The Experimental MR-DWI Study On Therapeutic Effect Of Allogeneic Mononuclear Bone Marrow Cells Transplantation To Acute Hepatic Injury

PartⅠ:The experimental study on therapeutic effect of allogeneic mononuclear bone marrow cells transplantation to rabbit liver with acute hepatic injuryObjective:To investigate the therapeutic effect of mononuclear bone marrow cells (MBMCs) transplantation to rabbit liver with acute hepatic injury (AHI) induced by D-galactosamine (D-GaIN)Materials and Methods:The prepared D-GaIN solution was injected sterilely into the hypogastrium of each rabbit at a dosage of 10g/kg to establish 66 AHI rabbit models. AHI rabbits (n=66)were randomly divided into 2 groups, a transplanted group (n=32) and a control group(n=34). MBMCs were derived from bone marrow obtained from all limb long bones of male albino rabbits.Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites'. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension. The survival rates were observed and liver functions (ALT, AST, ALB) were estimated for the two groups.7 model rabbits from each group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively, to be carried out pathological examination of liver and pathological score. All data were analyzed by SPSS 11.0 software package.Results:Survival rate of the transplanted group during the first week after MBMCs transplantation was higher than that of the control group(p<0.05). However, there was not significant difference of the survival rate between the two groups during the second week and the fourth week after MBMCs transplantation (p> 0.05). With regard to liver function, the liver functions of the two groups both got improvement gradually, but the level of liver enzyme ALT and AST significantly decreased in the transplanted group compared to the control group at any same time point observed after MBMCs transplantation (p<0.05).There was also a significant improvement of serum albumin (ALB) in the transplanted group compared to the control group (p<0.05)except the observed time point of the 24th hour and 28th day after AHI establishment and MBMCs transplantation. Results of the histopathological scores showed that the histopathological scores of the two groups both gradually decreased over time, but the histopathological score of the transplanted group was better than that of the control group at any time point observed(p<0.05).Conclusions:Direct injection of MBMCs suspension into liver at multiple sites can effectively improved the liver function of the acute liver injury in D-GalN-induced rabbits. Direct injection of MBMCs suspension into liver at multiple sites is a simple and effective route of MBMCs transplantation in experimental model.PartⅡ:Evaluation of MVD and VEGF protein expression of liver tissue in the treatment of acute hepatic injury with allogeneic MBMCs transplantation into liverObjective:To investigate the dynamical changes of VEGF (Vascular endothelial cell growth factor) protein expression and the average value of average MVD(micro-vessel density) in acute hepatic injury model after transplantation of MBMCs (mononuclear bone marrow cells). To investigate the correlation between the average value of MVD and liver function.Materials and Methods:In this part,there were 42 model rabbits of acute liver injury,which were chosen to carry out pathological examination of liver and pathological score in the first part of the thesis. So, in this part there were two groups too,the transplanted group and the control group.There were 21 model rabbits in each group. Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension.7 model rabbits from each group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively. A series of comparative assays performed were including:functional assay of liver, immunohistochemical pathological examination of liver sections, VEGF-expressing cells count and quantification of the average MVD value. At the same time,we investigated the the correlation between the average MVD value and liver function.Results:Before MBMCs transplantation, the difference of liver function was not significant between the transplanted group and the control group. But after MBMCs transplantation,the liver functions of model rabbits in the transplanted group were significantly better than those of the control group at all time points observed (p<0.05). More VEGF-expressing cells and higher average MVD value were observed in the transplanted group than the control group (p<0.05). In the transplanted group, the count of VEGF-expressing cells gradually reduced, the count on day 7 was higher than that on day 14 (p>0.05), while both were significantly higher than the count on day 28 (p<0.05). In contrast, the average value of MVD increased gradually in the transplanted group, the value on day 28 was higher than the one on day 14 (p>0.05), while both were significantly higher than the one on day 7 (p<0.05). In the control group, the count of VEGF-expressing cells also gradually reduced, the VEGF-expressing cell number on