Studies On Bone Marrow Mesenchymal Stem Cells Transplantation For The Treatment Of Cirrhotic Rats

Objective:To establish the standard rat models of cirrhosis and explore whether the transplanted BMSCs affect the regeneration of remnant liver with partial hepatectomy(PH),and whether BMSCs transplantation can treat the cirrhosis rats without PH.To observe the planting and homing of transplanted BMSCs in the liver.Method:1. The rat models of cirrhosis were prepared with compound factors.(intraperitoneal injection of olive oil which contained 40% CCl4,sole drinking water contained 20% alcohol, high-fat and low-protein diet),whether the cirrhosis models was successfully established was detected by pathologic histology section, the detection of biochemistry and Collegen IV(IV-Co) aimed at knowing the physical condition of the model rat.2. Bone marrow was obtained by surgery from 2-weeks-old rats. BMSCs were isolated and cultivated by Whole Bone Marrow Adherent Culture Method.The cell surface antigens(CD29,CD34,CD44,CD90) of BMSCs were detected by flow cytometry and immunofluorescence. BMSCs were labeled by using CM-Dil in vitro.Both labeling rate and survival rate of the cells were detected.3. The rat models were divided into four groups randomly: Group A, partial hepatectomy(PH)+ cells transplantation group(n=18):38% liver were resected and transplantated with lml BMSCs(1×106) via portal vein;Group B, partial hepatectomy(PH) group(n=18):38% liver were resected and transplantated with lml PBS via the portal vein;Group C, cells transplantation group(n=18):lml BMSCs(1×106) were transplantated into liver via portal vein;Group D,control group(n=18):lml PBS was transplantated into liver via portal vein;All the rats were feed with ordinary food and water after surgery.three to four rats were selected from Group C and Group D randomly,them were sacrificed on 1,3,7,14 days and 1,2months post transplantation.Liver function and IV-Co of the sacrificed rats were detected,and HE and Masson staining were detected to know the pathologic change of the liver.Frozen sections were observed to know the planting and homing of the transplantated BMSCs in the liver.Results:1.The rat models of cirrhosis were successfully established by compound factors in eight weeks.HE and Masson staining showed: the structure of normal rats’ liver lobules altered to disordered structure,majority of the liver tissue were bestrewed with fibres, pseudolobuli was observed.Blood detections showed: the titre of aminopherase,bilirubin and IV-Co were increased, and the titre of albumin was reduced.2.BMSCs obtained from 2-weeks-old rats grew well.They grew as fish scales or vortexes,with uniformly long fusiform or irregular shape. Immunofluorescence analysis showed: CD34+,CD44-.Flow cytometry analysis showed: the positive rate of CD29 was 94.3%, CD90 was 97.5% and CD44 was 2.39%.The labeling rate of BMSCs by CM-Dil was 99.8%,and the survival rate of the labeled BMSCs was 96.7%.3.All of the rats from Group A and B died in a short time after the PH surgery,the main cause of death was ileus which caused by serious adhesion of peritoneal cavity.ALT,AST,ALB and IV-Co of the rats in Goup C began to be improved 2 weeks post BMSCs transplantion.(P<0.05),the value of TBIL obviously declined less than one month after the cells transplantations(P<0.05),compared with Group D the changes was statistical(P<0.05). Histopathology showed: the degree of necrosis, steatosis and fibrosis improved gradually.The Laeannec grading Score of hepatic fibrosis showed significant differences between Group C and Group D(P<0.05).4. As the rats of Group C were transplantated BMSCs,the fluorescent tracer technique showed:fluorescence mainly appeared in portal area after one day,fluorescence mainly appeared nearby fibrotic septum of liver after 1 week,fluorescence mainly appeared inner hepatic lobule after 2 weeks,fluorescence dispersally appeared in hepatic lobule after 2 months,but the intensity weakened.There wasn’t fluorescence in Group D.Conclusion:1. Allogeneic BMSCs transplantion can improve the liver function of cirrhosis rats and alleviate the degree of liver fibrosis.2.The method of labeling CM-Dil can be used for observing the planting and homing of transplanted BMSCs in the liver.