Effect Of Feridex-GFP Double Labeling On Bone Marrow Mesenchymal Stem Cell Colonized In The Damaged Liver In Constant Magnetic Field By Invitro

Background and Objective]Progressive liver fibrosis is a variety of chronic liver disease develop liver cirrhosis primary pathogenesis. It makes the over-deposition of extracellular matrix and reduction of functi-onal liver cells at the same time, especially in acute liver injury. In recent years, it became a hot point to supplement the lost of liver cells. The stem cell transplantation is one of the most cutting-edge and hottest medic-al technologies in the world. Researches shown, the autologous stem cell transplantation is effective for the treatment of ascites due to cirrhosis. It had the value to promote in clinical practice, known as the second ultimate way of cirrhosis treatment.Bone marrow mesenchymal stem cells (BMSCs) are which derive-ed from mesoderm, which have multi-differentiation capacity, and exist in connective tissue, especially in bone marrow. BMSCs can be induced and differentiated into neural cells, hematopoietic cells, fat cells and so on. BMSCs have strong capacity of self-replication and different-iation potential, but are low immunogenic. They can be induced to differentiate into liver cells in vitro and in vivo. Animal experiments and clinical studies shown, autologous stem cell transplantation can improve liver function and was expected to be the new cell source in the treatment of liver diseases.The transplant way of BMSCs is an important factor to affect the colonization of stem cells into liver. It is common to transplant BMSCs through portal vein and hepatic artery in clinical treatment. The ideal way should be minimally invasive, easily to operate and highly colonizable. Feridex is a magnetic targeted and can positione in the target organ. It can improve the accuracy of stem cells transplant.In our study is to transplant Feridex-GFP double labeling BMSCs is applied to transplant into rats with acute liver injury via peripheral vein, portal vein and hepatic artery under effect of constant magnetic field. The colonization of BMSCs through different way into liver was analyzed and the liver function recovery status was tested, especially constant magnetic field in vitro. Minimally invasive, easily operated and high colonizated way should be found for BMSCs transplant.Method(1) The isolation and purification of BMSCs. BMSCs were isolated by density gradient centrifugation and adherent cell co-culture method. Purified BMSCs were gained through continuous cell co-culture and verified by flow cytometry through characteristic surface markers of BMSCs.(2) Induced to differentiation of BMSCs. Purified BMSCs were treated by using hepatocyte growth factor (HGF) and basic fibroblast factor-4(FGF-4) for28days, and induced to liver cells.(3) Feridex-GFP double labeled BMSCs in vitro. BMSCs were treated by the Feridex magnetization and the GFP recombinant retrovirus. The labeled BMSCs were observed by prussian blue staining, fluorescence microscopy and MTT test.(4) The establishment of rats model with acute liver injury. The normal female Wistar rats were intraperitoneal injected with10%CC14. Albumin (ALB), alanine transaminase (ALT), aspertate aminotransferase (AST) were tested and the liver biopsy was made (HE staining).(5) The transplant of BMSCs into liver through different ways. Feridex-GFP double labeling BMSCs were transplanted into liver through six different ways:peripheral vein, portal vein and hepatic artery and those in effect of constant magnetic field in vitro. The rats were sacrificed at the1st,2nd,3rd and4th week after the transplant. ALB, ALT, AST were tested. The liver, kidney and lung tissue biopsy were made. GFP-positive cells in liver were observed by fluorescence microscopy.Results(1) Using density gradient centrifugation and adherent cell co-culture method, BMSCs were obtained in high purity and activity in vitro proliferation. (2) Purified BMSCs were high expression of CD29and CD44, but no expression of CD11b and CD45through flow cytometry.(3) Feridex-GFP double labeled BMSCs in vitro. BMSCs had stable expression of GFP by fluorescence microscopy and Fe3+expression by prussian blue staining. There were no significant difference between double lableled and unlableled BMSCs by MTT.(4) HE staining showed the successful establishment of rats model with acute liver injury by CCl4. Serum was detected and showed significant changes in ALB, ALT and AST (P<0.05).(5) BMSCs were transplanted into liver through6groups, the rats were sacrificed at the1st,2nd,3rd and4th week after the transplantion. Liver pathology results were improved, but no remarkable difference among groups. The GFP expression was found in liver at the4th week by fluorescence microscopy. It indicated BMSCs were colonized in liver after tranplantion of6groups, especially portal vein+constant magnetic field group and hepatic artery+constant magnetic field group with high GFP expression in liver but low in lung and kidney.(6) Serum was detected in ALB, ALT and AST at the1st,2nd,3rd and4th week after the transplantion. There were significant difference in decreased ALT, AST and increased ALB at the1st week. And it was found significant difference in decreased ALT, AST and increased ALB of portal vein+constant magnetic field group and hepatic artery+constant magnetic field group at the2nd week, but no difference among6groups at the4th week. It indicated the2groups of translation could inhibit the liver damages earlier than other4groups.Conclusion(1) BMSCs were obtained in high purity and activity in vitro prolif-eration by density gradient centrifugation and adherent cell co-culture method.(2) BMSCs were transplanted into liver through6groups and all can found GFP expression in liver at the4th week by fluorescence microscopy. BMSCs were colonized in liver after tranplantion, especially portal vein+constant magnetic field group and hepatic artery+constant magnetic field group with high colonized expression. (3) Based on long term efficacy similarly, portal vein+constant magnetic field group and hepatic artery+constant magnetic field group promote the early recovery of liver function.(4) Compare colonization rate and efficacy, combined with clinically relevant technical difficulty and feasibility of the operation, we initially considered for BMSCs transplantation for acute liver damage, you may prefer portal vein+constant magnetic field group ways, hepatic artery+constant magnetic field group ways, the second choice peripheral vein ways or peripheral vein+constant magnetic field group ways.There are totally12figures,5tables and93references.