day 7 was higher than the one on day 14 and day 28 (p<0.05), and the number on day 14 was higher than the one on day 28 (p>0.05). The average MVD value increased with significance between each time points observed over time (p<0.05). The correlation between the average MVD value and the ALT or AST was negative (the transplanted group:-0.913,-0.918, the control group:-0.776,-0.678) (p<0.05), at the same time, the correlation between the average MVD value and the ALB was positive (the transplanted group:0.924, the control group:0.729) (p<0.05).Conclusions:Transplantation of MBMCs promotes the expression of VEGF and also up-regulates the average MVD value in the acute injury livers. This facilitates the recovery of liver function.PartⅢ:The study of magnetic resonance diffusion-weighted imaging(MR-DWI) to the treatment of acute hepatic injury with allogeneic MBMCs transplantation into liverObjective:By analyzing the dynamic changes of the mean ADC values of the injury livers after MBMCs transplantation and the correlation between the mean ADC values and the injury liver histopathological score or MVD values,to investigate the feasibility that evaluated the functional recovery of acute hepatic injury model with MR-DWI and the degree of the influence of the histopathological score and blood perfusion to the mean ADC value of the injury liver. Materials and Methods:In this part,there were 10 healthy rabbits and 42 model rabbits with acute liver injury,which were chosen to carry out pathological examination of liver and pathological score in the first part of the thesis. So, in this part there were three groups:the normal control group(n=10), the transplanted group(n=21) and the control group(n=21). Each rabbit of the transplanted group was directly infused 5 ml MBMCs suspension (about containing 2×107 MBMCs) into its liver at multiple sites. All manipulations to each rabbit in the control group were as same as those in the transplanted group except that 5 ml of D-Hanks solution was injected instead of MBMCs suspension.7 model rabbits from the transplanted group and the control group were killed on day 7, day 14 and day 28 of AHI establishment after transplantation of MBMCs, respectively. A series of comparative assays performed were including:immunohistochemical pathological examination of liver sections, quantification of pathological score and the average MVD value. Before the rabbits killed,each model rabbit was carried out MR-DWI scan and calculated the mean ADC value of the injury liver. Each rabbit of the normal control group was only carried out MR-DWI scan and calculated the mean ADC value of the normal liver.Results:On day 7 of AHI establishment after MBMCs transplantation, the mean ADC values of the injury livers of the transplanted group and the control group were significant lower than those of the normal control group (p<0.05). The mean ADC values of the injury livers of the transplanted group and the control group increased to the mean ADC values of the normal control group over time. At the same time point observed, The mean ADC values of the injury livers of the transplanted group were significant higher than those of the control group when the b value was same.No matter the b value was 100 s/mm2 or 400 s/mm2, there was negative correlation between the histopathological scores and the mean ADC values in the transplanted group and the control group (the transplanted group:-0.805,-0.883; the control group:-0.628,-0.762) (p<0.05), but there was positive correlation between the average MVD values and the mean ADC values in the transplanted group and the control group (the transplanted group:0.946,0.840; the control group:0.884,0.654) (p<0.05). When the b value was 100 s/mm2, the correlation coefficient between the histopathological scores and the mean ADC values was lower than that between the average MVD values and the mean ADC values,at the same time, it also was lower than that between the histopathological scores and the mean ADC values when the b value was 400 s/mm2. When the b value was 100 s/mm2, the correlation coefficient between the average MVD values and the mean ADC values was higher than that when the b value was 400 s/mm2. When the b value was 400 s/mm2, the correlation coefficient between the histopathological scores and the mean ADC values was higher than that between the average MVD values and the mean ADC valuesConclusions:The recovery of the inury liver can be detected with observing dynamic change of its mean ADC value. The lower the b value, the higher the correlation coefficient between the average MVD values and the mean ADC values, which meant the influence of blood perfusion became more obvious to the mean ADC values of the injury livers with the decrease of b value. The higher the b value, the higher the correlation coefficient between the histopathological scores and the mean ADC values, which meant the mean ADC values became more truly reflect the diffusion of water molecules themselves in the injury liver with the increase of the b value.