunofluorescence(IF) staining of type I collagen in frozen sections, the distribution of CM-Di L labeled cells were observed under a fluorescent microscope. 5. Ki-67 and CD31 expressions were detected by IHC staining of liver tissues to detect the cell proliferation and neovascularization. Hepatocyte growth factors(HGF) and vascular endothelial growth factor(VEGF) gene expressions were analyzed by q RT-PCR.Results: 1. Multiple and repeated injection of CCl4 could lead to chronic liver fibrosis accompanying with increased serum level of glutamic oxalacetic transaminase(AST) and glutamic pyruvic transaminase(ALT) and but decreased level of albumin(ALB). 2. Bone marrow cells expressed high level of CD31, CD133 and KDR after HD culture. Meanwhile, in vitro tubular formation assay confirmed that cells from HD culture formed obvious tubular network but not in the cells from RD culture, indicating that cells from HD culture contains more endothelial progenitor cells(EPC).3. Both AST and ALT were increased in the PBS treated group, but not in the HD and RD groups. ALB level was decreased in the PBS group but not in the other two cell-transplanted groups. HD cells performed better than RD cells in the treatment.4. Hemotoxylin & Eosin staining of liver section showed that liver remodeling and pseudolobule formation occurred after CCl4 injection. Reduction of pseudolobule formation was observed in the HD and RD groups but not in PBS group. Picric sirius red and Masson staining of liver tissues confirmed extracellular collagen deposition of HD group was significantly decreased than that of RD group and PBS group. Meanwhile, q RT-PCR analyses of liver tissues demonstrated that α2-procollagen, α-SMA and fibronectin gene expression of HD group was lower than RD group.5. IHC staining showed that, more CD31 positive blood vessels and Ki-67 positive cells were observed in the HD group than those in the RD and PBS groups. Further q RT-PCR analysis of relative gene expression of HGF and VEGF showed that a higher level of VEGF and HGF expression was observed in livers transplanted with HD cultured cells.Conclusion: We demonstrated that HD cultured bone marrow cells played a more effective role in amelioration of rat liver fibrosis, functional recovery, and hepatic regeneration. This simple and cost-effective culture system provides an effective way for expansion of bone marrow cells for future clinical applications.