Study On Differentiation Of Multipotent BMSCs Into Hepatocyte-like Cell In Vitro And MRI Tracking Of Labeled Cells After Transplantation Into Injured Liver

It is always a focus in research as well as a difficulty in clinical practice to regenerate hepatocyte and repair liver function after hepatic injury due to various causes. Because the shortage of liver donators, it is difficulty to survive after implantation and high cost limited undesirably the therapies of liver graft and hepatic stem cell transplantatoin. Recently, however, embryonic stem cells (ESC) and bone marrow stem cells (BMSCs) have been highly thought of for their easy acquisition and convenient amplification in vitro, let alone their ability to differentiate to hepatocyte under some circumstances and involve in the regeneration of hepatocyte and the recovery of liver function.But the application of mesenchymal stem cells instead of mature liver cells in transplantation will be a breakthrough for the treatment of liver diseases. However, in vivo image of transplant cells and further investigation would be a trouble to be faced with. Since Weissleder proposed conception of molecular imaging (MI) in 1999, M I had become the focus in radiology and relative clinical medicine. However, it is unclear how BMSCs locate, immigrate and differentiate in the recipient liver in vivo after allogeneil graft. Moreover, the previous methods in the experiment cannot be applied to human research for their fatal trauma on tested objects for obtaining of liver specimens from the objects. So a labeling reagent is needed to label and track the allogeneilly grafted BMSCs in vivo in a convenient way.Meanwhile, it is also expected to make it easy to understand the location, immigration, differentiation and ultimately the therapeutic efficacy on hepatic diseases as well as to develop an effective labeling method and even provide a solid ground for tracking the similar role of other cells. Objective: To investigate characteristics of BMSCs derived from young rats bone marrow and evaluate the feasibility of constructing tissue-engineering cell.This study aim to establish a method of isolation and expandation mesenchymal stem cells, to optimize and standardize the condition of inducing BMSCs to differentiate into hepatocyte-like cells in vitro, and to investigate the induced BMSCs transplantation for the treatment of acute and chronic liver injury treated with CCl₄. The exploration will be beneficical for clinical application.Method: BMSCs were isolated, expanded and purified by density gradient centrifugation combined with adherent culture; BMSCs were evaluated cell surface characterization; Oil RedO distinguishs that BMSCs could induced to differentiate into the adipocytes; Under the induction of DEX, V_C. andβ-Gly,levels of ALP were elevated and BMSCs differentiated into osteoblasts. which were Von Kossa stain positive; 3.10%FBS f 10% injured liver homogenate in vitro induced BMSCs to morphological change and differentiated character detectived albumin by immunocytochemistry and RT-PCR could evaluates the expression of AFP,ALB;The model rats were randomly divided into control groups and treatment groups.The labelled BMSCs on 21th day after induction were transplanted into the rats by portal vein, liver topical, respectively. PBS was transfused into rats in various control groups.Result The isolated BMSCs were mononuclear cells in bone marrow their living behaviores was quite stable in L-DMEM containing 10% FBS and BMSCs were quite similar exhibiting a large expensive potential and the typical fibroblast-like morphology. BMSCs are pure and well-organized cell groups;Passages 3 express high levels of cell surface characterization CD105:The Horse serum could induce BMSCs to differentiate into the adipocytes. After induction, lipid droplets appearance and the cells displayed a perinuclear accumulation of lipid vacuoles gradually, and their shape from long fusiform cells to round or polygonal. Then Oil Red O stain positive.Under the induction of dexamethasone (DEX), ascorbate-phosphate (Vita min C), andβ-glycerophosphate (β-Gly), theBMSCs gradually changed their shape from polygonal, triangle, or long fusiform cells, to cubiform, then elliptic, and even rotundity. Under induction, levels of ALP were elevated and the morphological changes of osteoblast.phases of proliferation, aggregate- ion, nodulation and mineralization were observed.That meant the BMSCs differentiated into osteoblasts, which were Von Kossa stain positive. Passage 3 differentiate into hepatocyte-like cells in L-DMEM containi- ng 10% FBS and 10% injured liver homogenate Albumin immunocytochemic- al result was positive. RT-PCR was used to study the expression of AFP, ALB. The cells underwent MR imaging with TiW, T2W sequences. The signal intensity change could be observed on T1W, T2W.Successfully labeled BMSCs were respectively transplanted through portal vein, local liver parenchyma of the rats. MRI examination was conducted on the rat livers. MRI showed that no remarkably low signal change in the control group However, the images of the group with graft via portal vein showed remarkably low signal changes within 24 after transplantation.For the group grafted by way of local liver parenchyma, remarkably low signal changes were seen to come up within 24h after transplantation, and the signal change more remarkably than the group injected through portal vein.Conclusion 1. The isolated BMSCs were mononuclear cells in bone marrow their living behaviores was quite stable. BMSCs were quite similar exhibiting a large expensive potential in vitro.2. Under the induction of Horse serum, BMSCs were induced to differentiate into the adipocytes.That established a model for BMSCs differentiate into the adipocytes. The potential of adipocytes differentiation was an important criterion for distinguishing the bone marrow-derived mesenchymal stem cells from hematopoietic stem cells.BMSCs cultured in vitro have strong proliferation ability and osteogenic capacity; DEX, Vc,β-Gly were important factor for inducing BMSCs differentiation into osteoblasts.3. Passage 3 differentiate into hepatocyte-like cells in L-DMEM containing 10% FBS and 10% injured liver homogenate,so we obtain the evidence that BMSCs cure hepatic disease by intrahepatic transplantation.4. MRI tracing in vivo after the cell transplantation: within 24h:no significant differences can be observed in normal control group. The decreasing MR signal can be detected in normal control group within 24h after transplanted into left lobe of liver. The decreasing signal can be clearly observed in T1WI, T2WI sequences in the labeled stem cells team, labeled stem cells transplanted into left lobe of liver can be detected remarkable low signal changes.