The Research On The Hepatocyte Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Induced By Liver Homogenates Of Acute Alcoholic Liver Injury Mode

Background and objective:Acute liver damage characterized by a short period of time liver cells degeneration and necrosis and inflammatory cells infiltration,the treatment of which is clinical thorny problem.Acute and chronic liver injury,and even liver failure caused by viruses, drugs, bacteria, alcohol or genetic factors,does great harm to people’s health.Currently accepted therapy and the most effective method for acute severe and end-stage liver disease is liver transplantation.Due to lack of liver source,expensive price and immune rejection reactions after surgery,the transplantation is limited.Bone marrow mesenchymal stem cells is derived from a group of mesoderm heterogeneous Cells,with self-renewal and multi-differentiation potentials,and which can differentiate into osteoblasts, cartilage and fat cells under the appropriate microenvironment,and even myocardial cells,liver cells and nerve cells.For the characteristics of easily obtained,easily amplified,low immune rejection,no ethics controversy and low costs,BMSCs becomes tissue engineering and cell therapy research focus.To explore the feasibility of BMSCs in pathology microenvironment to liver cell differentiation,know the possible mechanism of BMSCs treatment of acute liver disease,and provide a theory basis for stem cell transplantation in the treatment of acute liver disease,acute alcoholic liver injury rat model was prepared by alcohol toxicity,and then rat BMSCs was induced by damage liver tissue homogenates to differentiate into hepatocyte,and the normal liver tissue homogenates as control.Methods:(1)BM cells were obtained by flushing out the femurs of4-week-old Sprague-Dawley male rats,then BMSCs were cultivated,purified and proliferated in LG-DMEM Medium containing10%FBS by adherence wall culture technique.Morphological characteristics of BMSCs were observed under an inverted microscope;MTT assay tested the3rd generation BMSCs and the growth curve was drawn;the expression of CD29,CD34and CD105was detected by flow cytometry.(2)10Sprague-Dawley male rats weight400-500g,were randomly divided into the liver injury group and control group,5in each group.After fasting12h but provide water,liver injury group rats were disposed with56°one-time Red Star Erguotou Irrigation stomach method10ml/kg (equivalent to the alcohol4.5g/kg),and the control group rats received equivalent saline.Rats were sacrificed after24hours.Liver tissue was taken for histological examination.(3) According the proportion of liver wet weight O.1g plus lml LG-DMEM medium with10%FBS prepared normal and injury liver homogenates on the ice. The3rd generation BMSCs was cultured by damage liver tissue homogenates,and the normal liver tissue homogenates as control.The expression of AFP and ALB was detected by RT-PCR and western blot at the0,3rd,7th,10th,14th and21th dayResults:(1)The medium of primary cells was changed firstly after72h,and a small amount of long spindle cells could be seen.The cells grew rapidly and started to fusion after8-9days.Passage cells grew rapidly with form homogeneous,swirled and fish-like growth.Growth curve showed that the cells is in the incubation period at lst-2nd days, the4th-6th days in the proliferation period with rapidly growth, and the7th-8th days into platform period with slowly growth.The expression of surface antigen CD29and CD105was positive,and CD34was negative.(2)Liver pathology of liver damage model rats showed hepatic steatosis,and liver tissue of the control group with normal morphology.(3)Under the culture of normal and injury liver homogenates,BMSCs gradually changed the long spindle shape into the polygonal shape,and with hepatocyte-like change at the14th days in both groups.The expression of AFP from the3th days was detected in both groups,and peaked at the7th days,then gradually decreased until disappeared.ALB expression was detected at the7th days,and was gradually increased with time.The expression of mRNA levels and protein levels were consistent. However,the expression levels of AFP and ALB between damage and normal liver tissue homogenates groups were no significant differences at each time point (P>0.05).Conclusion:(1)The adherence wall culture method could obtain BMSCs with high purity,strong activity and proliferated.(2)Acute alcoholic liver injury model could be successfully prepared by56°Red Star Erguotou one-time irrigation stomach after fastting12hours.(3)The liver tissue homogenates of acute alcoholic liver injury rat model could induce BMSCs to differentiate into hepatocyte,but the result is similar to the normal liver tissue homogenates.